Treatment and prevention of microbial infections

Abstract

The present invention provides methods for treating or preventing microbial (e.g., bacterial) infections and means for practicing such methods. In particular, the present invention enables the treatment of infections requiring rapid and sustained therapy, such as for treating acute conditions such as sepsis, sepsis, SIRS, or septic shock. The present invention is also particularly useful for treating microbial infections, for example, for environmental, food, and beverage use. The present invention relates, inter alia, to methods for controlling microbial corrosion (MIC) or biofouling of industrial or domestic substrates or fluids. The present invention is also useful for treating pathogenic bacterial infections in subjects undergoing transplantation or treatment for a disease or condition, such as treatment for cancer, viral infection, or autoimmune disease.

Description

[Technical Field]

[0001] The present invention provides methods for treating or preventing microbial (e.g., bacterial) infections and means for practicing such methods. In particular, the present invention allows for the treatment of infections requiring rapid or sustained therapy, such as for treating acute conditions such as septicemia, sepsis, SIRS, or septic shock. The present invention is also particularly useful for the treatment of microorganisms, for example, for environmental, food, and beverage uses. The present invention relates, inter alia, to methods for controlling microbiologically influenced corrosion (MIC) or biofouling of substrates or fluids in industrial or domestic systems.

[0002] The present invention is also useful for treating pathogenic bacterial infections in subjects undergoing transplantation or treatment for a disease or condition, such as treatment for cancer, viral infection, or autoimmune disease. [Background technology]

[0003] Sepsis is an acute and serious blood infection. Sepsis is also known as bacteremia or blood poisoning. Sepsis occurs when a bacterial infection elsewhere in the body, such as in the lungs or skin, enters the bloodstream. This is dangerous because bacteria and their toxins can be carried throughout the subject's body via the bloodstream. Sepsis can quickly become a life-threatening condition. Sepsis must be treated promptly, such as in a hospital. If left untreated, sepsis can progress to sepsis.

[0004] Sepsis and sepsis are not the same. Sepsis is a serious complication of sepsis. Sepsis occurs when inflammation occurs throughout the body. This inflammation can cause blood clots, blocking oxygen from reaching vital organs and leading to organ failure. According to the National Institutes of Health (NIH), it is estimated that more than one million Americans suffer from severe sepsis each year. 28 to 50 percent of these patients may die from the condition. When inflammation occurs and blood pressure becomes very low, it is called septic shock. Septic shock is often fatal.

[0005] The increasing average age of the population, more people with chronic diseases, people on immunosuppressive medications, and an increasing number of invasive procedures being performed are linked to an increasing rate of sepsis. People over 65 years of age, especially those with health problems, are even more susceptible to sepsis than any other group. According to a study published in 2006, people aged 65 and older make up approximately 12% of the U.S. population and account for 65% of sepsis cases in hospitals.

[0006] Sepsis is caused by an infection in a part of the body. The infection is typically acute. Many types of bacteria can lead to sepsis. The exact source of the infection often cannot be determined. The most common infections leading to sepsis are: · Urinary tract infection Lung infections such as pneumonia · Kidney infection Infections in the abdominal area

[0007] The bacteria in these infections enter the bloodstream and multiply rapidly, causing acute infection and immediate symptoms.

[0008] People who are already hospitalized for something else, such as surgery, are at higher risk of developing sepsis. Secondary infections may occur during hospitalization. These infections are often more dangerous because the bacteria may already be resistant to antibiotics. A subject is at higher risk of developing sepsis if they: Have severe wounds or burns Being very young or very old Have a compromised immune system, which may result from diseases such as HIV or leukemia - Having a urinary or intravenous catheter Being on a mechanical ventilator · You are undergoing medical treatment that weakens your immune system, such as chemotherapy or steroid injections.

[0009] Symptoms of sepsis usually begin very rapidly. Even in the first stage of the illness, an individual may appear very ill. Symptoms of sepsis may occur after trauma, surgery, or another localized (e.g., restricted to one location) infection such as pneumonia. The most common early symptoms are: Chills High body temperature (fever) Very rapid breathing Tachycardia

[0010] More severe symptoms will begin to appear as sepsis progresses if not treated appropriately. These symptoms include: Confusion or inability to think clearly Nausea and vomiting Red spots appearing on the skin Reduced urine volume Inadequate blood flow (shock)

[0011] Sepsis, which begins to affect organ or tissue function, is an acute medical emergency. Sepsis must be treated promptly in a hospital. Many people with sepsis enter a hospital's ICU for treatment and recovery. It is recommended not to take a "wait and see" approach or try to treat the problem at home. If a subject shows signs of sepsis, it is important to rush to a hospital immediately. Sepsis has several serious complications that can be fatal if left untreated or if treatment is delayed for too long.

[0012] Septic shock One complication of sepsis is a severe drop in blood pressure. This is called septic shock. Toxins released by bacteria in the bloodstream can cause very low blood flow, which can lead to organ or tissue damage. Septic shock is an acute medical emergency. People with septic shock are usually cared for in a hospital intensive care unit (ICU). In cases of septic shock, patients may need to be placed on a ventilator, a breathing machine.

[0013] Acute respiratory distress syndrome (ARDS) Another complication of sepsis is acute respiratory distress syndrome (ARDS), a life-threatening condition that prevents enough oxygen from reaching the lungs and blood. According to the National Heart, Lung, and Blood Institute (NHLBI), ARDS is fatal in about one-third of cases. ARDS often results in some level of permanent lung damage. ARDS can also damage the brain, which can lead to memory problems.

[0014] Sepsis Sepsis occurs when the body mounts a strong immune response to an infection. This leads to widespread inflammation throughout the body. When it leads to organ failure, it is called severe sepsis. People with chronic diseases such as HIV or cancer are at higher risk of sepsis because their immune systems are weaker and unable to fight off infections on their own. Sepsis causes millions of deaths worldwide each year and is the most common cause of death among hospitalized people. The global incidence of sepsis is estimated to be 18 million cases per year. In the United States, sepsis affects approximately 3 in 1,000 people, and severe sepsis contributes to more than 200,000 deaths per year. Sepsis occurs in 1-2% of all hospitalizations and accounts for as much as 25% of ICU bed occupancy.

[0015] Early diagnosis and appropriate management of sepsis are necessary because prompt initiation of therapy is key to reducing mortality from severe sepsis. Within the first 3 hours of suspected sepsis, diagnostic studies should include a white blood cell count, measurement of serum lactate, and obtaining appropriate cultures before initiating antibiotics, unless delaying the use of diagnostic studies for more than 45 minutes.

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

[0017] Speed ​​of treatment is essential. Two sets of blood cultures (aerobic and anaerobic) should be obtained without delaying the initiation of antibiotics. Cultures may also be obtained from other sites, such as respiratory secretions, urine, wounds, cerebrospinal fluid, and catheter insertion sites (in situ for longer than 48 hours) if infection originating from these sites is suspected. Broad-spectrum antibiotics (usually two broad-spectrum beta-lactam antibiotics or a broad-spectrum carbapenem combined with a fluoroquinolone, macrolide, or aminoglycoside) are traditionally used in severe sepsis and septic shock. However, antibiotic combinations are not recommended for the treatment of sepsis in immunocompromised individuals without shock, unless the combination is used to broaden antibacterial activity. Antibiotic administration is critical in determining an individual's survival. Some recommend administration within one hour of diagnosis, and each hour of antibiotic administration delay is associated with a 6% increase in mortality.

[0018] Early goal-directed therapy (EGDT) is a strategy for managing severe sepsis during the first 6 hours after diagnosis. It is a stepwise approach, and the physiological goals are to optimize cardiac preload, afterload, and contractility. This strategy involves the early administration of antibiotics.

[0019] Neonatal sepsis is difficult to diagnose because newborns may be asymptomatic. If a newborn exhibits signs and symptoms suggestive of sepsis, antibiotics are initiated immediately, either modified to target specific organisms identified by diagnostic tests, or discontinued after infectious causes of the symptoms have been ruled out.

[0020] Approximately 20-35% of people with severe sepsis and 30-70% of people with septic shock will die. The Surviving Sepsis Campaign (SSC) is a global initiative bringing together expert organizations to reduce sepsis mortality. Antibiotics are administered within two hours of admission / diagnosis. After the onset of septic shock, every hour a patient is denied antibiotic therapy reduces the patient's chances of survival by 7.9% (Survivesepsis.org 2005).

[0021] Therefore, there is a need for rapid treatment of acute microbial infections, such as bacterial infections associated with septicemia, sepsis, or septic shock. It would be advantageous if treatment could last for hours. Rapid and sustained treatment of microorganisms is also desirable for controlling microbial corrosion (MIC) or biofouling of substrates in industrial and domestic systems. [Prior art documents] [Patent documents]

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[0024] Acute bacterial infections can, in some circumstances, be health-threatening or even life-threatening. This may be the case, for example, in cancer patients, organ transplant patients, or other subjects. The need for treatment of bacterial infections can be urgent and, indeed, is the focus of immediate medical care. It would be useful to provide a method for treating such pathogenic bacterial infections that does not adversely affect the effectiveness of cancer therapy or other separate therapies to which the patient also needs to respond. [Means for solving the problem]

[0025] The present invention provides a solution by using the action of programmable nuclease cleavage of microbial genomes. This is distinct from other mechanisms of action used by metabolic inhibitors and beta-lactams and other conventional antibiotics to treat infectious diseases. Targeted cleavage provides selective microbial kill or reduction in growth or proliferation to treat or prevent infectious diseases. Furthermore, the inventors have surprisingly found that in some embodiments, substantial kill (several logs) can be achieved very quickly (e.g., within 15 minutes) and a sustainable effect can be achieved (e.g., for more than one hour, even up to about three hours, after initiation of treatment). Accordingly, the present invention provides the following compositions:

[0026] First configuration A programmable nuclease for use in a method for treating a microbial infection in a subject, wherein the microbial infection is caused by a first species or strain of microorganism, and the nuclease is programmable to cleave a target site contained by the genome of the microorganism that has infected the subject, thereby killing the first species or strain of microorganism or reducing its growth or proliferation, and the method of treatment comprises contacting the subject with the nuclease, wherein the nuclease is programmed to cleave the target site, thereby cleaving the genome of the microorganism contained by the subject and treating the microbial infection in the subject.

[0027] Second Configuration a plurality of viruses (e.g., phages or phagemids for producing phages) for use with a programmable nuclease in a method for treating a microbial infection in a subject, wherein the microbial infection is caused by a first species or strain of microorganism, and the nuclease is programmable to cleave a target site contained by the genome of the microorganism infecting the subject, thereby killing or reducing the growth or proliferation of the first species or strain of microorganism; the method of treatment includes contacting the subject with the nuclease and the virus, and the nuclease is programmed to cleave the target site, thereby cleaving the genome of the microorganism contained by the subject and treating the microbial infection in the subject; each virus comprises a copy of a nucleic acid encoding an RNA for expressing the RNA in the subject, the RNA complexing with a nuclease and programming the nuclease to cleave a target site in a microorganism contained by the subject; A virus is a virus capable of infecting and delivering nucleic acid to a microorganism contained by a subject.

[0028] Third Configuration A composition comprising a plurality of nucleic acids for programming a programmable nuclease in a method for treating a microbial infection in a subject, wherein the microbial infection is caused by a first species or strain of microorganism, and the nuclease is programmable to cleave a target site contained by the genome of the microorganism infecting the subject, thereby killing or reducing the growth or proliferation of the first species or strain of microorganism, the method of treatment comprising contacting the subject with the nuclease and the nucleic acid, and the nuclease is programmed to cleave the target site, thereby cleaving the genome of the microorganism contained by the subject and treating the microbial infection in the subject; A composition wherein each nucleic acid encodes an RNA for expressing the RNA in a subject, wherein the RNA complexes with a nuclease and programs the nuclease to cleave a target site in a microorganism contained by the subject.

[0029] Fourth Configuration A CRISPR / Cas system comprising a nuclease according to the invention for use in a method of treatment, wherein the nuclease is a Cas nuclease (e.g., Cas3 or Cas9), and the system comprises one or more guide RNAs or DNA encoding one or more guide RNAs, each guide RNA being capable of programming the Cas nuclease to cleave a target site contained by the genome of a microorganism.

[0030] Fifth Configuration A method for treating a microbial infection in a subject, wherein the microbial infection is caused by a first species or strain of microorganism, and a nuclease is programmable to cleave a target site contained by the genome of the microorganism that has infected the subject, thereby killing the first species or strain of microorganism or reducing its growth or proliferation, the method of treatment comprising contacting the subject with the nuclease, wherein the nuclease is programmed to cleave the target site, thereby cleaving the genome of the microorganism contained by the subject and treating the microbial infection in the subject.

[0031] Sixth Configuration A method for treating a microbial infection in a subject, wherein the microbial infection is caused by a first species or strain of microorganism, and a nuclease is programmable to cleave a target site contained by the genome of the microorganism that has infected the subject, thereby killing the first species or strain of microorganism or reducing its growth or proliferation, the method of treatment comprising contacting the subject with a nuclease and a plurality of viruses, wherein the nuclease is programmed to cleave the target site, thereby cleaving the genome of the microorganism contained by the subject and treating the microbial infection in the subject, each virus containing a copy of a nucleic acid encoding an RNA for expressing the RNA in the subject, the RNA complexing with the nuclease and programming the nuclease to cleave the target site in the microorganism contained by the subject, and the viruses are capable of infecting and delivering the nucleic acid to the microorganism contained by the subject.

[0032] Seventh Configuration A method for treating a microbial infection in a subject, wherein the microbial infection is caused by a first species or strain of microorganism, and a nuclease is programmable to cleave a target site contained by the genome of the microorganism infecting the subject, thereby killing or reducing the growth or proliferation of the first species or strain of microorganism, the method of treatment comprising contacting the subject with a nuclease and a plurality of nucleic acids, wherein the nuclease is programmed to cleave the target site, thereby cleaving the genome of the microorganism contained by the subject and treating the microbial infection in the subject, wherein each virus contains a copy of a nucleic acid encoding an RNA for expressing the RNA in the subject, the RNA complexes with the nuclease and programs the nuclease to cleave the target site in the microorganism contained by the subject, and each nucleic acid encodes an RNA for expressing the RNA in the subject, the RNA complexes with the nuclease and programs the nuclease to cleave the target site in the microorganism contained by the subject.

[0033] Eighth Configuration Use of a nuclease, a plurality of viruses, a system, a guide RNA, a DNA or a vector of the invention in the manufacture of a composition for carrying out a method of treatment as defined herein, wherein the subject is an organism other than a human or an animal.

[0034] 9th Configuration Use of a nuclease, multiple viruses, systems, guide RNAs, DNA, or vectors of the present invention in the manufacture of a composition for performing an ex vivo or in vitro method for treating a microbial infection of a substrate, wherein the microbial infection is caused by a first species or strain of microorganism, and the nuclease is programmable to cleave a target site contained by the genome of the microorganism that has infected the subject, thereby killing the first species or strain of microorganism or reducing its growth or proliferation, and the method of treatment comprises contacting the subject with the nuclease, wherein the nuclease is programmed to cleave the target site, thereby cleaving the genome of the microorganism contained by the subject and treating the acute microbial infection of the substrate.

[0035] 10th Configuration Use of a programmable nuclease in the manufacture of a composition for performing an ex vivo method for treating a microbial infection of a substrate, wherein the microbial infection is caused by a first species or strain of microorganism, and the nuclease is programmable to cleave a target site contained by the genome of the microorganism that has infected the subject, thereby killing the first species or strain of microorganism or reducing its growth or proliferation, and the method of treatment includes contacting the subject with the nuclease, wherein the nuclease is programmed to cleave the target site, thereby cleaving the genome of the microorganism contained by the subject and treating the acute microbial infection of the substrate.

[0036] In any configuration, for example, the infection is an acute infection. For example, the infection is an acute infection that is rapidly treated. For example, the infection is rapidly treated, e.g., the method includes reducing the infection by at least 100-fold within the first 30 minutes of treatment (e.g., within the first 15 minutes). For example, the treatment is sustained, e.g., the reduction in infection persists for at least 30 minutes immediately after the first 30 minutes of treatment. Also, optionally, the at least 100-fold or 1000-fold reduction in infection is maintained for at least 60 minutes (e.g., at least 120 minutes) after initiation of treatment. Surprisingly, examples demonstrating this are provided below, including rapid kill sustained for about 3 hours after initiation of treatment. For example, the method improves survival of a subject or improves the survival rate of a human or human patient suffering from an infection caused by a first species or strain of microorganism.

[0037] The present invention also provides a solution to the need for effective treatment of pathogenic bacterial infections in subjects undergoing cancer or other separate therapies that must also be effective. Accordingly, the present invention further provides:

[0038] 11th Configuration A method for treating a pathogenic bacterial infection in a human or animal subject caused by a first species or strain of bacteria (first bacterium), comprising selectively killing the first bacterium contained by the subject by cleaving a target site contained by the genome of the first bacterium, wherein the cleavage is carried out using a programmable nuclease that is programmed to cleave the target site, wherein the subject is suffering from an additional disease or condition other than the pathogenic bacterial infection, and the method comprises administering a therapy to the subject to treat or prevent the additional disease or condition, wherein the nuclease treats the infection and the therapy, in the presence of the programmed nuclease, is effective in treating or preventing the disease or condition.

[0039] 12th Configuration A method for treating a pathogenic bacterial infection in a cancer patient caused by a first species or strain of bacteria (first bacterium), comprising selectively killing the first bacterium contained by the subject by cleaving a target site contained by the genome of the first bacterium, wherein the cleavage is carried out using a Cas nuclease programmed to cleave the target site using a guide RNA, the method also comprising administering immunotherapy to the subject to treat the patient's cancer, wherein the nuclease treats the infection and the immunotherapy, in the presence of the programmed nuclease, is effective in treating the cancer.

[0040] 13th Configuration Programmable nucleases for use in the methods of the present invention.

[0041] 14th Configuration 13. A CRISPR / Cas system comprising a nuclease according to a thirteenth configuration for use in the method of the eleventh or twelfth configuration, wherein the nuclease is a Cas nuclease (e.g., Cas3 or Cas9), and the system comprises one or more guide RNAs (gRNAs) or DNA encoding the one or more guide RNAs, each guide RNA capable of programming the Cas nuclease to cleave a target site contained by the genome of a first bacterium.

[0042] 15th Configuration A guide RNA or DNA encoding a guide RNA for use in a system or method for treating a pathogenic bacterial infection.

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

[0044] 17th Configuration A pharmaceutical composition comprising a first nucleic acid vector(s) encoding a nuclease and a second nucleic acid vector(s) encoding a guide RNA. [Brief explanation of the drawings]

[0045] [Figure 1] Figure 1 shows the time-kill curve of Escherichia coli (EHEC) ATCC43888 strain harboring the CGV system. (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 for up to 60 minutes. (b) 30 minutes after induction, a dilution series (101-106) of E. coli ATCC43888 harboring the CGV system was spotted (5 μl) on an LB agar plate. [Figure 2] CRISPR killing of the target strain Escherichia coli (EHEC) ATCC43888 in Galleria mellonella larvae. G. mellonella larvae were delivered by injecting bacteria behind the terminal left protruding leg. Approximately 1 hour after injection, the CRISPR inducer was administered behind the terminal right protruding leg. Larvae were incubated at 37°C for 2 hours and then sacrificed. A control group was also injected with control bacteria carrying an off-target single-stranded guide RNA plasmid. [Figure 3] Figure 1 shows the CRISPR-killing curve of Escherichia coli (EHEC) ATCC43888 in Galleria mellonella. G. mellonella larvae were delivered by injecting bacteria behind the terminal left protruding leg. Approximately 1 hour after injection, the CRISPR inducer was administered behind the terminal right protruding leg. Larvae were incubated at 37°C and sacrificed at 0, 1, and 2 hours after induction. [Figure 4] Kaplan-Meier survival curves for Galleria mellonella larvae infected with Escherichia coli (EHEC) ATCC 43888. CRISPR induction significantly improved larval survival (black line) compared to the off-target control (dotted line) harboring an off-target single-stranded guide RNA plasmid. [Figure 5]Figure 1 shows the time-kill curve of Escherichia coli Nissle 1917 harboring the pks-targeting CGV system. (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 E. coli Nissle 1917 harboring the CGV system 15 minutes after induction on an LB agar plate, drop-spotting (5 μl). [Figure 6] Time-kill curves of Escherichia coli Nissle 1917 harboring 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) of E. coli Nissle 1917 harboring a CGV system on an LB agar plate 15 minutes after induction. [Figure 7] Figure 1 shows complete killing of transconjugant C. difficile. Figure 1 shows complete and precise killing of Clostridium difficile using gRNA-encoding CRISPR arrays delivered from a probiotic carrier bacterial species by a conjugative plasmid as a vector. Carrier bacteria (E. coli donor strain containing the vector) mated with Clostridium difficile were killed upon delivery of the indicated array. This utilized the endogenous Cas3 machinery of Clostridium difficile. 100% killing of Clostridium difficile cells was achieved, as shown in this figure. [Figure 8]Antibiotic treatment during ICI therapy has been associated with fatal outcomes. Kaplan-Meier curves for overall survival in a validation cohort at Memorial Sloan Kettering Cancer Center, including n=239 patients with advanced NSCLC treated with anti-PD-L1 / anti-PD-1 mAbs who either received (ATB, n=68) or did not receive (no ATB, n=171) antibiotics 2 months before immune checkpoint blockade injection. Median overall survival in the absence of antibiotic treatment was 21.9 months, compared with 9.8 months with antibiotic treatment. Thus, median overall survival for patients treated with classical antibiotics was <50% (or >12 months shorter) than that for patients not receiving antibiotic treatment. [Figure 9A] This figure shows that the gut microbiome modulates the efficacy of anti-PD-1 blockade in melanoma patients (Gopalakrishnan et al., Science 2018, 359, 97-103). [Figure 9B] This figure shows that the gut microbiome modulates the efficacy of anti-PD-1 blockade in melanoma patients (Gopalakrishnan et al., Science 2018, 359, 97-103). DETAILED DESCRIPTION OF THE INVENTION

[0046] The approach of the present invention differs from conventional antibiotic approaches. It employs targeted cleavage of microbial genomes using programmed nucleases, whereas conventional antibiotics rely on metabolic processes and cellular replication cycles and their inhibition for their activity. By focusing instead on nuclease cleavage, the present invention surprisingly achieves very rapid, efficient, and remarkably durable microbial kill. This is demonstrated in the following experiments with different microorganisms, different nucleases, and different delivery techniques. Surprisingly, typically 99-100% kill was repeatedly observed. Furthermore, 3-4 log kill was achieved very rapidly and over a long period of time.

[0047] The present invention provides methods for treating or preventing microbial (e.g., bacterial) infections and means for practicing such methods. In particular, infections requiring rapid therapy, such as for treating acute conditions such as sepsis, sepsis, SIRS, or septic shock, can be treated. As described herein, rapid response is critical in addressing microbial infections in many settings. Speed ​​is of the essence in many infectious disease scenarios, such as acute infections requiring hospitalization. The benefits of the present invention may be one or more of: reducing the spread, severity, or progression of the infection in a subject; reducing the onset, severity, or progression of symptoms of the infection (e.g., sepsis or septic shock); or increasing the chances of survival of a human or animal patient.

[0048] The present invention utilizes programmable nuclease cleavage of microbial genomes. Targeted cleavage provides selective microbial kill or growth or proliferation reduction to treat or prevent infection, as opposed to the more broad-spectrum microbial kill of several different species seen with conventional antibiotics. Selective kill advantageously leaves beneficial microorganisms that are not the target of treatment intact, which can be beneficial to the patient. Furthermore, the inventors have surprisingly found that in some embodiments, substantial (multiple log) kill can be achieved very quickly (e.g., within 15 minutes) and sustained effects can be achieved (e.g., for longer than one hour). As exemplified below, the inventors have surprisingly achieved remarkably rapid and sustained kill over a period of approximately 2-3 hours.

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

[0050] A programmable nuclease for use in a method for treating a microbial infection (e.g., an acute microbial infection) in a subject, wherein the microbial infection is caused by a first species or strain of microorganism, and the nuclease is programmable to cleave a target site contained by the genome of the microorganism that has infected the subject, and the first species or strain of microorganism is killed or the growth or proliferation of the microorganism is reduced, the method of treatment comprising contacting the subject with the nuclease, and the nuclease being programmed to cleave the target site, thereby cleaving the genome of the microorganism contained by the subject and treating the microbial infection in the subject.

[0051] Another aspect provides a programmable nuclease for use in a method for rapidly treating an acute microbial (e.g., bacterial) infection in a subject, wherein the microbial infection is caused by a first species or strain of microorganism, and the nuclease is programmable to cleave a target site contained by the genome of the microorganism infecting the subject, thereby killing or reducing the growth or proliferation of the first species or strain of microorganism, and the method of treatment includes contacting the subject with the nuclease, wherein the nuclease is programmed to cleave the target site, thereby cleaving the genome of the microorganism contained by the subject and rapidly treating the acute microbial infection in the subject.

[0052] Another aspect provides a programmable nuclease for use in a method for treating a microbial (e.g., bacterial) infection in a subject, wherein the microbial infection is caused by a first species or strain of microorganism, and the nuclease is programmable to cleave a target site contained by the genome of the microorganism that has infected the subject, thereby killing or reducing the growth or proliferation of the first species or strain of microorganism, and the method of treatment comprises contacting the subject with the nuclease and a nucleic acid that programs the nuclease to recognize and cleave the target site, thereby cleaving the genome of the microorganism contained by the subject and treating the microbial infection in the subject.

[0053] Another aspect provides a programmable nuclease for use in a method for rapidly treating an acute microbial (e.g., bacterial) infection in a subject, wherein the microbial infection is caused by a first species or strain of microorganism, and the nuclease is programmable to cleave a target site contained by the genome of the microorganism that has infected the subject, thereby killing or reducing the growth or proliferation of the first species or strain of microorganism, and the method of treatment comprises contacting the subject with the nuclease and a nucleic acid that programs the nuclease to recognize and cleave the target site, thereby cleaving the genome of the microorganism contained by the subject and rapidly treating the acute microbial infection in the subject.

[0054] Another aspect provides a programmable nuclease for use in a method for persistently treating a microbial (e.g., bacterial) infection in a subject, wherein the microbial infection is caused by a first species or strain of microorganism, and the nuclease is programmable to cleave a target site contained by the genome of the microorganism that has infected the subject, thereby persistently killing or reducing the growth or proliferation of the first species or strain of microorganism, and the method of treatment includes contacting the subject with the nuclease, wherein the nuclease is programmed to cleave the target site, thereby cleaving the genome of the microorganism contained by the subject and treating the microbial infection in the subject.

[0055] Another aspect provides a programmable nuclease for use in a method for persistently treating a microbial (e.g., bacterial) infection in a subject, wherein the microbial infection is caused by a first species or strain of microorganism, and the nuclease is programmable to cleave a target site contained by the genome of the microorganism that has infected the subject, thereby persistently killing or reducing the growth or proliferation of the first species or strain of microorganism, and the method of treatment comprises contacting the subject with the nuclease and a nucleic acid that programs the nuclease to recognize and cleave the target site, thereby cleaving the genome of the microorganism contained by the subject and treating the microbial infection in the subject.

[0056] Another aspect provides a programmable nuclease for use in a method for persistently treating an acute microbial (e.g., bacterial) infection in a subject, wherein the microbial infection is caused by a first species or strain of microorganism, and the nuclease is programmable to cleave a target site contained by the genome of the microorganism that has infected the subject, and the first species or strain of microorganism is persistently killed or the growth or proliferation of the microorganism is reduced, the method of treatment comprising contacting the subject with the nuclease and a nucleic acid that programs the nuclease to recognize and cleave the target site, thereby cleaving the genome of the microorganism contained by the subject and treating the acute microbial infection in the subject.

[0057] Surprisingly, as exemplified below, when using nucleases (as opposed to conventional means for conventional antibiotic killing), a sustained effect of several logs (e.g., 3 or 4 logs) was observed approximately 3 hours after the first contact of the programmed nuclease with the bacteria. Thus, this aspect of the invention allows for dosing regimens for less frequent contact with the programmed nuclease (i.e., less frequent administration of programmed nucleases, programmable nucleases, and / or nucleic acids to program the nucleases). For example, a Cas and gRNA (or DNA encoding the gRNA) for programming a nuclease can be administered to a subject at a first time point (T1) and a second time point (T2) along with a programmable nuclease (e.g., Cas9 or Cas3), or a gRNA (or DNA encoding the gRNA) can be administered at T1 and T2 to program an endogenous Cas nuclease (e.g., Cas9 or Cas3) of said first species or strain of bacteria, where the programmed endogenous Cas cleaves the bacterial genome, killing the bacteria or reducing their growth or proliferation, thus treating the infection. Such less frequent dosing is convenient for health care workers and patients, as well as providing an economical therapy. Thus, optionally, the nuclease and / or nucleic acid is administered to the subject at T1 and T2, where T2 is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 24 hours after T1. For example, T2 is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 24 hours after T1. For example, T2 is 2 to 7 hours after T1. 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 nucleic acid that programs the nuclease to recognize and cleave a target site is administered to the subject at T1 and T2, where T2 is at least 1 hour (e.g., 1, 1.5, 2, 2.5, or 3 hours) after T1.

[0059] Another aspect is a Cas nuclease for use in a method for treating a microbial (e.g., bacterial) infection in a subject, wherein the microbial infection is caused by a first species or strain of microorganism, and the nuclease is programmable with a guide RNA (gRNA) to cleave a target site contained by the genome of the microorganism infecting the subject, thereby killing or reducing the growth or proliferation of the first species or strain of microorganism, the method of treatment comprising administering to the subject the nucleic acid, wherein the nucleic acid is a gRNA or DNA encoding the gRNA, thereby programming the nuclease to recognize and cleave the target site in the microorganism contained by the subject, thereby cleaving the genome of the microorganism and treating the microbial infection in the subject, the method comprising administering to the subject the nucleic acid at a first time point (T1) and a second time point (T2), whereby the subject contacts the programmed nuclease at T1 and T2, providing the Cas nuclease, wherein T2 is one or more hours after T1.

[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; optionally, T2 is 12 hours or more after T1; optionally, T2 is 13 hours or more after T1; optionally, T2 is 14 hours or more after T1; optionally, T2 is 24 hours or more after T1. Additionally or alternatively, optionally, T2 is 7 hours or less after T1; optionally, T2 is 12 hours or less after T1; optionally, T2 is 24 hours or less 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; optionally, 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 hour after T1. For example, T2 is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 24 hours after T1. For example, T2 is 1 to 7 hours after T1; or T2 is 2 to 7 hours after T1; or T2 is 3 to 7 hours after T1; or T2 is 4 to 7 hours after T1; or T2 is 5 to 7 hours after T1; or T2 is 6 to 7 hours after T1.

[0061] Optionally, the method includes reducing infection by at least 100-fold within the first 30 minutes of treatment (e.g., within the first 15 minutes). Optionally, the method includes reducing infection by at least 1000-fold within the first 30 minutes of treatment (e.g., within the first 15 minutes). Optionally, the method includes reducing infection by at least 10,000-fold within the first 30 minutes of treatment (e.g., within the first 15 minutes).

[0062] Optionally, the method includes reducing infection such that the reduction in infection is sustained for 30 minutes immediately after the first 30 minutes of treatment. Optionally, the method includes reducing infection such that a reduction in infection by at least 100-fold is sustained for 30 minutes immediately after the first 30 minutes of treatment. Optionally, the method includes reducing infection such that a reduction in infection by at least 1000-fold is sustained for 30 minutes immediately after the first 30 minutes of treatment. Optionally, the method includes reducing infection such that a reduction in infection by at least 10,000-fold is sustained for 30 minutes immediately after the first 30 minutes of treatment.

[0063] Optionally, the method includes reducing infection by at least 100-fold within the first 30 minutes of treatment (e.g., within the first 15 minutes), wherein the at least 100-fold reduction in infection persists for 30 minutes immediately after the first 30 minutes of treatment. Optionally, the method includes reducing infection by at least 1000-fold within the first 30 minutes of treatment (e.g., within the first 15 minutes), wherein the at least 1000-fold reduction in infection persists for 30 minutes immediately after the first 30 minutes of treatment. Optionally, the method includes reducing infection by at least 10,000-fold within the first 30 minutes of treatment (e.g., within the first 15 minutes), wherein the at least 10,000-fold reduction in infection persists for 30 minutes immediately after the first 30 minutes of treatment.

[0064] Optionally, the method includes maintaining at least a 100-fold reduction in infection for at least 60 minutes (e.g., at least 120, 145, or 180 minutes) after contacting the subject with the programmed nuclease. Optionally, the at least a 100-fold reduction in infection is maintained for at least 60 minutes (e.g., at least 120, 145, or 180 minutes) after contacting the subject with the programmed nuclease. Optionally, the method includes maintaining at least a 1000-fold reduction in infection for at least 60 minutes (e.g., at least 120, 145, or 180 minutes) after contacting the subject with the programmed nuclease. Optionally, the at least a 1000-fold reduction in infection is maintained for at least 60 minutes (e.g., at least 120, 145, or 180 minutes) after contacting the subject with the programmed nuclease. Optionally, the method includes maintaining at least a 10,000-fold reduction in infection for at least 60 minutes (e.g., at least 120, 145, or 180 minutes) after contacting the subject with the programmed nuclease. Optionally, the at least a 10,000-fold reduction in infection is maintained for at least 60 minutes (e.g., at least 120, 145, or 180 minutes) after contacting the subject with the programmed nuclease.

[0065] Optionally, the method includes reducing infection by at least 100-fold within the first 30 minutes (e.g., within the first 15 minutes) of treatment, wherein the at least 100-fold reduction in infection is maintained for at least 60 minutes (e.g., at least 120, 145, or 180 minutes) after contacting the subject with the programmed nuclease. Optionally, the method includes reducing infection by at least 1000-fold within the first 30 minutes (e.g., within the first 15 minutes) of treatment, wherein the at least 1000-fold reduction in infection is maintained for at least 60 minutes (e.g., at least 120, 145, or 180 minutes) after contacting the subject with the programmed nuclease. Optionally, the method includes reducing infection by at least 10,000-fold within the first 30 minutes (e.g., within the first 15 minutes) of treatment, and the at least 10,000-fold reduction in infection is maintained for at least 60 minutes (e.g., at least 120, 145, or 180 minutes) after contacting the subject with the programmed nuclease.

[0066] Optionally, the method includes reducing infection by at least 10,000-fold within the first 15 minutes of treatment, and the at least 10,000-fold reduction in infection is maintained for at least 45 minutes after contacting the subject with the programmed nuclease, as exemplified below.

[0067] In examples, the infection is treated persistently, and at least a 100-fold reduction in infection is maintained for at least 60 minutes (e.g., at least 120, 145, or 180 minutes) after treatment begins. In examples, the infection is treated persistently, and at least a 1000-fold reduction in infection is maintained for at least 60 minutes (e.g., at least 120, 145, or 180 minutes) after treatment begins. In examples, the infection is treated persistently, and at least a 10,000-fold reduction in infection is maintained for at least 60 minutes (e.g., at least 120, 145, or 180 minutes) after treatment begins.

[0068] Optionally, the infection is reduced by at least 100,000 fold by the first 30 or 45 minutes of treatment. Optionally, the infection is reduced by at least 100,000 fold by the first 30 or 45 minutes of treatment, and the reduction is maintained through 60 minutes of treatment.

[0069] Optionally, the infection is reduced by at least 1,000,000 fold by the first 30 or 45 minutes of treatment. Optionally, the infection is reduced by at least 1,000,000 fold by the first 30 or 45 minutes of treatment, and the reduction is maintained through 60 minutes of treatment.

[0070] Optionally, infection is reduced by at least 100-fold by the first 15 minutes of treatment. Optionally, infection is reduced by at least 1000-fold by the first 15 minutes of treatment. Optionally, infection is reduced by at least 100-fold by the first 15 minutes of treatment, and by at least 1000-fold by the first 30 minutes of treatment.

[0071] For example, the reduction is maintained for at least an additional 15 minutes, e.g., the infection is reduced by at least 100-fold or at least 1000-fold by the first 15 minutes of treatment, and the reduction is maintained from 15-30 minutes of treatment, or 15-45 minutes of treatment, or 15-60 minutes of treatment.

[0072] For example, the infection is reduced by at least 100 fold, or at least 1000 fold, or at least 10,000 fold by the first 15 minutes of treatment, and the reduction is maintained from 15-30 minutes or 15-45 minutes of treatment.

[0073] Optionally, the method includes reducing infection by at least 100-fold within the first 30 minutes of treatment (eg, within the first 15 minutes).

[0074] Optionally, the method includes reducing infection by at least 1000-fold within the first 30 minutes of treatment (eg, within the first 15 minutes).

[0075] Optionally, the method includes reducing infection by at least 10,000 fold within the first 30 minutes of treatment (eg, within the first 15 minutes).

[0076] Optionally, the method includes reducing infection so that the reduction in infection persists for 30 minutes immediately after the first 30 minutes of treatment; for example, the reduction may persist for at least 60 minutes after the first 30 minutes of treatment. If treatment is administered at time zero (T0), the reduction in infection may be present at 60 minutes after T0, and may actually persist 60 minutes after that. In Figures 1(a), 5(a), and 6(a), for example, the reduction is seen 60-180 minutes after T0. Optionally, the reduction in infection persists for at least 30 minutes after the first 30 minutes of treatment.

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

[0078] To determine the kill or reduction in growth or proliferation of a target microorganism, for example, one can determine the difference in the number of microorganisms of a first species or strain in (i) a sample (e.g., a blood, intestine, or leaf sample) taken from the subject immediately before the start of treatment, and (ii) a sample (the same type as the sample in (i), e.g., a blood, intestine, or leaf sample, respectively) taken from the subject 30 minutes into treatment. For example, if the microorganism is a bacterium, the samples may be evaluated for the difference in colony forming units (CFU) / ml sample, for example, when the samples are plated on agar in corresponding Petri dishes and incubated under identical conditions. In another example, microscopic counting of microorganisms in a sample or other routine methods known to those skilled in the art may be used.

[0079] In examples, at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% kill of microorganisms is achieved within the first 30, 60, 90, or 120 minutes of treatment (e.g., within the first 30 minutes or within the first 120 minutes). For example, if the subject is a human or animal, kill is determined by measuring the prevalence of microorganisms (e.g., bacteria) in a blood sample taken immediately before treatment begins (e.g., by standard colony counting on an agar plate) compared to a sample taken after the first 15 or 30 minutes of treatment. In examples, at least 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% kill of microorganisms is achieved within the first 0.5, 1, or 2 hours of treatment. In examples, at least 99% kill of microorganisms is achieved within the first 30 minutes of treatment. In examples, at least 99% kill of microorganisms is achieved by the first 2 hours of treatment. In examples, 100% kill is achieved. These are exemplified below. In one embodiment, less than 100% of microorganisms are killed.

[0080] Examples of bacterial killing are shown below. Surprisingly, when programmed nucleases are used to target selected bacteria, specific cleavage results in rapid killing of the target bacteria. At least 3 or 4 log kills (i.e., 1000-fold or 10,000-fold kills) can be observed in a very short period of time. Surprisingly, these are maintained for at least 1 hour. Optionally, infection is reduced by at least 1000-fold within the first 15, 30, or 45 minutes of treatment. Optionally, infection is reduced by at least 1000-fold within the first 15, 30, or 45 minutes of treatment, and the reduction is maintained until 60, 120, or 180 minutes of treatment. Optionally, infection is reduced by at least 10,000-fold within the first 15, 30, or 45 minutes of treatment. Optionally, the infection is reduced by at least 10,000 fold by the first 15, 30, or 45 minutes of treatment, and the reduction is maintained through 60, 120, or 180 minutes of treatment. See, e.g., the example in Figure 5a.

[0081] In examples, 100% kill is achieved by 24 hours after initiation of treatment.

[0082] In examples, infection is reduced by at least 1000-fold over a period of 2 hours or longer (e.g., 2-3 hours), and optionally, infection is reduced by at least 1000-fold by the first 15 or 13 minutes of treatment.

[0083] In examples, infection is reduced by at least 10,000 fold over a period of 2 hours or longer (e.g., 2-3 hours), and optionally, infection is reduced by at least 10,000 fold by the first 15 or 13 minutes of treatment.

[0084] In examples, infection is reduced by at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% over one hour, or over one hour or longer, or over two hours or longer (e.g., 2-3 hours). Optionally, infection is reduced by at least 90% over one hour, or over one hour or longer, or over two hours or longer (e.g., 2-3 hours), and optionally by the first 30 minutes of treatment (e.g., by the first 15 minutes). Optionally, infection is reduced by at least 90% over one hour or longer, and by the first 30 minutes of treatment (e.g., by the first 15 minutes). Optionally, infection is reduced by at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% by the first 15 or 13 minutes of treatment. Optionally, the infection is reduced by at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% by the first 15 or 13 minutes of treatment, and the reduction is maintained for 1 hour or longer (e.g., 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). An example infection is provided below, where the bacteria is E. coli.

[0085] Optionally, the subject is a human or animal, the microorganism is a bacterium (e.g., E coli or C dificile), and the bacterial blood infection in the subject is reduced by at least 100-fold within the first 30 minutes of treatment (e.g., within the first 15 minutes). Optionally, the subject is a human or animal, the microorganism is a bacterium (e.g., E coli or C dificile), and the bacterial blood infection in the subject is reduced by at least 1000-fold within the first 30 minutes of treatment (e.g., within the first 15 minutes). Optionally, the subject is a human or animal, the microorganism is a bacterium (e.g., E coli or C dificile), and the bacterial blood infection in the subject is reduced by at least 10,00-fold within the first 30 minutes of treatment (e.g., within the first 15 minutes). Optionally, the E coli is EHEC E coli.

[0086] Optionally, the programmed nuclease (e.g., Cas9 or Cas3) is capable of cleaving a target site contained by E. coli (EHEC) ATCC43888. Optionally, the programmed nuclease (e.g., Cas9 or Cas3) is capable of cleaving a target site contained by E. coli Nissle. Optionally, the invention kills E. coli and / or Klebsiella (e.g., K. pneumoniae) that produce extended-spectrum beta-lactamases (ESBLs). Thus, the invention may be for treating or preventing an ESBL bacterial infection in a subject, e.g., a human. For example, the infection is a urinary tract infection (UTI) or sepsis. In examples, the bacteria is resistant to one or all of cephalosporins, penicillins, fluoroquinolones, trimethoprim, and tetracyclines. In examples, the bacteria is carbapenem-resistant E coli or Klebsiella.

[0087] Optionally, the subject's blood is collected immediately prior to treatment. 7 ~10 12 CFU / ml (e.g., 107 ~10 11 , 10 7 ~10 10 , 10 7 ~10 9 , or 10 7 ~10 8 CFU / ml) of bacteria.

[0088] The following examples demonstrate improved survival using the methods of the invention in in vivo models. Thus, in the examples, the methods of the invention are for improving survival in a subject by treating an acute microbial infection in the subject. In the examples, the programmed nucleases herein are capable of killing a first species or strain of bacteria in a Galleria mellonella larval in vivo infection model.

[0089] The nuclease may be, for example, a DNase (e.g., Cpf1, Cas9, or Cas3) or an RNase (e.g., Cas13b). In examples, the nuclease is a class 1 nuclease. In examples, the nuclease is a class 2 nuclease. In examples, the nuclease is a type I nuclease, such as a type IA, IB, IC, ID, IE, IF, or IU nuclease. In examples, the nuclease is a type II nuclease, such as a type IIA, IB, or IC nuclease, such as Cas9, spCas9, or saCas9. In examples, the nuclease is a type III nuclease, such as a type IIIA, IIIB, IIIC, or IIID nuclease. In examples, the nuclease is a type IV nuclease, such as a type IVA or IVB nuclease. In examples, the nuclease is a type V nuclease, such as cpf1. In examples, the nuclease is a type VI nuclease, such as Cas13, such as Cas13a, Cas13b, Cas13c, or Cas13d.

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

[0091] For example, the nuclease is a type IB Cas3, e.g., a Clostridium (e.g., C dificile) Cas3.

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

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

[0094] For example, the nuclease is an IE-type Cas3, such as an E coli or Pseudomonas (e.g., P aeruginosa) Cas3.

[0095] For example, the nuclease is an IF-type Cas3, such as a Pseudomonas (e.g., P. aeruginosa) Cas3.

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

[0097] In examples, the nuclease is Cas6, e.g., Cas6f.

[0098] In examples, the nuclease is an isolated or recombinant nuclease. For example, the nuclease is a synthetic or non-naturally occurring nuclease. In examples, the nuclease is a nickase.

[0099] In examples, the nuclease is ex vivo, e.g., in vitro. In examples, the nucleic acid is ex vivo. In examples, the guide RNA or DNA encoding the guide RNA herein is ex vivo, e.g., 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), or zinc finger nuclease. In examples, the Cas is Streptococcus (e.g., pyogenes or aureus) Cas9, Clostridium (e.g., S. dificile), Salmonella (e.g., S. typhimurium), or E. coli Cas3. For example, the Cas is spCas. In examples, the Cas9 is combined with a tracrRNA or DNA encoding the tracrRNA that is operable with the Cas. For example, the tracrRNA is of the same species as the Cas, e.g., S. pyogenes tracrRNA or DNA encoding it.

[0101] In examples, the nuclease is a Cas3 encoded by a nucleic acid comprising SEQ ID NO: 9, or a sequence at least 80, 85, 90, 95, 96, 97, 98, or 99% identical thereto. Also optionally, the bacterium is a Clostridium (e.g., C. dificile) bacterium or any Clostridium species listed in Table 1, as exemplified below.

[0102] In examples, the nuclease is a Cas9 encoded by a nucleic acid comprising SEQ ID NO: 10, or a sequence at least 80, 85, 90, 95, 96, 97, 98, or 99% identical thereto. In examples, the nuclease is a Cas9 encoded by a nucleic acid comprising SEQ ID NO: 11, or a sequence at least 80, 85, 90, 95, 96, 97, 98, or 99% identical thereto. Also optionally, the bacterium is a Clostridium bacterium (e.g., C. dificile) or any Clostridium bacterium listed in Table 1. Also optionally, the bacterium is an E. coli bacterium (e.g., EHEC), as exemplified below.

[0103] Optionally, the method includes administering to the subject RNA or a nucleic acid (e.g., DNA) encoding the RNA for expression in the subject, wherein the RNA complexes with a nuclease and programs the nuclease to cleave a target site in a microorganism contained by the subject.

[0104] Optionally, the nuclease is administered to the subject simultaneously or sequentially with the RNA or nucleic acid.

[0105] Optionally, the subject contains a nuclease before administering the RNA or nucleic acid to the subject. For example, the nuclease is a Cas nuclease that is an endogenous Cas nuclease of the bacterial cells of the first species or strain contained by the subject. Thus, in this example, the RNA or nucleic acid can be administered to the subject and introduced into the bacterium to program the endogenous Cas contained by the bacterium, thereby forming a programmed Cas nuclease that cleaves the target site in the bacterial genome, thereby killing the bacterium or reducing its growth or proliferation, and thus treating or preventing the infection.

[0106] Optionally, multiple viruses (e.g., phages or phagemids) are administered to the subject, each virus containing a copy (e.g., one or more, e.g., multiple copies) of a nucleic acid, and the viruses infect and deliver the nucleic acid to a microorganism contained by the subject. For example, the viruses herein are phage or phagemids that infect (or are capable of infecting) a first species or strain of bacteria.

[0107] Optionally, the ratio of administered virus to microorganisms contained by the subject is 10 to 150. For example, if the microorganisms are bacteria, the ratio is 10 to 100, i.e., a multiplicity of infection (MOI) of 1 to 100, e.g., 10 to 100 (e.g., if the virus is replicable, e.g., a phage and not a phagemid). The ratio can be determined, for example, by using a sample (e.g., a blood sample or intestinal sample) from a human or animal subject immediately prior to treatment and determining the number of microorganisms (e.g., bacteria per ml of blood sample or intestinal sample). The amount of virus to be administered can then be calculated according to the determination using the sample.

[0108] Optionally, the microorganism is a bacterium. Alternatively, the microorganism is an archaea. Alternatively, the microorganism is a virus. Alternatively, the microorganism is a fungus. Alternatively, the microorganism is an algae. Alternatively, the microorganism is a protozoan.

[0109] In examples, the subject is a human and the infection is a hospital-acquired infection. In examples, the subject is a plant, yeast, protozoan, or amoeba.

[0110] Optionally, the subject is a human (e.g., an adult, child, newborn, infant, teenager, male, or female) or an animal (e.g., a dog, cat, horse, cow, sheep, goat, salmon, chicken, turkey, pig, companion animal, or livestock animal).

[0111] In an example, the subject is a human or animal, and optionally the infection is a lung, abdominal, or urinary tract infection. In an example, the subject has a urinary tract infection, a lung infection such as pneumonia, a kidney infection, or an abdominal infection. In an example, the subject is a surgical patient. In an example, the subject is a burn patient. In an example, the subject has an infected wound (e.g., a bacterially infected wound). In an example, the patient has AIDS or is infected with HIV. In an example, the subject has a blood cancer, such as leukemia, e.g., AML, or CML, or CLL, or a cancer such as lymphoma. In an example, the subject is a tissue or organ transplant patient, e.g., a hematopoietic stem cell transplant patient or a bone marrow organ transplant patient. In an example, the subject has a urinary catheter or an intravenous catheter. In an example, the subject is on a mechanical ventilator. In an example, the subject is receiving an immunosuppressant. In an example, the subject has pneumonia. In an example, the subject is an intensive care unit (ICU) patient. In an example, the subject is an acute respiratory distress syndrome (ARDS) patient. In an example, the subject is suffering from meningitis, a pregnancy infection, a ruptured gallbladder (a ruptured gallbladder is a medical condition in which the gallbladder leaks or ruptures. Rupture is typically caused by inflammation of the gallbladder), abortion-induced septic shock (abortion-induced septic shock is an acute, life-threatening illness), endometritis (endometritis is an inflammatory condition of the lining of the uterus, usually due to an infection), acute respiratory distress syndrome (acute respiratory distress syndrome is a lung condition that occurs when the alveoli of the lungs fill with fluid), or cellulitis.

[0112] The increasing average age of the population, more people with chronic diseases, under immunosuppressive medication, and an increasing number of invasive procedures being performed are linked to an increased rate of sepsis. Optionally, the subject has undergone surgery, is under immunosuppressive medication, and / or suffers from a chronic disease.

[0113] Optionally, the subject is a human older than 60, 65, 70, 75, or 80 years old, or a pediatric patient. Alternatively, the subject is a pediatric patient (e.g., a human infant or child) or an adolescent. In an example, the method treats or prevents neonatal sepsis in a subject. In an example, the subject is an immunocompromised human or animal suffering from an acute viral infection, such as, for example, an HIV infection, or the subject is suffering from cancer, e.g., a blood cancer, such as leukemia, or the patient is a transplant patient who has undergone, for example, an organ transplant, tissue transplant, or bone marrow transplant. In an example, the subject is a human or animal positive for gram-negative bacterial lipopolysaccharide or lipid A. In an example, the subject is a human or animal positive for gram-positive bacterial cell wall lipoteichoic acid.

[0114] Optionally, the method treats or prevents sepsis and / or sepsis (eg, septic shock) in a subject.

[0115] SIRS (systemic inflammatory response syndrome) criteria are used to define sepsis. SIRS involves the presence of two or more of the following: abnormal body temperature, heart rate, respiratory rate, or blood gases, and white blood cell count. Sepsis, for example, is SIRS in response to an infectious process. Severe sepsis, for example, is sepsis with sepsis-induced organ dysfunction or tissue hypoperfusion (manifesting as low blood pressure, elevated lactate, or decreased urine output). Septic shock, for example, is severe sepsis plus persistently low blood pressure despite administration of intravenous fluids.

[0116] In embodiments, the method prevents or slows the progression of end organ dysfunction in a subject (where the subject is a human or animal).

[0117] Examples of end-organ dysfunction include: ·Lungs: Acute respiratory distress syndrome (ARDS) (PaO2 / FiO2<300) Cerebral: Encephalopathy symptoms including agitation, confusion, and coma; causes include ischemia, hemorrhage, microvascular clot formation, microabscesses, and multifocal necrotizing leukoencephalopathy. Liver: Disruption of protein synthesis manifests acutely as a progressive disruption of blood clotting due to an inability to synthesize clotting factors, while disruption of metabolic function leads to impaired bilirubin metabolism, resulting in elevated unconjugated serum bilirubin levels. Renal: Hypo- and anuria, electrolyte abnormalities, or volume overload Heart: Systolic and diastolic heart failure likely due to chemical signals that impair muscle cell function, and cell damage manifests as troponin leakage (although not necessarily ischemic in nature).

[0118] More specific definitions of end-organ dysfunction exist for pediatric SIRS. Cardiovascular dysfunction (after fluid resuscitation with at least 40 ml / kg of crystalloids) Hypotension, as manifested by a blood pressure <5th percentile for age or a systolic blood pressure <2 standard deviations below normal for age; or · The need for vasopressors, or Two of the following criteria: Metabolic acidosis of unknown etiology with a base deficit >5 mEq / L Lactic acidosis: serum lactate twice the upper limit of normal Oliguria (urine output <0.5 ml / kg / h) Long-term capillary refill time of >5 seconds Core-to-peripheral temperature difference >3°C Respiratory dysfunction (in the absence of cyanotic heart disease or known chronic lung disease) The ratio of arterial oxygen tension to the fraction of oxygen in the inspired gas (PaO2 / FiO2) is <300 (definition of acute lung injury), or Arterial carbon dioxide partial pressure (PaCO2) >65 torr (20 mmHg) relative to baseline PaCO2 (evidence of hypercapnic respiratory failure), or The need for supplemental oxygen greater than FiO20.5 to maintain oxygen saturation ≥ 92% Neurological dysfunction Glasgow Coma Score (GCS) ≤ 11, or Mental status changes in individuals with developmental delay / intellectual disability, manifested by a GCS decline of 3 points or greater Hematological dysfunction ·Platelet count<80,000 / mm 3 Or a 50% decrease from the maximum chronic thrombocytopenia International normalized ratio (INR) > 2 ·Disseminated intravascular coagulation Renal dysfunction Serum creatinine ≥ 2 times the upper limit of normal for age or a 2-fold increase in baseline creatinine in people with chronic kidney disease Liver dysfunction (only applicable to infants >1 month old) Total serum bilirubin ≥ 4 mg / dl, or Alanine aminotransferase (ALT) ≥ 2 times the upper limit of normal

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

[0120] Optionally, the method reduces one or more symptoms of the patient selected from fever, hypothermia, rapid breathing, elevated heart rate, confusion, confusion, metabolic acidosis, respiratory alkalemia, hypotension, blood clotting dysfunction (such as blood clotting in one or more organs or subcutaneous bleeding), and edema. Optionally, the method reduces septic shock. Optionally, the sepsis is severe sepsis.

[0121] Optionally, at the start of treatment, the subject (e.g., human) has a body temperature of <36°C or >38°C, a heart rate of >90 / min, a respiratory rate of >20 breaths / min or a PaCO2 of <4.3 kPa, and a CO2 of <4000 / mm 3 or >12,000 / mm 3 have a white blood cell count of

[0122] Optionally, at the start of treatment, the subject (e.g., human) exhibits the presence of two or more of an abnormal body temperature, an abnormal heart rate, an abnormal respiratory rate, an abnormal blood gas, and an abnormal white blood cell count.

[0123] Optionally, the subject is a plant. In an example, the subject is a protist, such as an amoeba. Optionally, in this example, the microorganism is a virus (e.g., a large or giant virus, such as a mimivirus). The nuclease, for example, Cas, can be programmed using a guide RNA delivered by a virophage that infects the viral microorganism.

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

[0125] Preferably, the microorganism is a bacterium. Optionally, the bacterium is a gram-positive bacterium. Optionally, the bacterium is a Staphylococcus, Streptococcus, Enterococcus, Legionella, Haemophilus, Ghonnorhea, Acinetobacter, Escherichia, Klebsiella, Pseudomonas, or Stenotrophomonas bacterium (e.g., E coli (e.g., EHEC E coli), C dificile, V cholera, Staphylococcus (e.g., S aureus or MRSA), Streptococcus pyogenes, Acinetobacter baumannii, Legionella, Pseudomonas aeruginosa, Klebsiella pneumoniae bacterium).

[0126] Optionally, the first species is selected from the species in Table 1.

[0127] Optionally, the first species is enterohemorrhagic E. coli (EHEC), E. coli serotype O157:H7, or Shiga toxin-producing E. coli (STEC). In examples, the bacterium is selected from: Shiga toxin-producing E. coli (STEC) (STEC is sometimes also called verotoxin-producing E. coli (VTEC)), Enterohemorrhagic E. coli (EHEC) (this pathotype is the one most commonly seen and heard about in the news in relation to foodborne outbreaks), ·Toxigenic E. coli (ETEC), ·Enteropathogenic E. coli (EPEC), ·Enteroaggregative E. coli (EAEC), Enteroinvasive E. coli (EIEC), and · Diffuse-adherent E. coli (DAEC).

[0128] Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a human pathogen responsible for global epidemics of bloody diarrhea and hemolytic uremic syndrome (HUS). Conventional antibacterial agents trigger the EHEC SOS response, which promotes the release of potent Shiga toxins, which are responsible for much of the morbidity and mortality associated with EHEC infection. Cattle are the natural host of EHEC, and approximately 75% of EHEC outbreaks are associated with the consumption of contaminated bovine products. EHEC causes disease in humans but is asymptomatic in adult ruminants. E. coli serotype O157:H7 (EHEC) infection is characterized by abdominal cramps and bloody diarrhea, as well as the life-threatening complication hemolytic uremic syndrome (HUS). Currently, there is a need for treatments for EHEC infections (Goldwater and Bettelheim, 2012). The use of conventional antibiotics exacerbates Shiga toxin-mediated cytotoxicity. In an epidemiological study conducted by the Centers for Disease Control and Prevention, patients treated with antibiotics for EHEC enteritis were at higher risk of developing HUS (Slutsker et al., 1998). Additional studies support the contraindication of antibiotics in EHEC infections, and children receiving antibiotic therapy for EHEC-associated hemorrhagic colitis showed an increased chance of developing HUS (Wong et al., 2000; Zimmerhackl, 2000; Safdar et al., 2002; Tarr et al., 2005). Conventional antibiotics promote Shiga toxin production by enhancing the replication and expression of stx genes encoded within chromosomally integrated lambdoid prophage genomes. The present approach relies on nuclease cleavage. Stx induction also promotes phage-mediated lysis of the EHEC cell envelope, allowing the release and dissemination of Shiga toxin into the environment (Karch et al., 1999; Matsushiro et al., 1999; Wagner et al., 2002). Advantageously, therefore, the present invention provides an alternative means for treating EHEC in human and animal subjects, as exemplified below with surprising results regarding the speed and duration of anti-EHEC action produced by nuclease action (as opposed to conventional antibiotic action).

[0129] In an example, a subject (eg, a human) has or is at risk for hemolytic uremic syndrome (HUS), for example, the subject has an E coli infection, such as an EHEC E coli infection.

[0130] An aspect of the present invention provides a plurality of viruses (e.g., phages or phagemids for producing phages) for use with the nucleases of the invention in methods of treatment, each virus containing a copy of a nucleic acid described herein, and the virus capable of infecting and delivering the nucleic acid to a microorganism contained by a subject.

[0131] An aspect of the invention provides a plurality of viruses (e.g., phages or phagemids for producing phages) for use with a programmable nuclease in a method for treating a microbial infection in a subject, wherein the microbial infection is caused by a first species or strain of microorganism, and the nuclease is programmable to cleave a target site contained by the genome of the microorganism infecting the subject, thereby killing or reducing the growth or proliferation of the first species or strain of microorganism, the method of treatment comprising contacting the subject with the nuclease, wherein the nuclease is programmed to cleave the target site, thereby cleaving the genome of the microorganism contained by the subject and treating the microbial infection in the subject; each virus comprises a copy of a nucleic acid encoding an RNA for expressing the RNA in the subject, the RNA complexing with a nuclease and programming the nuclease to cleave a target site in a microorganism contained by the subject; Viruses provide a number of viruses that are capable of infecting and delivering nucleic acid to microorganisms contained by a subject.

[0132] Optionally, the method is for continuous treatment, eg, as described herein, and / or optionally, the infection is an acute infection.

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

[0134] Optionally, the nuclease is according to any nuclease of the invention herein. Optionally, the nucleic acid is according to any nucleic acid of the invention herein.

[0135] Optionally, the nuclease is according to any nuclease of the invention herein. Optionally, the nucleic acid is according to any nucleic acid of the invention herein.

[0136] Alternatively, when the microorganism is a virus, the plurality of viruses are phages capable of infecting a host cell harboring the microorganism, and the nucleic acid is introduced into the host cell to express the RNA therein. The RNA complexes with a nuclease in the host cell and guides the nuclease to cleave a target site in the microorganism (i.e., cleave the viral RNA or DNA), thereby inactivating the viral microorganism. For example, the microorganism is a virus (e.g., in an amoeba or in a human, animal, or plant cell), and a virus of the plurality of viruses can target the microorganism, thereby programming the nuclease to cleave the microorganism (e.g., in the amoeba or in the cell).

[0137] An aspect of the invention provides a composition comprising a plurality of nucleic acids for programming a nuclease of the invention in a method of treatment, each nucleic acid being a nucleic acid as defined herein.

[0138] An embodiment of the present invention provides a composition comprising a plurality of nucleic acids for programming a programmable nuclease in a method for treating a microbial infection in a subject, wherein the microbial infection is caused by a first species or strain of microorganism, and the nuclease is programmable to cleave a target site contained by the genome of the microorganism that has infected the subject, thereby killing or reducing the growth or proliferation of the first species or strain of microorganism, and the method of treatment comprises contacting the subject with the nuclease and the nucleic acids, and the nuclease is programmed to cleave the target site, thereby cleaving the genome of the microorganism contained by the subject and treating the microbial infection in the subject, and each nucleic acid encodes an RNA for expressing the RNA in the subject, and the RNA complexes with the nuclease and programs the nuclease to cleave the target site in the microorganism contained by the subject.

[0139] Optionally, the method is for continuous treatment, eg, as described herein, and / or optionally, the infection is an acute infection.

[0140] Optionally, the method is for rapid treatment, eg, as described herein, and / or optionally, the infection is an acute infection.

[0141] Optionally, the nuclease is according to any nuclease of the invention herein. Optionally, each nucleic acid is according to any nucleic acid of the invention herein.

[0142] Optionally, the composition is a pharmaceutical composition comprising the nucleic acid and a pharmaceutically acceptable diluent, carrier, or excipient. Optionally, the composition is for oral, intravenous, pulmonary, rectal, topical, buccal, ocular, intranasal, or subcutaneous administration to a human or animal subject. Optionally, the composition is a herbicide or insecticide or insecticide or nematodicide or aracnicide. Optionally, the composition is toxic to yeast. Optionally, the composition is toxic to giant viruses.

[0143] An aspect of the present invention provides a CRISPR / Cas system comprising a nuclease according to the invention for use in a method of treatment, wherein the nuclease is a Cas nuclease (e.g. Cas3 or Cas9 or any other Cas mentioned herein), and the system comprises one or more guide RNAs or DNA encoding one or more guide RNAs, each guide RNA being capable of programming the Cas nuclease to cleave a target site contained by the genome of a microorganism.

[0144] In examples, each guide RNA referred to herein is a single-stranded guide RNA (i.e., a chimeric guide RNA). In another example, each guide RNA comprises a crRNA hybridized with a tracrRNA.

[0145] In examples, the target sites referred to herein are comprised by a bacterial essential gene, a virulence gene, or an antibiotic resistance gene. In examples, the target sites referred to herein are comprised by a multicopy sequence (i.e., a sequence that is present in one or more (e.g., 2, 3, 4, 5, 6, 7, 8, or 9 or more) copies in each bacterial genome). For example, the target site is comprised by a ribosomal RNA gene. In examples, the target sites referred to herein are comprised by a ribosomal RNA gene (e.g., the 23S ribosomal RNA gene), a yapH gene, or a pks gene, or a homolog or ortholog thereof.

[0146] Optionally, each guide RNA herein can hybridize to a protospacer sequence comprising a target site, wherein the protospacer sequence is 15-45 nucleotides in length, e.g., 15-25, 18-21, 20, or about 20 nucleotides in length. Optionally, each guide RNA herein comprises a spacer sequence that is 15-45 nucleotides in length, e.g., 15-25, 18-21, 20, or about 20 nucleotides in length.

[0147] Optionally, each guide RNA herein is cognate to a 5'-NGG protospacer adjacent motif (PAM), e.g., when the bacterium is E. coli. Optionally, each guide RNA herein is cognate to a 5'-CCA or 5'-CCT protospacer adjacent motif (PAM), e.g., when the bacterium is C. dificile.

[0148] An aspect of the invention provides a guide RNA or DNA encoding a guide RNA for use in a system of the invention for use in a method for treating an acute microbial infection in a subject, such as sepsis or sepsis.

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

[0150] Optionally, the vector is a phage, phagemid, viriophage, virus, plasmid (e.g., conjugative plasmid), or transposon. The example below shows that nearly complete killing can be achieved using conjugative plasmids as vectors. Thus, in embodiments, each vector is a conjugative plasmid delivered from a carrier bacterium, e.g., a probiotic carrier bacterium, for administration to a human or animal subject. In examples, the carrier bacterium is Lactobacillus (e.g., L. reuteri) or E. coli. This is exemplified below, where complete (100%) killing is achieved.

[0151] An aspect of the invention provides an anti-sepsis or anti-sepsis composition for administration to a human or animal to treat sepsis or septicemia, the composition comprising a plurality of vectors, each vector comprising a vector of the invention.

[0152] Aspects of the present invention provide methods for treating (e.g., for rapid and / or sustained treatment of) an acute microbial infection in a subject, the method being as defined herein.

[0153] An aspect of the present invention provides a method for treating (e.g., for rapid and / or sustained treatment of) an acute microbial infection in a subject, wherein the microbial infection is caused by a first species or strain of microorganism, and a nuclease is programmable to cleave a target site contained by the genome of the microorganism that has infected the subject, thereby killing or reducing the growth or proliferation of the first species or strain of microorganism, the method of treatment comprising contacting the subject with the nuclease, wherein the nuclease is programmed to cleave the target site, thereby cleaving the genome of the microorganism contained by the subject, and treating (e.g., for rapid and / or sustained treatment of) the acute microbial infection in the subject.

[0154] An embodiment of the present invention provides a method for treating (e.g., for rapid and / or sustained treatment) an acute microbial infection in a subject, wherein the microbial infection is caused by a first species or strain of microorganism, and a nuclease is programmable to cleave a target site contained by the genome of the microorganism infecting the subject, thereby killing or reducing the growth or proliferation of the first species or strain of microorganism, the method of treatment comprising contacting the subject with a nuclease and a plurality of viruses, wherein the nuclease is programmed to cleave the target site, thereby cleaving the genome of the microorganism contained by the subject, and treating (e.g., for rapid and / or sustained treatment) the acute microbial infection in the subject, wherein each virus contains a copy of a nucleic acid encoding an RNA for expressing the RNA in the subject, wherein the RNA complexes with the nuclease and programs the nuclease to cleave the target site in the microorganism contained by the subject, and the viruses are capable of infecting and delivering the nucleic acid to the microorganism contained by the subject.

[0155] Optionally, the nuclease is according to any nuclease of the invention herein. Optionally, the nucleic acid is according to any nucleic acid of the invention herein.

[0156] An aspect of the invention is a method for treating (e.g., for rapid and / or sustained treatment of) an acute microbial infection in a subject, wherein the microbial infection is caused by a first species or strain of microorganism, and a nuclease is programmable to cleave a target site contained by the genome of the microorganism that has infected the subject, thereby killing or reducing the growth or proliferation of the first species or strain of microorganism, the method of treatment comprising contacting the subject with a nuclease and a plurality of nucleic acids, wherein the nuclease is programmed to cleave the target site, thereby and a method for treating (e.g., rapidly and / or sustainably treating) an acute microbial infection in a subject, wherein each virus comprises a copy of a nucleic acid encoding an RNA for expressing the RNA in the subject, the RNA complexing with a nuclease and programming the nuclease to cleave a target site in the microorganism contained by the subject, and each nucleic acid encodes an RNA for expressing the RNA in the subject, the RNA complexing with a nuclease and programming the nuclease to cleave a target site in the microorganism contained by the subject.

[0157] Optionally, the nuclease is according to any nuclease of the invention herein. Optionally, each nucleic acid is according to any nucleic acid of the invention herein.

[0158] In examples, the invention is for medical, or dental, or opthalmic use (e.g., for treating or preventing an infection in an organism, or for limiting the spread of an infection in an organism).

[0159] In examples, the present invention is for cosmetic use (eg, use in cosmetic products such as make-up), or hygiene use (eg, use in hygiene products such as soap).

[0160] In examples, the vector and / or nuclease prior to administration to a subject is comprised by a composition (wherein host refers to a first species or strain of microorganism) as any of the following: In examples, the composition is a medical composition, an opthalmic composition, a dental composition, or a pharmaceutical composition (e.g., comprised by an anti-host vaccine). In examples, the composition is an antibacterial composition, e.g., an antibiotic or antiviral agent, e.g., a medicine, a disinfectant, or a mouthwash. In examples, the composition is a cosmetic composition (e.g., a face or body makeup composition). In examples, the composition is a herbicide. In examples, the composition is an insecticide (e.g., where the host is a Bacillus (e.g., thuringiensis) host). In examples, the composition is a beverage (e.g., beer, wine, or alcoholic beverage) additive. In examples, the composition is a food additive (e.g., where the host is an E. coli, Salmonella, Listeria, or Clostridium (e.g., botulinum) host). In examples, the composition is a water additive. In an example, the composition is an additive for an acquatic animal environment (e.g., a fish tank). In an example, the composition is or is included in an oil or petrochemical composition (e.g., where the host is a sulfate-reducing bacteria, e.g., Desulfovibrio host). In an example, the composition is an oil or petrochemical additive. In an example, the composition is a chemical additive. In an example, the composition is a disinfectant (e.g., for human or animal use, e.g., for use in surgery or medicine, or for sterilizing equipment for infant feeding). In an example, the composition is a personal hygiene composition for human or animal use. In an example, the composition is a composition for environmental use, e.g., soil treatment or environmental decontamination (e.g., where the host is a sulfate-reducing bacteria, e.g., Desulfovibrio host, e.g., from sewage, or from oil, petrochemicals, or chemicals). In an example, the composition is a plant growth stimulant. In an example, the composition is a composition for use in oil extraction, petrochemical extraction, metal extraction, or mineral extraction. In an example, the composition is a fabric treatment or additive. In an example, the composition is an animal hide, leather, or suede treatment or additive.In an example, the composition is a dye additive. In an example, the composition is a beverage (e.g., beer or wine) brewing or fermentation additive (e.g., where the host is a Lactobacillus host). In an example, the composition is a paper additive. In an example, the composition is an ink additive. In an example, the composition is an adhesive additive. In an example, the composition is an anti-human, animal, or plant parasite composition. In an example, the composition is an air additive (e.g., where the host is a Legionella host, for example, for air in or provided by an air conditioning device). In an example, the composition is an anti-freeze additive (e.g., where the host is a Legionella host). In an example, the composition is an eye wash or opthalmic composition (e.g., contact lens solution). In an example, the composition is comprised by a dairy food product (e.g., where the host is a Lactobacillus, Streptococcus, Lactococcus, or Listeria host, for example, where the composition is in or is milk or a dairy product). In an example, the composition is comprised by or is a household or industrial cleaning product (e.g., where the host is an E. coli, Salmonella, Listeria, or Clostridium (e.g., botulinum) host). In an example, the composition is comprised by a fuel. In an example, the composition is comprised by a solvent (e.g., other than water). In an example, the composition is a baking additive (e.g., a food baking additive). In an example, the composition is a laboratory reagent (e.g., for use in biotechnology or recombinant DNA or RNA technology). In an example, the composition is comprised by a textile soak. In an example, the composition is for use in a vitamin synthesis process. In an example, the composition is an anti-grain or plant spoilage composition (e.g., where the host is a saprophytic bacterium).In examples, the composition is a corrosion inhibitor composition, e.g., for preventing or reducing metal corrosion (e.g., for use in reducing or preventing corrosion of oil extraction, processing, or containment equipment; metal extraction, processing, or containment equipment; or mineral extraction, processing, or containment equipment, e.g., where the host is a sulfate-reducing bacterium, e.g., a Desulfovibrio host). In examples, the composition is an agricultural or farm composition, or is included in such a composition. In examples, the composition is a silage additive. The invention provides a CRISPR array, gRNA-encoding nucleotide sequence, vector, or vectors described herein for use in any of the compositions described in this paragraph, or for use in any of the applications described in this paragraph, e.g., where the host cell is a bacterial or archaeal cell. The invention provides methods for any of the applications described in this paragraph, comprising combining a CRISPR array, gRNA-encoding nucleotide sequence, vector, or vectors of the invention with a host cell (e.g., a bacterial or archaeal cell). In embodiments, the host cell is not present in or on a human (or human embryo) or animal.

[0161] Any embodiment of the invention may be for or used in a method for industrial or domestic use, for example, agriculture, oil and petroleum industry, food or beverage industry, clothing industry, packaging industry, electronics industry, computer industry, environmental industry, chemical industry, aeronautics industry, automotive industry, biotechnology industry, medical industry, healthcare industry, dental industry, energy industry, consumer products industry, pharmaceutical industry, mining industry, cleaning industry, forestry industry, fishing industry, leisure industry, recycling industry, cosmetics industry, plastics industry, pulp or paper industry, textile industry, clothing industry, leather or suede or animal hide industry, tobacco industry, or steel industry.

[0162] A host cell herein refers to a microorganism of a first species or strain. Optionally, any host cell herein is a bacterial cell or an archaeal cell. In examples, the cell is in stationary phase. In examples, the cell is in exponential growth phase. In examples, the cell is in lag phase. In examples, for example, the cell is a wild-type cell or a naturally occurring cell, such as comprised by a naturally occurring microbiome of a human, animal, plant, soil, water, ocean, waterway, or environment. In examples, the cell has been artificially genetically modified.

[0163] In an example, a plurality of vectors of the invention are introduced into a plurality of said host cells, and the host cells are comprised by, for example, an ex vivo, in vivo, or in vitro bacterial population. In an example, the host cells are comprised by a biota population comprised by an organism or environment (e.g., a waterway microbiota, aquatic microbiota, a human or animal gut microbiota, a human or animal oral microbiota, a human or animal vaginal microbiota, a human or animal skin or hair microbiota, or a human or animal axillary microbiota), and the population comprises a first bacterium that is symbiotic or mutualistic with the organism or environment, and a second bacterium that comprises said host cells, and the host cells are harmful (e.g., pathogenic) to the organism or environment. In an embodiment, the population is ex vivo. In an example, the ratio of the first bacterial subpopulation to the second bacterial subpopulation is increased. In an example, the first bacterium is a Bacteroides (e.g., B fragalis and / or B thetaiotamicron) bacterium. Optionally, the Bacteroides comprises one, two, three, or more Bacteroides species selected from caccae, capillosus, cellulosilyticus, coprocola, coprophilus, coprosuis, distasonis, dorei, eggerthii, faecis, finegoldii, fluxus, fragalis, intestinalis, melaninogenicus, nordii, oleiciplenus, oralis, ovatus, pectinophilus, plebeius, stercoris, thetaiotaomicron, uniformis, vulgatus, and xylanisolvens. For example, the Bacteroides is or comprises B thetaiotaomicron. For example, the Bacteroides is or comprises B fragalis.

[0164] In examples, the host, first, or second cell is any bacterial species disclosed in U.S. Patent Application Publication No. 20160333348, GB Patent No. 1609811.3, PCT / EP2017 / 063593, and all U.S. equivalent applications, the disclosures of such species (including, in particular, Table 1 of PCT / EP2017 / 063593) are incorporated herein in their entirety and potentially encompassed by one or more disclosures herein in one or more claims herein.

[0165] In examples, the host cells or bacterial population are contained by a beverage or water for human consumption (e.g., waterway water or drinking water). In examples, the host cells or said population are contained by a composition (e.g., a medicament (e.g., a bacterial gut transplant), a beverage, a mouthwash, or a food) for administration to a human or non-human animal to establish and rebalance its gut or oral microbiota (e.g., the use of the medicament is to treat or prevent a disease or condition in a human or animal). In examples, the host cells or said population are on a solid surface or contained by a biofilm (e.g., an intestinal biofilm or a biofilm on an industrial device). In examples of the invention for in vitro treatment of industrial or medical fluids, solid surfaces, devices, or containers (e.g., for food, consumer products, cosmetics, personal healthcare products, petroleum or oil production), or for treating waterways, water, beverages, foodstuffs, or cosmetics, the host cells may be contained by or on the fluid, surface, device, container, waterway, water, beverage, foodstuff, or cosmetic.

[0166] In an example, the invention provides a container for medical or nutritional use, comprising a vector for use in the method, for example the container is a sterile container, such as an inhaler, or is connected to a syringe or IV needle.

[0167] In examples, the vector or composition is for (or is) administered to a human or non-human animal subject by mucosal, enteral, oral, nasal, rectal, vaginal, ocular, or buccal administration.

[0168] Optionally, each host cell is a strain or species found in the human microbiome, and optionally the host cells are mixed with cells of different strains or species, where the different cells are Enterobacteriaceae or bacteria that are probiotic, commensal, or mutualistic with humans (e.g., in the human gut). In examples, the host cells are E. coli or Salmonella cells.

[0169] The present invention is optionally for altering the relative ratios of a first and a second bacterial subpopulation in a mixed population of bacteria, for example for altering a human or animal microbiome, such as for altering the proportion of Bacteroidetes (e.g., Bacteroides, e.g., fragalis, and / or thetaiotamicron), Firmicutes, and / or Gram-positive or Gram-negative bacteria in the human microbiome.

[0170] In examples, the vector or composition of the invention comprises a nucleotide sequence for expressing an endolysin in a host cell to lyse the host cell, and optionally the endolysin is a phage phi11, phage Twort, phage P68, phage phiWMY, or phage K endolysin (e.g., MV-L endolysin or P-27 / HP endolysin).

[0171] In examples, the target site is contained by the chromosome of the respective microbial host cell, e.g., the sequence is contained by an antibiotic resistance gene, a virulence gene, or an essential gene of the host cell. An example provides a vector of the invention combined with an antibiotic agent (e.g., a beta-lactam antibiotic), e.g., the vector targets a protospacer sequence contained by an antibiotic resistance gene contained by the host cell genome or an episome (e.g., a plasmid contained by the host cell). In examples, the episome is a plasmid, transposon, mobile genetic element, or viral sequence (e.g., a phage or prophage sequence).

[0172] In examples, the target is a chromosomal sequence, an endogenous host cell sequence, a wild-type host cell sequence, a non-viral chromosomal host cell sequence, but not an exogenous sequence and / or a non-phage sequence (i.e., one or all of these), e.g., the sequence is a wild-type host chromosomal cell sequence, such as an antibiotic resistance gene sequence or an essential gene sequence contained by a host cell chromosome. In examples, the sequence is a host cell plasmid sequence, e.g., an antibiotic resistance gene sequence.

[0173] Optionally, the nuclease is a Cas, and the target site is encompassed by a protospacer sequence that is flanked by NGG, NAG, NGA, NGC, NGGNG, NNGRRT, or NNAGAAW protospacer adjacent motifs (PAMs), e.g., AAAGAAA or TAAGAAA PAMs (these sequences are written 5' to 3'). In embodiments, the PAM is immediately adjacent to the 3' end of the protospacer sequence. In examples, the Cas is S. aureus Cas, S. theromophilus Cas, or S. pyogenes Cas. In examples, the Cas is Cpfl and / or the PAM is TTN or CTA. Optionally, the Cas is a type I (e.g., type IA, type IB, type IC, type ID, type IE, or type IF) CRISPR-based Cas. Optionally, the Cas is a type II CRISPR-based Cas. Optionally, the Cas is a type III CRISPR-based Cas. Optionally, the Cas is a type IV CRISPR system Cas. Optionally, the Cas is a type V CRISPR system Cas. Optionally, the Cas is a type VI CRISPR system Cas.

[0174] Optionally, the nuclease is CAS, and each vector comprises a homogeneous CRISPR array comprising multiple copies of the same spacer for targeting a target site. Optionally, a vector or vectors of the invention are provided, wherein the vector comprises multiple CRISPR arrays of said gRNA-encoding sequences for targeting host cell protospacer sequences, wherein the protospacers comprise target sites. Optionally, each vector comprises two, three, or more copies of a nucleic acid sequence encoding a crRNA (e.g., gRNA), wherein the copies comprise the same spacer sequence for targeting a host cell target site (e.g., a site contained in a toxic gene sequence, a resistance gene sequence, or an essential gene sequence).

[0175] In examples, at least two target sequences are modified by Cas, e.g., an antibiotic resistance gene and an essential gene. Multiple targeting in this manner can be useful in reducing the evolution of escape mutant host cells.

[0176] In examples, the Cas is a wild-type endogenous host cell Cas nuclease. In examples, target site cleavage is performed by a dsDNA Cas nuclease (e.g., Cas9, e.g., spCas9 or saCas9), whereby repair of the cleavage is by non-homologous end joining (NHEJ), and alternatively, the Cas is an exonuclease or Cas3.

[0177] In examples, the array, gRNA coding sequence, or vector is not combined with a Cas endonuclease coding sequence that is naturally found in a cell along with the repeat sequences of the array or gRNA coding sequence.

[0178] The tracrRNA sequence may be omitted from the arrays or vectors of the invention, e.g., in the case of types of Cas systems that do not use tracrRNA, or endogenous tracrRNA may be used in conjunction with the cRNA encoded by the vector.

[0179] In examples, the host target site is encompassed by at least 5, 6, 7, 8, 9, 10, 20, 30, or 40 contiguous nucleotides.

[0180] In examples, each vector contains an exogenous promoter functional for transcribing the crRNA or gRNA in the microorganism.

[0181] Optionally, each vector is a plasmid, cosmid, virus, virion, phage, phagemid, or prophage. For example, the present invention provides a plurality of bacteriophages comprising a plurality of vectors of the present invention, for example, when the vectors are identical. In an example, the vector is a viral vector. Viral vectors have particularly limited capacity for exogenous DNA insertion, and therefore viral packaging capacity must be taken into consideration. It is necessary to leave room for sequences encoding essential viral functions, such as for expressing coat proteins and polymerases. In an example, the vector is a phage vector or an AAV or lentiviral vector. When the host is a bacterial cell, a phage vector is useful. In an example, the vector is a virus capable of infecting an archaeal host cell.

[0182] Optionally, the vector components are comprised by a transposon capable of moving into and / or between host cells. The transposon may be a transposon such as those described in U.S. Patent Application Publication No. 20160333348, GB Patent No. 1609811.3, and all U.S. equivalent applications, the disclosures of which, including the disclosures of such specific transposons, are incorporated herein in their entirety and potentially encompassed by one or more disclosures herein in one or more claims herein.

[0183] In examples, the respective vectors are provided by nanoparticles or in liposomes.

[0184] In examples, each host cell (or first and / or second bacterium) is a Gram-positive bacterial cell. In examples, each host cell is an Enterobacteriaceae, e.g., a Salmonella, Yersinia pestis, Klebsiella, Shigella, Proteus, Enterobacter, Serratia, or Citrobacter cell. Optionally, each cell is an E. coli (e.g., E. coli K12) or Salmonella (e.g., S. enteric serovar typhimurium) cell. Optionally, each host cell (or first and / or second bacterium) is a Gram-negative bacterial cell.

[0185] Optionally, the host (or the first and / or second bacterium) is a mycoplasma, chlamydiae, spirochete, 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. Optionally, the host (or the first and / or second bacterium) is an E. coli (e.g., O157:H7) host. Optionally, the host (or the first and / or second bacterium) is a Pseudomonas (e.g., aeruginosa) host. Optionally, the host (or the first and / or second bacterium) is a Vibro (e.g., cholerae (e.g., O139) or vulnificus) host. Optionally, the host (or the first and / or second bacterium) is a Neisseria (e.g., gonorrhoeae or meningitidis) host. Optionally, the host (or the first and / or second bacterium) is a Bordetella (e.g., pertussis) host. Optionally, the host (or the first and / or second bacterium) is a Haemophilus (e.g., influenzae) host. Optionally, the host (or the first and / or second bacterium) is a Shigella (e.g., dysenteriae) host. Optionally, the host (or the first and / or second bacterium) is a Brucella (e.g., abortus) host. Optionally, the host (or the first and / or second bacterium) is a Francisella host. Optionally, the host (or the first and / or second bacterium) is a Xanthomonas host. Optionally, the host (or the first and / or second bacterium) is an Agrobacterium host. Optionally, the host (or the first and / or second bacterium) is an Erwinia host. Optionally, the host (or the first and / or second bacterium) is a Legionella (e.g., pneumophila) host.Optionally, the host (or the first and / or second bacterium) is a Listeria (e.g., monocytogenes) host. Optionally, the host (or the first and / or second bacterium) is a Campylobacter (e.g., jejuni) host. Optionally, the host (or the first and / or second bacterium) is a Yersinia (e.g., pestis) host. Optionally, the host (or the first and / or second bacterium) is a Borrelia (e.g., burgdorferi) host. Optionally, the host (or the first and / or second bacterium) is a Helicobacter (e.g., pylori) host. Optionally, the host (or the first and / or second bacterium) is a Clostridium (e.g., dificile or botulinum) host. Optionally, the host (or the first and / or second bacterium) is an Erlichia (e.g., chaffeensis) host. Optionally, the host (or the first and / or second bacterium) is a Salmonella (e.g., typhi or enterica, e.g., serovar Typhimurium, e.g., DT 104) host. Optionally, the host (or the first and / or 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 second bacterium) is a Corynebacterium (e.g., amycolatum) host. Optionally, the host (or the first and / or second bacterium) is a Klebsiella (e.g., pneumoniae) host. Optionally, the host (or the first and / or second bacterium) is an Enterococcus (e.g., faecalis or faecim, e.g., linezolid-resistant) host. Optionally, the host (or the first and / or second bacterium) is an Acinetobacter (eg, baumannii, eg, multidrug resistant) host.

[0186] Optionally, the invention is for reducing the growth or proliferation of host cells in an environmental microbiome (e.g., soil, composition comprising said host cells and yeast cells), a human microbiome, an animal microbiome, or a plant microbiome, which is useful, for example, when the microbiome is naturally occurring.

[0187] Optionally, the nuclease is Cas and the target is encompassed by a protospacer sequence comprising at least 5, 6, 7, 8, 9, or 10 consecutive nucleotides immediately 3' to a cognate PAM in the genome of the host cell, wherein the PAM is selected from AWG, AAG, AGG, GAG, and ATG.

[0188] Non-medical ex vivo and in vitro use, etc. In certain configurations, the present invention's observations of rapid and sustained microbial killing and growth or proliferation inhibition using nuclease cleavage find application in subjects other than humans and animals (e.g., for treating plants or yeast cultures), or for ex vivo or in vitro treatment of substrates such as industrial surfaces, fluids, and devices. Accordingly, the present invention further provides the following concepts: Any other features herein of the invention, its configurations, aspects, embodiments, options, and examples above and elsewhere herein may be combined with such concepts, mutatis mutandis (including to provide combinations of features in the claims herein).

[0189] The concept provides for the use of a nuclease, a plurality of viruses, systems, guide RNAs, DNA or vectors of the invention in the manufacture of a composition for carrying out the methods of treatment defined herein, wherein the subject is an organism other than a human or animal.

[0190] The concept provides for the use of a nuclease, multiple viruses, systems, guide RNAs, DNA, or vectors of the invention in the manufacture of a composition for carrying out an ex vivo or in vitro method for treating a microbial infection of a substrate, wherein the microbial infection is caused by a first species or strain of microorganism, and the nuclease is programmable to cleave a target site contained by the genome of the microorganism that has infected the substrate, thereby killing the first species or strain of microorganism or reducing its growth or proliferation, and the method of treatment comprises contacting a subject with the nuclease, wherein the nuclease is programmed to cleave the target site, thereby cleaving the genome of the microorganism contained by the subject and treating an acute microbial infection of the substrate.

[0191] As used herein, treating an infection on a substrate can refer to treating a bacterial population (e.g., one or more colonies) on the surface of the substrate and / or incorporated into the material of the substrate. For example, the treatment can be to kill bacteria on the surface of industrial equipment or equipment (e.g., medical equipment such as scalpels or medical devices or tubing). In another example, the substrate is a fluid (e.g., a liquid or gas), such as a medical fluid or a petroleum product in fluid form (e.g., an oil or hydrocarbon fluid or liquid).

[0192] The concept provides for the use of a programmable nuclease in the manufacture of a composition for performing an ex vivo method for treating a microbial infection of a substrate, wherein the microbial infection is caused by a first species or strain of microorganism, and the nuclease is programmable to cleave a target site contained by the genome of the microorganism that has infected the substrate, thereby killing the first species or strain of microorganism or reducing its growth or proliferation, and the method of treatment includes contacting a subject with the nuclease, wherein the nuclease is programmed to cleave the target site, thereby cleaving the genome of the microorganism contained by the subject and treating an acute microbial infection of the substrate.

[0193] Optionally, a nuclease (e.g., a programmed nuclease) and / or a nucleic acid that programs the nuclease to recognize and cleave a target site is administered to a subject or substrate at a first time point (T1) and a second time point (T2), where T2 is at least 1 hour after T1. T1 and T2 may be as defined herein.

[0194] Optionally, the infection is reduced by at least 100-fold within the first 30 minutes (e.g., within the first 15 minutes) of treatment. Optionally, the infection is maintained at least 100-fold for at least 60 minutes (e.g., at least 120 minutes) after contacting the subject with the programmed nuclease.

[0195] Optionally, the reduction in infection persists for at least 30 minutes immediately after the first 30 minutes of treatment.

[0196] Optionally, the method includes administering to the subject or substrate RNA, or a nucleic acid encoding RNA for expression of RNA in or on the subject or substrate, wherein the RNA complexes with a nuclease and programs the nuclease to cleave a target site in a microorganism contained by the subject or substrate.

[0197] Optionally, the nuclease is administered to the subject or substrate simultaneously or sequentially with the RNA or nucleic acid.

[0198] Optionally, the subject or substrate contains a nuclease prior to administering the RNA or nucleic acid.

[0199] Optionally, multiple viruses (e.g., phages) are administered to the subject or substrate, each virus containing a copy of the nucleic acid, and the viruses infect and deliver the nucleic acid to a microorganism contained by the subject or substrate.

[0200] Optionally, the ratio of virus:microorganism administered is between 10 and 150.

[0201] Optionally, infection is reduced by at least 90% over an hour or longer period, optionally by the first 30 minutes (eg, by the first 15 minutes) of treatment.

[0202] Optionally, infection is reduced by at least 100-fold by the first 30 minutes of treatment (e.g., by the first 15 minutes), and the at least 100-fold reduction in infection is maintained for at least 60 minutes (e.g., at least 120, 145, or 180 minutes) after contacting the subject or substrate with the programmed nuclease.

[0203] Optionally, the object is a plant, or the substrate is a metal substrate, a plastic substrate, a concrete substrate, a stone substrate, a wood substrate, a glass substrate, or a ceramic substrate. Optionally, the object is a fluid (e.g., a liquid or a gas).

[0204] Optionally, the microorganism is a bacterium or an archaea. Optionally, the bacterium is a gram-positive bacterium. Optionally, the bacterium is a Staphylococcus, Streptococcus, Enterococcus, Legionella, Haemophilus, Ghonnorhea, Acinetobacter, Escherichia, Klebsiella, Pseudomonas, or Stenotrophomonas bacterium (e.g., E coli (e.g., EHEC E coli), C dificile, V cholera, Staphylococcus (e.g., S aureus or MRSA), Streptococcus pyogenes, Acinetobacter baumannii, Legionella, Pseudomonas aeruginosa, Klebsiella pneumoniae bacterium).

[0205] Optionally, the nuclease is a Cas nuclease (e.g., Cas3 or Cas9), a meganuclease, a TALEN (transcription activator-like effector nuclease), or a zinc finger nuclease.

[0206] Reference is made to WO2016177682, which discusses aspects of microbial corrosion (MIC) or biofouling of substrates and discloses methods for controlling MIC or biofouling of substrates. The methods, nucleases, arrays, RNA, vectors, and viruses disclosed therein can be used in the present invention, for example, to carry out the methods or uses of the present invention, and disclosure of portions thereof, as well as the substrates and bacteria disclosed in WO2016177682, are incorporated herein by reference to potentially provide disclosure of features that can be used in one or more claims herein.

[0207] Optionally, the use of the present invention is for controlling microbial corrosion (MIC) or biofouling of a substrate in an industrial or domestic system (e.g., a system disclosed in WO2016177682, the disclosure of which is incorporated herein by reference). In an example, the system comprises an apparatus (e.g., for use in an industrial process) and the surface is a surface of said apparatus. In an example, the biofouling comprises the formation, growth, or maintenance of a microbial biofilm, and / or sludge. In an example, the microorganisms are sessile. In an example, "controlling" comprises preventing, reducing, or eliminating said MIC or biofouling, or reducing the spread of said MIC or biofouling in a system. Cell growth or proliferation or maintenance is, for example, a characteristic of cell viability. Thus, in an example, the method reduces microbial growth and / or maintenance.

[0208] Optionally, the microorganisms are contained by a microbial biofilm in contact with the substrate. Optionally, the surface and host cells are in contact with a fluid, such as an aqueous liquid (e.g., seawater, freshwater, standing water, or drinking water).

[0209] Freshwater is naturally occurring water found on the Earth's surface in ice sheets, ice caps, glaciers, icebergs, marshes, ponds, lakes, rivers, and streams, and underground as groundwater in aquifers and groundwater streams. Freshwater is generally characterized by low concentrations of dissolved salts and other total dissolved solids. The term specifically excludes seawater and brackish water, but includes mineral-rich waters such as iron-containing springs. In examples, the freshwater is any of these freshwater types. Drinking water is water for human or animal (e.g., livestock) consumption. In examples, the fluid is selected from industrial cooling water if the system is a cooling system; wastewater if the system is a wastewater treatment or storage system; drinking water if the system is a drinking water treatment, storage, transportation, or distribution system; paper water if the system is a paper manufacturing or treatment system; swimming pool water if the system is a swimming pool or swimming pool water treatment or storage system; fire water if the system is a fire suppression system; or industrial process water in any pipe, tank, pit, pond, or canal.

[0210] Optionally, the use is for controlling bacterial rancidity of a fluid in a reservoir or container, the fluid comprising a population of first host cells of a first microbial species that mediates said biofouling, and the method comprises: (i) contacting the population with a plurality of vectors capable of transforming or transducing cells, each vector comprising a CRISPR array, whereby the CRISPR array is introduced into the host cell; (a) each CRISPR array comprises one or more sequences for expressing crRNA and a promoter for transcribing the sequences in a host cell; (b) each crRNA is capable of hybridizing to a target sequence in a host cell and directing a Cas (e.g., a Cas nuclease) of the host cell to modify (e.g., cleave) the target sequence, the target sequence being a genetic sequence for mediating host cell viability; The method includes allowing expression of the cRNA in a host cell in the presence of Cas, thereby modifying a target sequence in the host cell, resulting in reduced host cell viability and control of the biofouling.

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

[0212] system Exemplary systems are selected from the group consisting of: petrochemical recovery, processing, storage, or transportation systems; hydrocarbon recovery, processing, storage, or transportation systems; crude oil recovery, processing, storage, or transportation systems; natural gas recovery, processing, storage, or transportation systems (e.g., oil wells, oil rigs, oil drilling equipment, oil pumping systems, oil pipelines, gas rigs, gas extraction systems, gas pumping systems, gas pipelines, oil tankers, gas tankers, oil storage facilities, or gas storage facilities); water treatment or storage facilities; water storage reservoirs (e.g., drinking water reservoirs); air or water conditioning (e.g., cooling or heating) equipment, such as coolant tubes, condensers, or heat exchangers; medical or surgical equipment; environmental (e.g., soil, waterway, or air) treatment equipment; papermaking or paper recycling equipment; power plants, e.g., thermal or nuclear power plants; fuel (e.g., hydrocarbon fuels, e.g., petroleum, diesel, or LPG) storage equipment; mining or metallurgy, mineral or fuel recovery systems, e.g., mine or mining equipment; engineering systems; shipping equipment; cargo or goods storage equipment (e.g., cargo containers); food or beverage manufacturing, processing, or packaging cleaning equipment (e.g., laundry equipment, e.g., washing machines or dishwashers); catering (e.g., domestic or commercial catering) equipment; farm equipment; construction (e.g., building, public infrastructure, or roadworks) equipment; aviation equipment; aerospace equipment; transportation equipment (e.g., motor vehicles (e.g., cars, trucks, or vans); rail vehicles; aircraft (e.g., airplanes) or marine or waterway vessels (e.g., boats, or ships, submarines, or hovercraft)); packaging equipment, e.g., consumer product packaging equipment; or food or beverage packaging equipment; electronics (e.g., computers or mobile phones or electronic components thereof); or electronics manufacturing or packaging equipment; dental equipment; industrial or domestic piping (e.g., undersea pipes) or storage tanks (e.g., water tanks or fuel tanks (e.g., gasoline tanks, e.g., vehicle gas tanks)); underground utilities; buildings (e.g., residences or offices or commercial premises or factories or power plants); roads; bridges; agricultural equipment; industrial systems; crude oil or natural gas exploration equipment; office systems; and household systems.

[0213] In examples, the system is used in industries and businesses selected from the group consisting of agriculture, oil and petroleum industry, food or beverage industry, clothing industry, packaging industry, electronics industry, computer industry, environmental industry, chemical industry, aerospace industry, automotive industry, biotechnology 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, plastics industry, pulp or paper industry, textile industry, clothing industry, leather or suede or animal hide industry, tobacco industry, and steel industry. In examples, the surface or fluid to be treated is a surface or fluid of equipment used in said selected industries. In examples, the system is used in the crude oil industry. In examples, the system is used in the natural gas industry. In examples, the system is used in the petroleum industry. In examples, the system is a marine container, platform, or rig (e.g., an oil or gas platform or rig for use at sea or offshore), a ship, or a boat. In embodiments, such systems are moored at sea, e.g., non-temporarily moored at sea, e.g., moored at sea for 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 (e.g., consecutive months). In embodiments, such systems are in national or state waters, e.g., non-temporarily moored at sea in such waters, e.g., moored at sea for 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 (e.g., consecutive months).

[0214] In examples, the substrate surface to be treated includes stainless steel, carbon steel, copper, nickel, brass, aluminum, concrete, plastic, or wood. In examples, the substrate is a metal weld or joint. In examples, the surface is a metal (e.g., steel or iron) or non-metal (e.g., plastic, concrete, asphalt, wood, rubber, or masonry) surface. In examples, the metal is an alloy (e.g., stainless steel, brass, or nickel alloy, zinc alloy, copper alloy, nickel alloy, or aluminum alloy). In examples, the surface is a man-made polymer surface. In examples, the surface is a substrate coating. In examples, the substrate is in contact with soil, fresh water, or seawater.

[0215] In examples, the fluid is drinking water; a waterway; brackish water; or a liquid fuel such as gasoline or diesel (e.g., in a car or motor vehicle), LPG, kerosene, alcohol (e.g., ethanol, methanol, or butanol), liquid hydrogen, or liquid ammonia), and in examples, the fuel is a stored liquid fuel. In examples, the fluid is oil or a non-aqueous liquid. In examples, the fluid is a liquid contained by a waterway, or water such as seawater, fresh water, drinking water, a river, a stream, a pond, a lake, a reservoir, standing water (e.g., in a water tank or a cooling system), groundwater, well water, water from a rock formation, soil water, or rainwater. In examples, the liquid is seawater. In examples, the substrate is in contact with the liquid referred to in this paragraph. In examples, the fluid or liquid is selected from the group consisting of: oil, aqueous solutions, hydraulic fracturing fluids, fuels, carbon dioxide, natural gas, oil / water mixtures, fuel / water mixtures, salt water, ocean or sea water, brackish water, freshwater sources, lakes, rivers, streams, marshes, ponds, bogs, runoff from melting snow or ice, springs, groundwater, aquifers, precipitation, any substance that is liquid at ambient temperature (e.g., at RTP) and is hydrophobic but soluble in organic solvents, hexane, benzene, toluene, chloroform, diethyl ether, vegetable oils, petrochemical oils, crude oil, refined petrochemicals, volatile aromatic oils, fossil fuels, gasoline, mixtures of hydrocarbons, jet fuel, rocket fuel, biofuels. In examples, the fluid is an oil / water mixture.

[0216] The term "microbial corrosion" or "MIC," as used herein, unless otherwise indicated, refers to a process in which any element of a system (substrate) is structurally disrupted by the action of at least one member of a microbial population, e.g., a bacterial or archaeal population. The term "biofouling," as used herein, unless otherwise indicated, refers to a process in which microorganisms (such as bacteria and / or archaea) accumulate on a substrate surface in contact with a fluid (e.g., water or an aqueous liquid, or a hydrocarbon, or a petrochemical). It also includes the undesirable accumulation and proliferation of microorganisms (such as bacteria and / or archaea) in a fluid (e.g., water or an aqueous liquid, or a hydrocarbon, or a petrochemical), i.e., "rancidity" of the fluid. In an example, bacteria are contained by ship or boat ballast water, where bacteria are environmentally undesirable. The term "substrate," as used herein, refers to any type of surface to which cells can attach, on which a biofilm can form and grow, or on which biofouling (e.g., slime or sludge formation) can occur. The substrate may be an "industrial" substrate, such as a petrochemical, fuel, crude oil, or gas pipe system equipment surface, or it may be a "non-industrial" (e.g., domestic, e.g., household or office) substrate, such as a kitchen counter or shower substrate, or a garden substrate.

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

[0218] Optionally, the fluid is an aqueous liquid (eg, seawater, fresh water, pooled water, or drinking water).

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

[0220] Optionally, the microorganisms are sulfate-reducing bacterial (SRB) cells (e.g., Desulfovibrio or Desulfotomaculum cells). In examples, the cells are selected from the group consisting of: Desulfotomaculum nigrificans, Desulfacinum infernum, Thermodesulfobacterium mobile, Thermodesulforhabdus norvegicus, Archaeoglobus fulgidus, Desulfomicrobium apsheronum, Desulfovibrio gabonensis, Desulfovibrio longus, Desulfovibrio vietnamensis, Desulfobacterium cetonicum, Desulfomaculum halophilum, Desulfobacter vibrioformis, and Desulfotomaculum thermocisternum cells. In examples, the population includes a mixture of two or more of these cell types.

[0221] Optionally, the surface or fluid is comprised by a crude oil, gas, or petrochemical recovery, processing, storage, or transportation facility. Crude oil is one of the most important energy resources in the world. Crude oil is used as a feedstock for numerous industries, including the refining-petrochemical industry, where it is refined through various technological processes into consumer products such as gasoline, oil, paraffin oil, lubricants, asphalt, household fuel oil, petrolatum, and polymers. Oil-derived products are also commonly used in many other chemical processes. In the alternative, the fluid is, or the surface is in contact with, such a consumer product.

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

[0223] Optionally, step (i) of the method includes providing a population of microbial cells of a second species (second host cells), the second cells comprising the vector, the vector being transferable from the second host cells to the first host cells; and combining the second host cells with the first host cells, thereby introducing the vector into the first host cells. In an example, the second cells are environmentally, industrially, or domestically tolerant in the environment (e.g., in an aquatic or soil environment), and the first host cells are environmentally untolerant.

[0224] Optionally, the first host cell is comprised by a mixture of microbial cells (e.g., comprised by a microbial biofilm) prior to contact with said vector, and the mixture comprises cells of said second species.

[0225] Optionally, the second species is a species of Bacillus or nitrate-reducing bacteria (NRB) or nitrate-reducing, sulfide-oxidizing bacteria.

[0226] Optionally, the NRB is selected from the group consisting of Campylobacter sp., Nitrobacter sp., Nitrosomonas sp., Thiomicrospira sp., Sulfurospirillum sp., Thauera sp., Paracoccus sp., Pseudomonas sp., Rhodobacter sp., and Desulfovibrio sp., or comprises at least two of the foregoing species.

[0227] Optionally, the NRB is selected from the group consisting of Nitrobacter vulgaris, Nitrosomonas europea, Pseudomonas stutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans, Sulfurospirillum deleyianum, and Rhodobacter sphaeroides.

[0228] Optionally, the method includes contacting the host cells of said first species with a biocide, either simultaneously or sequentially, and said vector. In an example, the vector and biocide are provided pre-mixed in a composition that is contacted with the host cells.

[0229] Optionally, the biocide is selected from the group consisting of tetrakishydroxymethylphosphonium sulfate (THPS), glutaraldehyde, chlorine monoxide, chlorine dioxide, calcium hypochlorite, potassium hypochlorite, sodium hypochlorite, dibromonitriloproprionamide (DBNPA), methylene bis(thiocyanate) (MBT), 2-(thiocyanomethylthio)benzothiazole (TCMTB), bronopol, 2-Bromo-2-nitro-1,3-propanediol (BNPD), tributyltetradecylphosphonium chloride (TTPC), taurinamide and its derivatives, phenols, quaternary ammonium salts, chlorine-containing agents, quinaldinium salts, lactones, organic dyes, thiosemicarbazones, quinones, carbamates, urea, salicylamides, carboxanilides, guanides, amidines, imidazolines, acetic acid, benzoic acid, sorbic acid, propionic acid, boric acid, dehydroacetic acid, sulfurous acid, vanillic acid, parahydroxybenzoic acid Esters, isopropanol, propylene glycol, benzyl alcohol, chlorobutanol, phenylethyl alcohol, formaldehyde, iodine and its solution, povidone-iodine, hexamethylenetetramine, noxthiolin, 1-(3-chloroallyl)-3,5,7-triazo-l-azoniaadamantane chloride, taurolidine, taurultam, N-(5-nitro-2-furfuridene)-1-amino-hydantoin, 5-nitro-2-furaldehyde semicarbazone, 3,4,4'-trichome chlorocarbanilide, 3,4',5-tribromosalicylanilide, 3-trifluoromethyl-4,4'-dichlorocarbanilide, 8-hydroxyquinoline, 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylic acid, 1,4-dihydro-1-ethyl-6-fluoro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylic acid, hydrogen peroxide, peracetic acid, sodium oxychlorosene, parachlorometaxylenol, 2,4,Examples of biocides include 4'-trichloro-2'-hydroxydiphenol, thymol, chlorhexidine, benzalkonium chloride, cetylpyridium chloride, silver sulfadiazine, silver nitrate, bromine, ozone, isothiazolones, polyoxyethylene(dimethylimino)ethylene(dimethylimino)ethylene dichloride, 2-(tert-butylamino)-4-chloro-6-ethylamino-5'-triazine (terbuthylazine), and combinations thereof. In an example, the biocide is tetrakishydroxymethylphosphonium sulfate (THPS). In an example, the biocide is a quaternary ammonium compound.

[0230] Optionally, the system is used in an industrial operation selected from the group consisting of: mining; shipping; oil, gas, or petrochemical recovery or processing; hydraulic fracturing; air or water heating or cooling; potable water production, storage, or distribution; hydrocarbon transportation; and wastewater treatment.

[0231] Optionally, the surface is a surface of an apparatus used in said selected industry or the fluid is a fluid contained by an apparatus used in said selected industry.

[0232] Optionally, the surface is a surface of a kitchen, bathroom or garden appliance, or the fluid is contained by a kitchen, bathroom or garden appliance, for example an appliance used in a domestic setting.

[0233] Optionally, the fluid is a potable liquid contained in a container (eg a water tank or bottle) and the surface is a surface of the container in contact with the liquid.

[0234] Optionally, each vector comprises a mobile genetic element (MGE), the MGE comprising an origin of transfer (oriT) and the CRISPR array, the MGE capable of transfer between the first species host cell and an additional microbial host cell of the industrial or domestic system. For example, the additional cell is environmentally, industrially, or domestically tolerant in the environment (e.g., in an aqueous or soil environment), and the first host cell is environmentally non-tolerant. Optionally, oriT is functional in the first and additional host cells.

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

[0236] Optionally, each MGE is or comprises an integrative and conjugative element (ICE), or each vector is a phage capable of infecting host cells of said first species, and each MGE is a phage nucleic acid capable of said transfer between cells. Optionally, each ICE is a transposon, e.g., a conjugative transposon. Optionally, each vector is a plasmid, optionally comprising an MGE as described herein. Optionally, the sequence is comprised by a conjugative transposon in the first cell and / or the further cell.

[0237] In examples, the method is a method for controlling microbial corrosion (MIC) or biofouling of a substrate contained by an oil, gas, or petrochemical recovery, processing, storage, or transportation facility (e.g., an oil tanker, an oil rig, or an oil drilling rig), wherein the surface of the substrate is in contact with a population of first host cells, the first host cells being a first species of sulfur- or sulfate-reducing bacteria (SRB), extracellular polymeric substance-producing bacteria (EPSB), acid-producing bacteria (APB), sulfur- or sulfide-oxidizing bacteria (SOB), iron-oxidizing bacteria (IOB), manganese-oxidizing bacteria (MOB), ammonia-producing bacteria (AmPB), or acetogenic cells (AcPB) that mediate MIC or biofouling of the substrate, and the surface and cell population are in contact with a fluid selected from seawater, freshwater, fracturing fluid, or well fluid (e.g., an oil well or natural gas well), and the method includes: (i) contacting the cell population with vectors by mixing the liquid with a plurality of vectors capable of transforming or transducing a first host cell, each vector comprising a CRISPR array, whereby the CRISPR array is introduced into the host cell; (a) each CRISPR array comprises one or more sequences for expressing crRNA and a promoter for transcribing the sequences in a host cell; (b) each crRNA is capable of hybridizing to a target sequence in a host cell and directing a Cas (e.g., a Cas nuclease, e.g., Cas9 or Cfp1) of the host cell to modify (e.g., cleave) the target sequence, the target sequence being a genetic sequence for mediating host cell viability; (c) each sequence of (a) comprises a sequence R1-S1-R1' for expressing and producing a corresponding crRNA in a first host cell, wherein R1 is a first CRISPR repeat, R1' is a second CRISPR repeat, R1 or R1' is optional, and S1 is a first CRISPR spacer comprising or consisting of a nucleotide sequence that is 70, 75, 80, 85, 90, 95% or more identical to a target sequence in the first host cell; and (ii) allowing expression of said cRNA in a host cell in the presence of Cas, thereby modifying a target sequence in the host cell, resulting in a reduction in host cell viability and control of MIC or biofouling of said substrate. In embodiments, R1 and R1' are both present.

[0238] In an example, the method is for controlling bacterial biofouling in ballast water of a ship or boat, the water comprising a population of first host cells of a first microbial species that mediates said biofouling, the method comprising: (i) contacting the population with a plurality of vectors capable of transforming or transducing cells, each vector comprising a CRISPR array, whereby the CRISPR array is introduced into the host cell; (a) each CRISPR array comprises one or more sequences for expressing crRNA and a promoter for transcribing the sequences in a host cell; (b) each crRNA is capable of hybridizing to a target sequence in a host cell and directing a Cas (e.g., a Cas nuclease) of the host cell to modify (e.g., cleave) the target sequence, the target sequence being a genetic sequence for mediating host cell viability; and (ii) allowing expression of said cRNA in a host cell in the presence of Cas, thereby modifying a target sequence in the host cell, resulting in a reduction in host cell viability and control of said biofouling.

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

[0240] Optionally, step (i) comprises mixing ballast water, for example, from the hull of a ship or boat, with the vector. Optionally, the ship or boat is a marine vessel and the water is seawater. Optionally, instead of a ship or boat, the ballast water is contained by an offshore container or drilling platform, for example, an oil platform or oil rig. In examples, the ship, boat, container, platform, or rig is moored at sea (i.e., not temporarily in place).

[0241] In an example, the method is for discharging ballast water from a ship or boat, wherein the discharged ballast water comprises water treated by the method. Optionally, the water is discharged into a body of water, such as a sea, ocean, or waterway (e.g., a river, canal, lake, or reservoir), or into a container.

[0242] paragraph The present invention provides the following paragraphs, which are supported by the following examples. 1. A programmable Cas (e.g., Cas3 or Cas9) nuclease for use in a method for treating an E. coli or C. dificile infection in a subject, wherein the Cas nuclease is programmable with a guide RNA to cleave a target site contained by the genome of E. coli or C. dificile bacteria infecting the subject, thereby killing the E. coli or C. dificile cells or reducing cell growth or proliferation; the method of treatment comprises contacting the subject with the Cas nuclease, wherein the nuclease is programmed with a guide RNA to cleave the target site, thereby cleaving the genome of the E. coli or C. dificile bacteria contained by the subject and reducing infection in the subject by at least 100-fold within the first 30 minutes of treatment (e.g., within the first 15 minutes). 2. A programmable Cas (e.g., Cas3 or Cas9) nuclease for use in a method for treating an E. coli or C. dificile infection in a subject (optionally described in paragraph 1), wherein the Cas nuclease is programmable with a guide RNA to cleave a target site contained by the genome of an E. coli or C. dificile bacterium infecting the subject; Thereby, the E. coli or C. dificile cells are killed or cell growth or proliferation is reduced, and the method of treatment includes contacting a subject with a Cas nuclease, where the nuclease is programmed with a guide RNA to cleave a target site, thereby cleaving the genome of E. coli or C. dificile bacteria contained by the subject and reducing infection in the subject, and the at least 100-fold reduction in infection is maintained for at least 60 minutes (e.g., at least 120, 145, or 180 minutes) after contacting the subject with the programmed nuclease. 3. The nuclease of paragraph 1 or 2, wherein at least 60% of the infection is reduced by 60 minutes after contacting the subject with the programmed nuclease. 4. The nuclease of any one of paragraphs 1 to 3, wherein the nucleic acid encoding the nuclease (e.g., a programmed nuclease) and / or guide RNA is administered to the subject at a first time point (T1) and a second time point (T2), wherein T2 is at least 1 hour after T1. 5. A nuclease described in any one of paragraphs 1 to 4, wherein the method includes a step of reducing the infection such that the reduction in infection is sustained for 30 minutes immediately after the first 30 minutes of treatment. 6. The nuclease of any one of paragraphs 1 to 5, wherein the method comprises administering to the subject RNA or a nucleic acid encoding the RNA for expressing the RNA in the subject, wherein the RNA complexes with the nuclease and programs the nuclease to cleave a target site in a microorganism contained by the subject. 7. The nuclease of any one of paragraphs 1 to 6, wherein the nuclease is administered to the subject simultaneously or sequentially with the RNA or nucleic acid encoding the RNA. 8. The nuclease of paragraph 7, wherein the subject comprises the nuclease before administering the RNA or nucleic acid to the subject. 9. The nuclease of any one of paragraphs 1 to 8, wherein multiple viruses (e.g., phages) are administered to a subject, each virus containing a copy of a nucleic acid encoding an RNA, and the viruses infect and deliver the nucleic acid to a microorganism contained by the subject. 10. The nuclease described in paragraph 9, wherein the ratio of administered virus to microorganisms contained by the subject is 10 to 150. 11. The nuclease of any one of paragraphs 1 to 10, wherein the subject is a human or an animal, and optionally the subject is over 65 years of age or a pediatric patient. 12. The nuclease described in paragraph 11, wherein the infection is a lung, abdominal, or urinary tract infection, or the subject has undergone surgery, is under immunosuppressant drug treatment, and / or suffers from a chronic disease. 13. The nuclease of any one of paragraphs 1 to 12, wherein infection is reduced by at least 90% over a period of 1 hour or longer, optionally within the first 30 minutes (e.g., within the first 15 minutes) of treatment. 14. The nuclease of any one of paragraphs 1 to 13, wherein the method includes reducing infection by at least 100-fold within the first 30 minutes of treatment (e.g., within the first 15 minutes), and wherein the at least 100-fold reduction in infection is maintained for at least 60 minutes (e.g., at least 120, 145, or 180 minutes) after contacting the subject with the programmed nuclease. 15. The nuclease of any one of paragraphs 11 to 14, wherein the method treats or prevents sepsis and / or sepsis (e.g., septic shock) in a subject. 16. At the start of treatment, the subject (e.g., human) must have a body temperature of <36°C or >38°C, a heart rate of >90 beats per minute, a respiratory rate of >20 breaths per minute or a PaCO2 of <4.3 kP, and a blood pressure of <4000 / mm 3 or >12,000 / mm 3 17. The nuclease of paragraph 16, having a white blood cell count of 17. The nuclease of paragraph 15 or 16, wherein at the start of treatment, the subject (e.g., human) exhibits the presence of two or more of: abnormal body temperature, abnormal heart rate, abnormal respiratory rate, abnormal blood gases, and abnormal white blood cell count. 18. The nuclease of any one of paragraphs 1 to 17, wherein the subject is a human or animal, the microorganism is a bacterium (e.g., E. coli or C. dificile), and the subject's blood infection caused by the bacterium is reduced by at least 100-fold or 1000-fold within the first 30 minutes of treatment (e.g., within the first 15 minutes). 19. The subject's blood was collected 10 7 ~10 12 19. The nuclease of any one of paragraphs 11 to 18, infecting bacteria at CFU / ml. 20. The nuclease of any one of paragraphs 1 to 10, wherein the subject is a plant. 21. The nuclease of any one of paragraphs 1 to 20, wherein the bacteria are comprised by the microbiome. 22. The nuclease of paragraph 21, wherein the microbiome comprises Lactobacillus and / or Streptococcus bacteria. 23. The nuclease of any one of paragraphs 1 to 22, wherein the E coli is EHEC E coli. 24. The nuclease of any one of paragraphs 1 to 23, wherein the nuclease is a Cas nuclease (e.g., Cas3 or Cas9), a meganuclease, a TALEN (transcription activator-like effector nuclease), or a zinc finger nuclease. 25. A plurality of viruses (e.g., phages or phagemids for producing phages) for use with a nuclease described in any one of paragraphs 1 to 24 in a method of treatment, each virus containing a copy of a nucleic acid encoding an RNA, and the virus capable of infecting and delivering the nucleic acid to a microorganism contained by a subject. 26. A composition comprising a plurality of nucleic acids for programming a nuclease described in any one of paragraphs 1 to 24 in a method of treatment, wherein each nucleic acid is a nucleic acid defined in any one of paragraphs 6 to 9. 27. A CRISPR / Cas system comprising the nuclease of any one of paragraphs 1 to 26, for use in a method of treatment, wherein the nuclease is a Cas nuclease (e.g., Cas3 or Cas9), and the system comprises one or more guide RNAs or DNA encoding one or more guide RNAs, wherein each guide RNA is capable of programming the Cas nuclease to cleave a target site contained by the genome of the microorganism. 28. A guide RNA or DNA encoding a guide RNA for use in the system described in paragraph 27 for use in a method for treating an acute microbial infection in a subject, such as sepsis or sepsis. 29. A nucleic acid vector comprising the guide RNA or DNA described in paragraph 27 or 28. 30. The vector of paragraph 29, wherein the vector is a phage, phagemid, viriophage, virus, plasmid (e.g., a conjugative plasmid), or transposon. 31. An anti-sepsis or anti-sepsis composition for administration to a human or animal to treat sepsis or septicemia, the anti-sepsis or anti-sepsis composition comprising a plurality of vectors, each vector being as described in paragraph 29 or 30. 32. A method for treating an acute microbial infection in a subject, the method being as defined by any one of paragraphs 1 to 31. 33. Use of a nuclease, a plurality of viruses, a system, a guide RNA, a DNA, or a vector according to any one of paragraphs 1 to 25 and 27 to 30 in the manufacture of a composition for carrying out a method of treatment as defined by any one of paragraphs 1 to 32, wherein the subject is an organism other than a human or an animal. 34. Use of a nuclease, multiple viruses, systems, guide RNAs, DNA, or vectors described in any one of paragraphs 1 to 25 and 27 to 30 in the manufacture of a composition for performing an ex vivo method for treating a microbial infection of a substrate, wherein the microbial infection is caused by a first species or strain of microorganism, and the nuclease is programmable to cleave a target site contained by the genome of the microorganism that has infected the substrate, thereby killing the first species or strain of microorganism or reducing its growth or proliferation, and the method of treatment comprises contacting a subject with the nuclease, wherein the nuclease is programmed to cleave the target site, thereby cleaving the genome of the microorganism contained by the subject and treating the acute microbial infection of the substrate. 35. Use of a programmable nuclease in the manufacture of a composition for performing an ex vivo method for treating a microbial infection of a substrate, wherein the microbial infection is caused by a first species or strain of microorganism, and the nuclease is programmable to cleave a target site contained by the genome of the microorganism that has infected the substrate, thereby killing the first species or strain of microorganism or reducing its growth or proliferation, and the method of treatment comprises contacting a subject with the nuclease, wherein the nuclease is programmed to cleave the target site, thereby cleaving the genome of the microorganism contained by the subject and treating the acute microbial infection of the substrate. 36. The use of any one of paragraphs 33, 34, and 35, wherein a nuclease (e.g., a programmed nuclease) and / or a nucleic acid that programs the nuclease to recognize and cleave a target site is administered to a subject or substrate at a first time point (T1) and a second time point (T2), wherein T2 is at least 1 hour after T1. 37. The use of any one of paragraphs 33 to 36, wherein infection is reduced by at least 100-fold within the first 30 minutes of treatment (e.g., within the first 15 minutes). 38. The use of any one of paragraphs 33 to 37, wherein the reduction in infection is maintained at least 100-fold for at least 60 minutes (e.g., at least 120 minutes) after contacting the subject with the programmed nuclease. 39. The use of any one of paragraphs 33 to 38, wherein the reduction in infection lasts for 30 minutes immediately after the first 30 minutes of treatment. 40. The use of any one of paragraphs 33 to 39, wherein the method comprises administering to the subject or substrate RNA or a nucleic acid encoding RNA for expression of RNA in or on the subject or substrate, wherein the RNA complexes with a nuclease and programs the nuclease to cleave a target site in a microorganism contained by the subject or substrate. 41. The use of paragraph 40, wherein the nuclease is administered to the subject or substrate simultaneously or sequentially with the RNA or nucleic acid. 42. The use of paragraph 40, wherein the subject or substrate comprises a nuclease prior to administering the RNA or nucleic acid. 43. The use of any one of paragraphs 40 to 42, wherein multiple viruses (e.g., phages) are administered to a subject or substrate, each virus containing a copy of a nucleic acid, and the viruses infect and deliver the nucleic acid to a microorganism contained by the subject or substrate. 44. The use described in paragraph 43, wherein the ratio of administered virus:microorganism is 10 to 150. 45. The use of any one of paragraphs 33 to 44, wherein infection is reduced by at least 90%, optionally by the first 30 minutes (e.g., by the first 15 minutes) of treatment, and for one hour or longer. 46. ​​The use of paragraph 44 or 45, wherein infection is reduced by at least 100-fold within the first 30 minutes of treatment (e.g., within the first 15 minutes), and the at least 100-fold reduction in infection is maintained for at least 60 minutes (e.g., at least 120, 145, or 180 minutes) after contacting the subject or substrate with the programmed nuclease. 47. The use of any one of paragraphs 33 to 46, wherein the object is a plant or the substrate is a metal substrate, a plastic substrate, a concrete substrate, a stone substrate, a wood substrate, a glass substrate, or a ceramic substrate. 48. The use of any one of paragraphs 33 to 47, wherein the microorganism is a bacterium. 49. The use according to paragraph 48, wherein the bacterium is a gram-positive bacterium. 50. The use according to paragraph 48 or 49, wherein the bacterium is a Staphylococcus, Streptococcus, Enterococcus, Legionella, Haemophilus, Ghonnorhea, Acinetobacter, Escherichia, Klebsiella, Pseudomonas, or Stenotrophomonas bacterium (e.g., E coli (e.g., EHEC E coli), C dificile, V cholera, Staphylococcus (e.g., S aureus or MRSA), Streptococcus pyogenes, Acinetobacter baumannii, Legionella, Pseudomonas aeruginosa, Klebsiella pneumoniae bacterium). 51. The use of any one of paragraphs 33 to 50, wherein the nuclease is a Cas nuclease (e.g., Cas3 or Cas9), a meganuclease, a TALEN (transcription activator-like effector nuclease), or a zinc finger nuclease.

[0243] Treatment of pathogenic bacterial infections Infectious complications are a significant cause of morbidity and mortality in cancer patients, especially those with underlying hematologic malignancies, with autopsy studies demonstrating that approximately 60% of deaths are infection-related. Although fewer data exist on infectious mortality in patients with solid organ tumors, it is estimated that approximately 50% of these patients have an infection as either the primary or associated cause of death (Epidemiology of Infections in Cancer Patients, Springer International Publishing, Switzerland, 2014). Most are bacterial infections. These infectious complications remain a significant limitation of cancer treatment modalities.

[0244] The adverse effects of traditional antibiotic treatment with broad-spectrum antibiotics have been demonstrated in cancer patients treated with immune checkpoint inhibitors (ICIs). Routy et al. investigated how the gut microbiome influences the efficacy of PD-1-based immunotherapy against epithelial tumors (Routy et al., Science 2018, Vol. 359, pp. 91–97). In this study, the authors also analyzed infection / antibiotic use datasets in patients with advanced NSCLC (n=140), renal cell carcinoma (n=67), or urothelial carcinoma (n=42) who received an ICI antibody against the PD-1 / PD-L1 interaction after one or several prior therapies. Among these patients, broad-spectrum antibiotics (beta-lactam inhibitors, fluoroquinolones, or macrolides) were prescribed within two months before or within one month after the first dose of PD-1 / PD-L1 mAb. Patients generally received oral antibiotics for common indications (dental, urinary, and pulmonary infections). Adverse effects were observed when treating infections with classical broad-spectrum antibiotics in cancer patients receiving ICI therapy. See Figure 8, which shows that antibiotic treatment during ICI therapy has a fatal outcome. The median overall survival was 21.9 months in the absence of antibiotic treatment, compared with an overall survival of 9.8 months with antibiotic treatment. Thus, the median overall survival of patients treated with classical antibiotics was <50% (or >12 months shorter) than that of patients not receiving antibiotic treatment.

[0245] Another recent example supporting the importance of a "healthy" microbiome in the outcome of immuno-oncology therapy is a study by Gopalakrishnan et al. (Gopalakrishnan et al., Science 2018, 359, 97-103). See Figures 9A and 9B. The gut microbiome was observed to modulate the efficacy of anti-PD-1 blockade in melanoma patients.

[0246] Several other studies add to the expanding evidence base of important associations between the microbiome and immuno-oncological outcomes. "Microbiota: a key orchestrator of cancer therapy," Nat. Rev. Cancer 2017, Vol. 17, pp. 271-285 Matson et al., Science 2018, 359, 104-108 · L. Derosa et al., Annals of Oncology 2018 (epub March 30, 2018) M. Vetizou et al., Science. 2015, 350, 1079-84 Sivan et al., Science 2015, 350, 1084-1089

[0247] Another report, claiming to be the first systematic review of infections in patients receiving immune checkpoint blockade for cancer therapy, investigated serious infections in melanoma patients treated with immune checkpoint inhibitors (anti-CTLA-4, PD-1, and / or PD-L1) (M. Del Castillo et al., Clin. Infect. Dis. 2016, 63, 1490–1493). Serious infections were defined as those requiring hospitalization or parenteral antimicrobial therapy. Of 740 patients (898 courses of immune checkpoint blockade), 54 patients developed serious infections (7.3%). Nine patients (17%) were considered to have died from the infection. Some patients developed more than one infection, bringing the total number of infections to 58. The majority of infections were bacterial in origin (approximately 80%; i.e., bacterial infections: 80% of 7.3%; 5.8% of patients). Pneumonia and blood infections were the two main types of bacterial infections.

[0248] Immune checkpoint blockers are associated with immune-related adverse effects (irAEs) related to upregulation of the immune system. Complications are managed with immunosuppressive medications such as steroids (immunosuppression is a risk factor for subsequent opportunistic infections). Of 740 patients, 46% received steroids during the course of treatment. The risk of serious infections was 13.5% in the cohort receiving corticosteroids or infliximab (compared to 7.3% in the overall population).

[0249] Yet another report investigated the potential risk of infectious disease in lung cancer patients receiving ICI therapy. Of 84 NSCLC patients receiving nivolumab (a PD-1 inhibitor), 20 patients (23.8%) developed infectious disease. Bacterial infections accounted for 75% of infections, meaning 18% of patients had bacterial infections. The most common type of bacterial infection was pneumonia. See K. Fujita et al., Eur. Resp. J. 2017, 50, OA1478.

[0250] The gram-negative rod E. coli is one of the most common causes of bacteremia in cancer patients. The all-cause 30-day mortality rate for this pathogen is high (approximately 15%) (YE Ha et al., Int. J. Antimicr. Agen. 2013, 42, 403–409). Published estimates of 30-day all-cause mortality in patients (cancer and non-cancer) with E. coli bacteremia vary from approximately 10 to 35% (JK Abernethy et al., Clin. Microbiol. Infect. 2015, 21, 251.e1–251.e8), clearly highlighting the high burden associated with this pathogen alone. Overall, in a study examining >100 bacteremia cases in cancer patients, the pathogens causing bacteremia were primarily gram-negative bacteria (65%), with E. coli (18.3%), P. aeruginosa (18.3%), and K. pneumoniae (17.3%) being the most common organisms encountered, accounting for 54% of bacteremia cases (G. Samonis et al., Support Care Cancer 2013, 21, 2521–2526). In-hospital mortality was 26.2% in this study. Similar figures can be found elsewhere. For example, a study of neutropenic and non-neutropenic adult cancer patients with blood infections examined 399 cases of blood infection in 344 cancer patients. The most common causative pathogen group was gram-negative bacilli (45%). Among clinical isolates, E. coli (35%) was 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, 25, 1-7). These three pathogens together accounted for 33% of bacteremia cases (or 74% of Gram-negative cases). The overall 30-day mortality rate in this study was 32%.Two other reports examined the causative agents of blood infections in patients with solid tumors and found that gram-negative bacteria were the predominant pathogen type (47–55% of infections across several hundred patients) (M. Marin et al., Medicine 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 the larger of these two studies (the one with more robust individual pathogen counts), the three predominant pathogens within the gram-negative group were again E. coli (55%), P. aeruginosa (18%), and Klebsiella spp. (11%). This corresponds to 92% of gram-negative cases or 51% of the total 528 blood infections studied.

[0251] The above data regarding specific causative infectious agents in cancer patients are summarized in Table 5 below.

[0252] Thus, available data on blood infections in cancer indicate that gram-negative pathogens are involved in 45–65% of infectious cases, with three important pathogens, E. coli, K. pneumoniae, and P. aeruginosa, responsible for the majority of gram-negative cases (73–92%).

[0253] Therefore, we have systematically described the oncologist's dilemma. The reduced efficacy of cancer therapy is likely due to the reduction in microbiome diversity caused by antibiotic therapy. At least one-third of patients on checkpoint inhibitors develop serious, life-threatening infections. · If these infections are not treated, they can lead to death (within 1-2 weeks). Treatment with classical antibiotics is associated with a reduction in 4-year progression-free survival from >40% to approximately 10%. The choice is to treat potentially fatal infections that pose a risk of seriously compromising cancer therapy and are of immediate need (must be addressed).

[0254] Thus, the inventors have recognized that a need exists for methods that can treat bacterial pathogenic infections in a different way that minimizes interference with cancer therapy. The inventors have recognized that this need will also be useful in other therapeutic settings, such as transplant settings, where microbiome compositions can modulate therapeutic outcomes.

[0255] While not wishing to be bound by any particular theory, the inventors believe that using the present invention to mitigate the adverse effects of conventional antibiotic therapy on overall survival in ICI patients could, in some embodiments, lead to a doubling of overall survival (or >12 months). Capturing a treatment effect of several months in terms of median overall survival is a significant achievement in this field. Indeed, effect sizes of this order are comparable to outcomes reported in ICI clinical trials (i.e., where benefit is typically measured in months rather than years). Additionally, PD-1 / PD-L1 drugs are predicted to dominate the ICI market. By 2023, PD-1 / PD-L1 drugs are projected to account for 94% of $46B USD in global ICI sales (CTLA-4 blockers account for only 6%). Source: "Landscape & Forecast: Immune Checkpoint Inhibitors," Decision Resources, December 2017. There is therefore a particularly urgent need in medicine for improved treatments using immune checkpoint inhibitors of PD-1 or PD-L1, and the inventors believe that the present invention will be of particular benefit in this regard.

[0256] In an example, the method eliminates the need to administer a classical antibiotic, such as a broad-spectrum antibiotic (or any other disclosed herein). In another example, the present invention reduces the amount or dosing frequency of a classical antibiotic, such as a broad-spectrum antibiotic (or any other disclosed herein), administered to a subject to treat an infection. For example, inducible nuclease cleavage can be used while administering a low-dose broad-spectrum antibiotic (e.g., 50, 40, 30, 20, 10%, or less of the conventional dose) to a subject, thus treating the infection in this setting. The present invention may be particularly beneficial for patients under immunosuppression, such as cancer patients, transplant patients, or patients suffering from a viral infection (e.g., HIV (human immunodeficiency virus), CMV (cytomegalovirus), or RSV (respiratory syncytial virus) infection).

[0257] The term "broad-spectrum antibiotic" can refer to antibiotics that act against the two major groups of bacteria, Gram-positive and Gram-negative, or any antibiotic that acts against a wide range of disease-causing bacteria. These medications are used when a bacterial infection is suspected but the bacterial group is unknown (also known as empirical therapy) or when infection with multiple bacterial groups is suspected. Powerful broad-spectrum antibiotics carry inherent risks, particularly in terms of interference with naturally occurring, common bacteria and the development of antibacterial resistance. Examples of commonly used broad-spectrum antibiotics include aminoglycosides (excluding streptomycin), ampicillin, amoxicillin, clavulanic acid (Augmentin), carbapenems (e.g., imipenem), piperacillin, tazobactam, quinolones (e.g., ciprofloxacin), tetracycline, chloramphenicol, ticarcillin, trimethoprim, and sulfamethoxazole (Bactrim). In veterinary medicine, examples are co-amoxiclav (eg, for small animals), penicillin, streptomycin, oxytetracycline, and potentiated sulfonamides.

[0258] Terms Thus, in one aspect, the present invention provides the following provisions relating to the treatment of pathogenic bacterial infections using programmed nucleases. 1. A method for treating a pathogenic bacterial infection in a human or animal subject caused by a first species or strain of bacteria (first bacterium), comprising selectively killing the first bacterium contained by the subject by cleaving a target site contained by the genome of the first bacterium, wherein the cleavage is carried out using a programmable nuclease that is programmed to cleave the target site, wherein the subject is suffering from an additional disease or condition other than the pathogenic bacterial infection, and the method comprises administering a therapy to the subject to treat or prevent the additional disease or condition, wherein the nuclease treats the infection and the therapy is effective in treating or preventing the disease or condition in the presence of the programmed nuclease.

[0259] In the example, section 1 provides: A method for treating a pathogenic bacterial infection in a cancer patient caused by a first species or strain of bacteria (first bacterium), comprising selectively killing the first bacterium contained by the subject by cleaving a target site contained by the genome of the first bacterium, wherein the cleavage is carried out using a Cas nuclease programmed by a guide RNA to cleave the target site, the method also comprising administering immunotherapy to the subject to treat the patient's cancer, wherein the nuclease treats the infection and the immunotherapy is effective in treating the cancer in the presence of the programmed nuclease. A method for treating a pathogenic bacterial infection in a cancer patient caused by a first species or strain of bacteria (first bacterium), comprising selectively killing the first bacterium contained by the subject by cleaving a target site contained by the genome of the first bacterium, wherein the cleavage is performed using a Cas nuclease programmed by a guide RNA to cleave the target site; the method also comprises administering immunotherapy to the subject to treat the patient's cancer, wherein the nuclease treats the infection and the immunotherapy is effective in treating the cancer in the presence of the programmed nuclease; (a) the immunotherapy optionally comprises administering to the patient an anti-PD-1 antibody selected from pembrolizumab (or KEYTRUDA™) and nivolumab (or OPDIVO™); (b) the cancer is selected from metastatic melanoma; renal cell carcinoma; bladder cancer; solid tumor; non-small cell lung cancer (NSCLC); head and neck squamous cell carcinoma (HNSCC); Hodgkin lymphoma; cancer that overexpresses PD-L1 and does not have an EGFR or ALK mutation; colorectal cancer, and hepatocellular carcinoma; (c) the first bacterium is selected from Pseudomonas aeruginosa, Klebsiella pneumoniae, E. coli, Salmonella (e.g., S. typhimurium), Clostridium dificile, Staphylococcus (e.g., S. aureus or S. epidermis), Streptococcus (e.g., S. viridans or S. thermophilus), Pneumococcus, and Enterococcus bacteria. A method for treating a pathogenic bacterial infection in a cancer patient caused by a first species or strain of bacteria (first bacterium), comprising selectively killing the first bacterium contained by the subject by cleaving a target site contained by the genome of the first bacterium, wherein the cleavage is performed using a Cas nuclease programmed by a guide RNA to cleave the target site; the method also comprises administering immunotherapy to the subject to treat the patient's cancer, wherein the nuclease treats the infection and the immunotherapy is effective in treating the cancer in the presence of the programmed nuclease; (a) the immunotherapy optionally comprises administering an anti-PD-L1 antibody selected from atezolimumab (or TECENTRIQ™), avelumab (or BAVENCIO™), and durvalumab (or IMFINZI™); (b) the cancer is selected from metastatic melanoma; renal cell carcinoma; bladder cancer; solid tumor; non-small cell lung cancer (NSCLC); head and neck squamous cell carcinoma (HNSCC); Merkel cell carcinoma; Hodgkin lymphoma; cancer that overexpresses PD-L1 and does not have an EGFR or ALK mutation; colorectal cancer, and hepatocellular carcinoma; (c) the first bacterium is selected from Pseudomonas aeruginosa, Klebsiella pneumoniae, E. coli, Salmonella (e.g., S. typhimurium), Clostridium dificile, Staphylococcus (e.g., S. aureus or S. epidermis), Streptococcus (e.g., S. viridans or S. thermophilus), Pneumococcus, and Enterococcus bacteria. A method for treating a pathogenic bacterial infection in a cancer patient caused by a first species or strain of bacteria (first bacterium), comprising selectively killing the first bacterium contained by the subject by cleaving a target site contained by the genome of the first bacterium, wherein the cleavage is performed using a Cas nuclease programmed by a guide RNA to cleave the target site; the method also comprises administering immunotherapy to the subject to treat the patient's cancer, wherein the nuclease treats the infection and the immunotherapy is effective in treating the cancer in the presence of the programmed nuclease; (a) immunotherapy comprises administering to the patient an anti-CD52 antibody, optionally alemtuzumab (or CAMPATH™); (b) the cancer is B-cell chronic lymphocytic leukemia (CLL); (c) the first bacterium is selected from Pseudomonas aeruginosa, Klebsiella pneumoniae, E. coli, Salmonella (e.g., S. typhimurium), Clostridium dificile, Staphylococcus (e.g., S. aureus or S. epidermis), Streptococcus (e.g., S. viridans or S. thermophilus), Pneumococcus, and Enterococcus bacteria. A method for treating a pathogenic bacterial infection in a cancer patient caused by a first species or strain of bacteria (first bacterium), comprising selectively killing the first bacterium contained by the subject by cleaving a target site contained by the genome of the first bacterium, wherein the cleavage is performed using a Cas nuclease programmed by a guide RNA to cleave the target site; the method also comprises administering immunotherapy to the subject to treat the patient's cancer, wherein the nuclease treats the infection and the immunotherapy is effective in treating the cancer in the presence of the programmed nuclease; (a) the immunotherapy comprises administering to the patient an anti-CD20 antibody, optionally ofatumumab (or ARZERRA™) or rituximab (or RITUXAN™); (b) the cancer is B-cell chronic lymphocytic leukemia (CLL) (e.g., refractory CLL) or non-Hodgkin's lymphoma; (c) the first bacterium is selected from Pseudomonas aeruginosa, Klebsiella pneumoniae, E. coli, Salmonella (e.g., S. typhimurium), Clostridium dificile, Staphylococcus (e.g., S. aureus or S. epidermis), Streptococcus (e.g., S. viridans or S. thermophilus), Pneumococcus, and Enterococcus bacteria. A method for treating a pathogenic bacterial infection in a cancer patient caused by a first species or strain of bacteria (first bacterium), comprising selectively killing the first bacterium contained by the subject by cleaving a target site contained by the genome of the first bacterium, wherein the cleavage is performed using a Cas nuclease programmed by a guide RNA to cleave the target site; the method also comprises administering immunotherapy to the subject to treat the patient's cancer, wherein the nuclease treats the infection and the immunotherapy is effective in treating the cancer in the presence of the programmed nuclease; (a) the immunotherapy comprises administering to the patient an anti-KIR antibody, optionally lirilumab; (b) the cancer is optionally acute myeloid leukemia or squamous cell carcinoma of the head and neck (SCCHN); (c) the first bacterium is selected from Pseudomonas aeruginosa, Klebsiella pneumoniae, E. coli, Salmonella (e.g., S. typhimurium), Clostridium dificile, Staphylococcus (e.g., S. aureus or S. epidermis), Streptococcus (e.g., S. viridans or S. thermophilus), Pneumococcus, and Enterococcus bacteria. A method for treating a pathogenic bacterial infection in a cancer patient caused by a first species or strain of bacteria (first bacterium), comprising selectively killing the first bacterium contained by the subject by cleaving a target site contained by the genome of the first bacterium, wherein the cleavage is performed using a Cas nuclease programmed by a guide RNA to cleave the target site; the method also comprises administering immunotherapy to the subject to treat the patient's cancer, wherein the nuclease treats the infection and the immunotherapy is effective in treating the cancer in the presence of the programmed nuclease; (a) the immunotherapy optionally comprises administering to the patient an anti-CD19 CAR-T selected from axicabtagene ciloleucel (or YESCARTA™) and tisagenlecleucel (or KYMRIAH™); (b) the cancer is selected from B-cell lymphoma (e.g., non-Hodgkin's lymphoma (NHL); erosive large B-cell 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; (c) the first bacterium is selected from Pseudomonas aeruginosa, Klebsiella pneumoniae, E. coli, Salmonella (e.g., S. typhimurium), Clostridium dificile, Staphylococcus (e.g., S. aureus or S. epidermis), Streptococcus (e.g., S. viridans or S. thermophilus), Pneumococcus, and Enterococcus bacteria.

[0260] Alternatively, the CAR-T is an anti-CD30, CD38, or CD22 CAR-T. In an example, the cancer is large B-cell lymphoma after failure of at least two other types of treatment. In an example, the cancer is high-grade B-cell lymphoma arising from follicular lymphoma and DLBCL. In an example, the cancer is relapsed / remitting B-cell acute lymphoblastic leukemia. In an example, the cancer is primary central nervous system lymphoma.

[0261] In examples, the nuclease treats the infection without causing a reduction in the efficacy of the therapy. In embodiments, "without causing a reduction in the efficacy of the therapy" refers to the efficacy of the therapy compared to the reduction caused in a patient by administration of a broad-spectrum antibiotic (or an antibiotic disclosed herein) that kills multiple different species, including the first species. In embodiments, "without causing a reduction in the efficacy of the therapy" means that the efficacy of the therapy is not reduced by more than 70, 80, 90, or 95% compared to administration of the therapy in the absence of treatment for the pathogenic bacterial infection (or compared to the therapy typically achieved in a patient suffering from and receiving said therapy for the disease or condition). This can be assessed, for example, by determining the subject's progression-free survival or the duration of treatment for the disease or condition, or the subject's overall survival, and / or by determining a reduction in one or more symptoms of the disease or condition.

[0262] In an example, the infection is completely or substantially completely treated. In another example, the infection is reduced (e.g., by at least 80, 90, or 95%, as determined by a marker of the infection or a symptom thereof). The marker may be, for example, CFU of a first species or strain of bacteria per ml of a blood sample taken from a patient after the method is performed, e.g., within 24 hours of performing the method, e.g., 1-12 hours or 1-24 hours after performing the method, or 1-12 hours or 1-24 hours after administering RNA or DNA encoding the RNA to program a nuclease in the subject. For example, the RNA is a guide RNA and the nuclease is Cas (e.g., Cas3 or Cas9). The reduction may be compared to a sample taken from the subject immediately before initiating the method. Alternatively, the sample may be a stool sample, saliva sample, or urine sample.

[0263] In examples, the invention increases the overall survival rate of a human subject (compared to the median overall survival rate of humans with the same cancer and receiving the same cancer therapy treatment (e.g., administration of the same immune checkpoint inhibitor, such as nivolumab, pembrolizumab, or another antibody disclosed herein). In examples, any composition or other product of the invention herein is provided for use in such methods of treatment.

[0264] In examples, the methods are performed on a population of human subjects, where the median overall survival of the population is 120-250% (e.g., 150-200%) of the median overall survival of humans with the same cancer and receiving the same cancer therapy treatment (e.g., administration of the same immune checkpoint inhibitor, such as nivolumab, pembrolizumab, or another antibody disclosed herein). In examples, any composition or other product of the invention herein is provided for use in such methods of treatment.

[0265] A "pathogenic bacterial infection" is an infection that threatens the health of a subject, e.g., a life-threatening infection. In embodiments, a pathogenic bacterial infection is one that requires hospitalization or parenteral antimicrobial agents. The infection may be an acute bacterial infection, such as a systemic infection or a localized infection. Bacterial pathogens often cause infection in specific areas of the body. Others are generalists. Pathogenic bacterial infections are in contrast to infections with commensal bacteria, such as commensal gut bacteria, in which case the bacteria do not immediately cause health-threatening or life-threatening events.

[0266] The infection (or its symptoms) may be any of the following: Bacterial vaginosis: This is caused by bacterial overgrowth that crowds out the Lactobacillus species that maintain a healthy vaginal microbial population, altering the vaginal microflora. · Bacterial meningitis: This is a bacterial inflammation of the meninges, the protective membranes that cover the brain and spinal cord. · Bacterial pneumonia: This is a bacterial infection of the lungs. Urinary tract infections: These are mainly caused by bacteria. Symptoms include a strong and frequent sensation or urge to urinate, pain during urination, and cloudy urine. The main causative agent is Escherichia coli. The bacteria can ascend to the bladder or kidneys, causing cystitis and nephritis. Bacterial gastroenteritis: This is caused by pathogenic bacteria in the intestine. These pathogenic species are usually different from the normally harmless bacteria of the normal intestinal flora. However, different strains of the same species can be pathogenic. Bacterial skin infections: Bacterial skin infections include: Impetigo, a highly contagious bacterial skin infection common in children, caused by Staphylococcus aureus and Streptococcus pyogenes. · Erysipelas, an acute streptococcal bacterial infection of the deeper skin layers that spreads via the lymphatic system. Cellulitis is a diffuse inflammation of connective tissue with severe inflammation of the dermal and subcutaneous layers of the skin. Cellulitis can be caused by normal skin flora or by infectious contact and usually occurs through open skin, cuts, blisters, cracks in the skin, insect stings, animal bites, burns, surgical wounds, intravenous drug injections, or intravenous catheter insertion sites. Most often, the skin of the face or lower extremities is affected, but cellulitis can also occur in other tissues.

[0267] In an example, the first bacterium is Streptococcus and the patient is suffering from a chest infection, cellulitis, or tonsillitis. In an example, the first bacterium is Enterococcus and the patient is suffering from a bladder infection or sepsis. In an example, the first bacterium is Pseudomonas aeruginosa and the patient is suffering from diarrhea. In an example, the first bacterium is E coli and the patient is suffering from diarrhea. 2. The method of claim 1, wherein the subject is a cancer patient and the therapy is a cancer therapy. 3. The method of paragraph 2, wherein the therapy comprises administration of a hematopoietic stem cell transplant, a chemotherapeutic agent, an immune checkpoint inhibitor, an immune checkpoint agonist, or an immune cell (e.g., T cell and / or NK cell) enhancer; adoptive cell therapy (e.g., CAR-T therapy); radiation; or surgery.

[0268] In examples, the therapy is immunotherapy. Examples of suitable immunotherapies are adoptive cell therapy (e.g., CAR-T therapy), administration of immune checkpoint inhibitors, immune checkpoint agonists, or immune cell (e.g., T cell and / or NK cell) enhancers. For example, administration of an anti-CTLA4, PD-1, PD-L1, PD-L2, LAG3, 0X40, CD28, BTLA, CD137, CD27, HVEM, KIR, TIM-3, VISTA, ICOS, GITR, TIGIT, or SIRPa antibody, such as administration of an antibody selected from ipilimumab (or YERVOY™), tremelimumab, nivolumab (or OPDIVO™), pembrolizumab (or KEYTRUDA™), pidilizumab, BMS-936559, durvalumab, and atezolizumab, or a CAR-T therapy such as axicabtagene ciloleucel (Yescarta™) or tisagenlecleucel (Kymriah™).

[0269] In an example, the immune enhancer comprises interleukin-2 (IL-2) or a fragment or deletion mutant thereof.

[0270] In examples, surgery involves the removal of necrotic or cancerous tissue.

[0271] In examples, the chemotherapy comprises administration of a platinum-containing chemotherapy agent. In examples, the chemotherapy comprises administration of gefitinib.

[0272] In examples, the therapy is cyclophosphamide, methotrexate, and 5-fluorouracil (CMF); 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 (MOPP). bleomycin, etoposide, and cisplatin (BEP); epirubicin, cisplatin, and 5-fluorouracil (ECF); or epirubicin, cisplatin, and capecitabine (ECX); methotrexate, vincristine, doxorubicin, and cisplatin (MVAC); cyclophosphamide, doxorubicin, and vincristine (CAV); or 5-fluorouracil, folinic acid, and oxaliplatin (FOLFOX).

[0273] In an example, the cancer is breast cancer and the therapy includes administering CMF or AC. In an example, the cancer is Hodgkin's lymphoma and the therapy includes administering TAC, ABVD, or MOPP. In an example, the cancer is non-Hodgkin's lymphoma and the therapy includes administering CHOP. In an example, the cancer is germ cell cancer and the therapy includes administering BEP. In an example, the cancer is gastric cancer and the therapy includes administering ECF or ECX. In an example, the cancer is bladder cancer and the therapy includes administering MVAC. In an example, the cancer is lung cancer and the therapy includes administering CAV. In an example, the cancer is colorectal cancer and the therapy includes administering FOLFOX. 4. The method of paragraph 3, wherein the therapy is an immune checkpoint inhibitor antibody.

[0274] Optionally, the antibody is an anti-CTLA4, PD-1, PD-L1, PD-L2, LAG3, 0X40, CD28, BTLA, CD137, CD27, HVEM, KIR, TIM-3, VISTA, ICOS, GITR, TIGIT, or SIRPa antibody. In examples, the antibody is an anti-PD-1 antibody. In examples, the antibody is a PD-L1 antibody. In examples, the antibody is an anti-CTLA4 antibody. 5. The method of paragraph 3, wherein the therapy is administration of an antibody selected from ipilimumab (or YERVOY™), tremelimumab, nivolumab (or OPDIVO™), pembrolizumab (or KEYTRUDA™), pidilizumab, BMS-936559, durvalumab, and atezolizumab.

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

[0276] In examples, the anti-PD-1 antibody used herein is selected from nivolumab, pembrolizumab, pidilizumab, OPDIVO®, KEYTRUDA®, AMP-514, REGN2810, CT-011, BMS 936559, MPDL3280A, and AMP-224.

[0277] In examples, the anti-CTLA4 antibody herein is selected from tremelimumab, YERVOY®, and ipilimumab.

[0278] In an example, the therapy is the administration of an anti-KIR antibody, such as lirilumab.

[0279] In examples, the checkpoint inhibitor is selected from an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR. In certain embodiments, the immune checkpoint inhibitor is a human programmed cell death 1 (PD-1) axis binding antagonist. In some embodiments, the PD-1 axis binding antagonist is selected from the group consisting of a PD-1 binding antagonist, a PD-L1 binding antagonist, and a PD-L2 binding antagonist. In certain embodiments, the PD-1 axis binding antagonist is a PD-1 binding antagonist. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and / or PD-L2.

[0280] In some embodiments, the immune checkpoint inhibitor is a PD-L1 antagonist, such as durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, or avelumab, also known as MSB00010118C. In certain aspects, the immune checkpoint inhibitor is a PD-L2 antagonist, such as rHIgM12B7. In some aspects, the immune checkpoint inhibitor is a LAG-3 antagonist, such as IMP321 or BMS-986016. The immune checkpoint inhibitor may also be an adenosine A2a receptor (A2aR) antagonist, such as PBF-509.

[0281] In some embodiments, an antibody described herein (e.g., an anti-PD-1 antibody, anti-PD-L1 antibody, or anti-PD-L2 antibody) further comprises a human or murine constant region. In a still further aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4. In a still further specific aspect, the human constant region is IgG1. In a still further aspect, the murine constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3. In a still further specific aspect, the antibody has reduced or minimal effector function. In a still further specific aspect, the minimal effector function results from production in prokaryotic cells, CHO cells, Cos cells, or HEK cells. In a still further specific aspect, the minimal effector function results from an "effector-free Fc mutation" or aglycosylation.

[0282] For example, the therapy includes hematopoietic stem cell transplantation, eg, bone marrow transplantation (such as when the patient is a cancer patient, eg, a blood cancer or leukemia patient).

[0283] For example, the therapy includes stem cell transplantation, skin transplantation, or organ transplantation, such as heart transplantation, liver transplantation, kidney transplantation, or lung transplantation. 6. The method of paragraph 1 or 2, wherein the therapy is tissue transplantation, organ transplantation, or cell transplantation. 7. The method of any one of paragraphs 1 to 6, wherein treating the bacterial infection is performed simultaneously with administering therapy to the subject. 8. The method of any one of paragraphs 1 to 6, wherein the treatment of the bacterial infection is carried out immediately before administering the therapy to the subject.

[0284] In examples, treating the bacterial infection is performed within 7, 6, 5, 4, 3, 2, or 1 day before or within 24, 12, 6, 5, 4, 3, 2, 1, or 0.5 hours after treating the additional disease or condition. In examples, treating the bacterial infection is performed within 7, 6, 5, 4, 3, 2, or 1 day after or within 24, 12, 6, 5, 4, 3, 2, 1, or 0.5 hours after treating the additional disease or condition.

[0285] Treatment of infection and administration of therapy may be performed simultaneously or sequentially. 9. The method of any one of paragraphs 1 to 6, wherein treating the bacterial infection is performed immediately after administering therapy to the subject. 10. The method of any one of paragraphs 1 to 9, wherein the method comprises administering to the subject an RNA (e.g., a gRNA) or a nucleic acid encoding the RNA for expressing the RNA in the subject, wherein the RNA complexes with a nuclease and programs the nuclease to cleave a target site in a first bacterium contained by the subject, thereby killing the first bacterium.

[0286] The RNA or nucleic acid is administered to the subject or patient, for example, orally, by IV injection, by subcutaneous injection, or by inhalation. 11. The method of any one of paragraphs 1 to 10, comprising administering to the subject a vector (e.g., a phage or plasmid), wherein the vector encodes a programmable nuclease.

[0287] The nuclease is administered to the subject or patient, for example, orally, by IV injection, by subcutaneous injection, or by inhalation. 12. The method of any one of paragraphs 1 to 10, wherein the programmable nuclease is an endogenous nuclease (e.g., a Cas nuclease) of the first cell. 13. The method of any one of paragraphs 1 to 12, wherein the efficacy of the therapy in the presence of the programmed nuclease is greater than the efficacy of the therapy in the presence of a broad-spectrum antibiotic.

[0288] In examples, greater efficacy is assessed by determining progression-free survival or duration of treatment for the disease or condition, and / or determining a reduction in one or more symptoms of the disease or condition, for example, compared to a similar determination in a patient suffering from the disease or condition and a bacterial infection and treated with a therapy and antibiotic (rather than nuclease killing of the first bacterium according to the present invention). 14. The method of any one of paragraphs 1 to 13, wherein the efficacy of therapy in the presence of a programmed nuclease is greater than the efficacy of therapy in the presence of an antibiotic selected from methicillin, vancomycin, linezolid, daptomycin, quinupristin, dalfopristin; teicoplanin; cephalosporins; carbapenems; fluoroquinolones; aminoglycosides; colistin; erythromycin; clindamycin; beta-lactams; macrolides; amoxicillin; azithromycin; penicillin; ceftriaxone; azithromycin; ciprofloxacin; isoniazid (INH); rifampicin (RMP); amikacin; kanamycin; capreomycin; trimethoprim; itrofurantoin; cephalexin; amoxicillin; metronidazole (MTZ); cefixime; tetracycline; and meropenem. 15. The first bacterium is (i) Staphylococcus aureus resistant to an antibiotic selected from methicillin, vancomycin, linezolid, daptomycin, quinupristin, dalfopristin, and teicoplanin; or (ii) Pseudomonas resistant to an antibiotic selected from cephalosporins, carbapenems, fluoroquinolones, aminoglycosides, and colistin. aeuroginosa; (iii) Klebsiella species resistant to carbapenems; (iv) Streptococcus species resistant to antibiotics selected from erythromycin, clindamycin, beta-lactams, macrolides, amoxicillin, azithromycin, and penicillin; (v) Salmonella species resistant to antibiotics selected from ceftriaxone, azithromycin, and ciprofloxacin; (vi) Shigella species resistant to ciprofloxacin or azithromycin; (vii) Mycobacterium resistant to antibiotics selected from resistance to isoniazid (INH), rifampicin (RMP), fluoroquinolones, amikacin, kanamycin, capreomycin, and azithromycin. (ix) Enterobacteriaceae species resistant to antibiotics selected from cephalosporins and carbapenems; (x) E coli resistant to antibiotics selected from trimethoprim, itorofurantoin, cephalexin, and amoxicillin; (xi) Clostridium species resistant to metronidazole (MTZ), fluoroquinolones, or carbapenems; (xii) Neisseria gonnorrhoeae resistant to antibiotics selected from cefixime, ceftriaxone, azithromycin, and tetracycline; (xiii) Acinetoebacter baumannii resistant to antibiotics selected from beta-lactams, meropenem, and carbapenems; and (xiv) Campylobacter species resistant to ciprofloxacin or azithromycin. 16. The method of any one of paragraphs 1 to 15, wherein treating an infection treats or prevents in a subject a condition selected from vaginosis, meningitis, pneumonia, urinary tract infection, cystitis, nephritis, gastroenteritis, skin infection, impetigo, erysipelas, dental infection, and cellulitis. 17. The method of any one of paragraphs 1 to 16, wherein treating an infection treats or prevents sepsis or sepsis in the subject.

[0289] In an example, the infection is a blood-borne infection. 18. The method of any one of paragraphs 1 to 17, wherein the further disease or condition is cancer; an autoimmune disease or condition; a viral infection or a GI tract disease or condition. In an example, the cancer is metastatic. In an example, the cancer is melanoma. In an example, the cancer is a solid tumor with mismatch repair deficiency or microsatellite instability. In an example, the cancer is NSCLC. In an example, the cancer is HNSCC. In an example, the cancer is Hodgkin's lymphoma. In an example, the cancer is urothelial carcinoma. In an example, the cancer is lung cancer. In an example, the cancer is head and neck cancer. In an example, the cancer is head cancer. In an example, the cancer is neck cancer. In examples, the viral infection is an HIV, CMV, or RSV infection. 19. The method of any one of paragraphs 1 to 18, wherein the subject comprises bacteria (second bacteria) of one or more strains or species different from the first strain or species, the genome of the second bacteria does not comprise the target site, and the genome of the second bacteria is not cleaved by the programmed nuclease in the subject, such that the second bacteria survives in the presence of the programmed nuclease in the patient, and the therapy is effective in the presence of the second bacteria. 20. The method of paragraph 19, wherein a reduction in the second bacterium in the patient (e.g., in the gut microbiome) is associated with reduced efficacy of the therapy.

[0290] Optionally, the therapy is effective in the presence of a second bacterium in the subject's intestine.

[0291] Optionally, the first and / or second bacteria are present in the intestine of the subject immediately prior to performing the method.

[0292] Optionally, the first and / or second bacteria are present in the subject's blood immediately prior to performing the method.

[0293] Optionally, the first bacterium is present in the subject's blood and the second bacterium is present in the subject's intestine immediately prior to performing the method.

[0294] Optionally, immediately prior to performing the method, the first bacterium is present in the intestine of the subject and the second bacterium is present in the blood of the subject.

[0295] Optionally, the first bacteria in the subject's blood is killed.

[0296] Optionally, the bacteria is a gram-positive bacterium. Optionally, the bacteria is a gram-negative bacterium.

[0297] Optionally, the first and second bacteria can be killed by the same antibiotic. Optionally, the method does not include administering an antibiotic to the subject. In examples, the antibiotic is selected from methicillin, vancomycin, linezolid, daptomycin, quinupristin, dalfopristin; teicoplanin; cephalosporins; carbapenems; fluoroquinolones; aminoglycosides; colistin; erythromycin; clindamycin; beta-lactams; macrolides; amoxicillin; azithromycin; penicillin; ceftriaxone; azithromycin; ciprofloxacin; isoniazid (INH); rifampicin (RMP); amikacin; kanamycin; capreomycin; trimethoprim; itorofurantoin; cephalexin; amoxicillin; metronidazole (MTZ); cefixime; tetracycline; and meropenem. In examples, the antibiotic is selected from an aminoglycoside, ampicillin, amoxicillin, amoxicillin or clavulanic acid, a carbapenem (e.g., imipenem), piperacillin or tazobactam, a quinolone (e.g., ciprofloxacin), a tetracycline, chloramphenicol, ticarcillin, trimethoprim or sulfamethoxazole, a penicillin, a streptomycin, an oxytetracycline, and a potentiated sulfonamide. In examples, the first bacteria is resistant to an antibiotic selected from an aminoglycoside, ampicillin, amoxicillin, amoxicillin or clavulanic acid, a carbapenem (e.g., imipenem), piperacillin or tazobactam, a quinolone (e.g., ciprofloxacin), a tetracycline, chloramphenicol, ticarcillin, trimethoprim or sulfamethoxazole, penicillin, streptomycin, oxytetracycline, and a potent sulfonamide. In alternative embodiments, the antibiotic is selected from a beta-lactam, a fluoroquinolone, and a macrolide.

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

[0299] Optionally, the first and second bacteria are bacteria of the same genus, but different species of that genus.

[0300] Optionally, the first and second bacteria are bacteria of the same family, but different genera within that family.

[0301] Optionally, the first and second bacteria are gram-positive bacteria.

[0302] Optionally, the first and second bacteria are gram-negative bacteria.

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

[0304] Optionally, a reduction in the second bacteria in the patient is associated with a reduction in the effectiveness of the therapy.Optionally, a reduction in the second bacteria in the patient reduces the effectiveness of the therapy.

[0305] Optionally, the presence of the second bacterium in the patient is associated with enhanced efficacy of the therapy. Optionally, the presence of the second bacterium in the patient enhances the efficacy of the therapy. For example, the enhanced efficacy is compared to the therapy in the absence or reduced presence of the second bacterium, such as in the presence of an antibiotic that kills the second bacterium.

[0306] In examples, the therapy is effective in the presence of the second bacterium and the subject's disease or condition (or symptoms thereof) is reduced by at least 20, 30, 40, 50, 60, 70, 80, 90, or 95%. In examples, the therapy is effective in the presence of the second bacterium and the progression of the subject's disease or condition (or symptoms thereof) is reduced by at least 20, 30, 40, 50, 60, 70, 80, 90, or 95%. In examples, the therapy is effective in the presence of the second bacterium and the disease progression-free period of the subject's disease or condition (or symptoms thereof) is reduced by at least 20, 30, 40, 50, 60, 70, 80, 90, or 95%. In examples, the therapy is effective in the presence of the second bacterium and the duration of the subject's disease or condition (or symptoms thereof) is reduced by at least 20, 30, 40, 50, 60, 70, 80, 90, or 95%. In examples, the therapy is effective in the presence of the second bacterium and the severity of the subject's disease or condition (or symptoms thereof) is reduced by at least 20, 30, 40, 50, 60, 70, 80, 90, or 95%. In examples, the therapy is effective in the presence of the second bacterium and the patient's disease or condition (or symptoms thereof) is reduced by at least 20, 30, 40, 50, 60, 70, 80, 90, or 95% for 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. In examples, the therapy is effective in the presence of the second bacteria and the patient's disease or condition (or symptoms thereof) is at least 20, 30, 40, 50, 60, 70, 80, 90, or 95% treated for 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. In examples, the therapy is effective in the presence of the second bacteria and the patient's disease or condition (or symptoms thereof) is at least 20, 30, 40, 50, 60, 70, 80, 90, or 95% undetectable for 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. 21. The method of claim 19 or 20, wherein the second bacterium is selected from the group consisting of Akkermansia, Alistipes, Bacteroides, Barnesiella, Bifidobacterium, Clostridium, Collinsella, Enterococcus, Fusobacterium, Lactobacillus, Propionibacterium, Ruminococcus, Segmented filamentous bacteria (SFB); Veillonella, Prevotella, Escherichia, and Streptococcus bacteria.

[0307] In some embodiments, the Clostridiales (e.g., Clostridium) bacteria comprise a first family and a second family. In some embodiments, the first family is selected from the group consisting of Ruminococcaceae, Christensenellaceae, Clostridiaceae, and Coriobacteriacease, and the second family is not the same as the first family. In the example, the second bacterium is Faecalibacterium prausnitzii, Ruminococcus albus, Ruminococcus bromii, Ruminococcus callidus, Ruminococcus flavefaciens, Ruminococcus champanellensis, Ruminococcus faecis, Ruminococcus gauvreauii, Ruminococcus gnavus, Ruminococcus hansenii, Ruminococcus hydrogenotrophicus, Ruminococcus lactaris, Ruminococcus luti, Ruminococcus obeum, Ruminococcus palustris, Ruminococcus pasteurii, Ruminococcus productus, Ruminococcus schinkii, Ruminococcus torques, Subdoligranulum variabile, Butyrivibrio fibrisolvens, Roseburia intestinalis, Anaerostipes caccae, Blautia obeum, Eubacterium nodatum, or Eubacterium oxidoreducen In certain embodiments, the second bacterium is Faecalibacterium prausnitzii. In examples, the second bacterium is a Firmicutes.

[0308] In certain embodiments, the first bacterium is a Bacteroidia or Prevotellaceae, such as a Bacteroidetes or Bacteroides.

[0309] In embodiments, the treatment results in or maintains a subject's microbiome (e.g., gut microbiome and / or blood microbiome) that is beneficial to immune checkpoint inhibition or other therapy. In examples, the microbiome comprises one or more bacterial species with high relative abundance from the phylum Firmicutes, class Clostridia, order Clostridiales, family Ruminococcaceae, genus Ruminococcus, genus Hydrogenoanaerobacterium, genus Faecalibacterium, phylum Actinobacteria, class Coriobacteriia, order Coriaobacteriales, family Coriobacteriaceae, domain Archaea, phylum Cyanobacteria, phylum Euryarchaeota, or family Christensenellaceae. Additionally or alternatively, the microbiome comprises bacteria with low relative abundance from the genus Dialister, family Veillonellaceae, phylum Bacteroidetes, class Bacteroida, order Bacteroidales, or family Prevotellaceae. Thus, a preferred microbial profile will have a higher relative abundance of one or more bacterial species from the phylum Firmicutes, class Clostridia, order Clostridiales, family Ruminococcaceae, genus Ruminococcus, genus Hydrogenoanaerobacterium, phylum Actinobacteria, class Coriobacteria, order Coriaobacteriales, family Coriobacteriaceae, domain Archaea, phylum Cyanobacteria, phylum Euryarchaeota, or family Christensenellaceae, and / or have a reduced abundance of one or more bacterial species from the genus Dialister, family Veillonellaceae, phylum Bacteroidetes, order Bacteroida, order Bacteroidales, and / or family Prevotellaceae.

[0310] For example, the microbiome comprises a higher relative abundance of Firmicutes compared to Bacteroidetes, Bacteroida, Bacteroidales, or Prevotellaceae. For example, the microbiome comprises a higher relative abundance of Firmicutes compared to Bacteroidetes, Bacteroida, Bacteroidales, and Prevotellaceae.

[0311] It contains 2 products: Akkermansia muciniphila;Alistipes shahii;Bacteroides fragilis;Bacteroides uniformis;Barnesiella intestinihominis;Bacteroides dorei;Bifidobacterium adolescentis;Bifidobacterium brief;Bifidobacterium longum;Clostridium orbiscindens;Clostridium novyi;Clostridium perfringens;Collinsella aerofaciens;Enterococcus hirae;Fusobacterium nucleatum;Lactobacillus casei Shirota;L. casei AO47;Lactobacillus rhamnosus;Propionibacterium granulosum;Ruminococcus gnavus;Segmented filamentous bacterium (SFB);Veillonella;Lactobacilli;Bacteroides;Clostridia;Prevotella;E. coli Nissle;Lactobacillus plantarum;Lactobacillus delbrueckii(from Bulgaricus);Lactobacillus paracasei;Lactobacillus acidophilus;Bifidobacterium infantis;and Streptococcus The microbiome in cancer immunotherapy: Diagnostic tools and therapeutic strategies: Lawrence Zitvogel, Science Published as of March 23, 2018: 359 members, 6382 members, range 1366~1370;

[0312] In examples, the second bacterium is a commensal bacterium in humans.

[0313] In examples, the first bacterium is comprised by gut microbiota, skin microbiota, oral microbiota, throat microbiota, hair microbiota, armpit microbiota, vaginal microbiota, rectal microbiota, anal microbiota, eye microbiota, nasal microbiota, tongue microbiota, lung microbiota, liver microbiota, kidney microbiota, genital microbiota, penile microbiota, scrotal microbiota, mammary microbiota, ear microbiota, urethral microbiota, labial microbiota, organ microbiota, or dental microbiota.

[0314] In examples, the second bacterium is comprised by gut microbiota, skin microbiota, oral microbiota, throat microbiota, hair microbiota, armpit microbiota, vaginal microbiota, rectal microbiota, anal microbiota, eye microbiota, nasal microbiota, tongue microbiota, lung microbiota, liver microbiota, kidney microbiota, genital microbiota, penile microbiota, scrotal microbiota, mammary microbiota, ear microbiota, urethral microbiota, labial microbiota, organ microbiota, or dental microbiota.

[0315] In examples, the first and / or second bacteria are blood-borne bacteria. 22. The method of any one of paragraphs 1 to 21, wherein the first bacterium is selected from the group consisting of Staphylococcus, Streptococcus, Enterococcus, Helicobacter, Legionella, Haemophilus, Ghonnorhea, Acinetobacter, Escherichia, Klebsiella, Pseudomonas, or Stenotrophomonas bacteria.

[0316] H pylori has been implicated in gastric cancer and gastric ulcers. Thus, in an example, the first bacterium is H pylori, and optionally the disease is cancer, such as gastric cancer. In an embodiment, the therapy is chemotherapy or immune checkpoint inhibitor (e.g., antibody) therapy. In an example, the first bacterium is H pylori, and the disease is gastric ulcers. In an embodiment, triple therapy for gastric ulcers is administered to the subject.

[0317] In examples, the first bacterium is a gram-negative bacterium, and optionally the infection is a blood-borne infection. In examples, the first bacterium is selected from E. coli, P. aeruginosa, and K. pneumoniae, and optionally the infection is a blood-borne infection. 23. The method of paragraph 22, wherein the first bacterium is selected from the group consisting of E coli (e.g., EHEC E coli), C dificile, V cholera, Staphylococcus (e.g., S aureus or MRSA), Streptococcus pyogenes, Helicobacter pylori, Acinetobacter baumannii, Legionella, Pseudomonas aeruginosa, and Klebsiella pneumoniae bacteria.

[0318] In examples, the subject is receiving or is on a course of immunosuppressant drugs, eg, steroids such as corticosteroids. 24. A programmable nuclease for use in the method of any one of paragraphs 1 to 23. 25. The method or nuclease of any one of paragraphs 1 to 24, wherein the nuclease is a Cas nuclease (e.g., Cas3 or Cas9), a meganuclease, a TALEN (transcription activator-like effector nuclease), or a zinc finger nuclease. 26. A CRISPR / Cas system comprising the nuclease of paragraph 24 or 25 for use in the method of any one of paragraphs 1 to 23, wherein the nuclease is a Cas nuclease (e.g., Cas3 or Cas9) and the system comprises one or more guide RNAs (gRNAs) or DNA encoding one or more guide RNAs, each guide RNA capable of programming the Cas nuclease to cleave a target site contained by the genome of the first bacterium. 27. A guide RNA or DNA encoding a guide RNA for use in the system described in paragraph 26. 28. A guide RNA or DNA encoding a guide RNA for use in a method for treating a pathogenic bacterial infection according to any one of paragraphs 1 to 23, wherein the guide RNA is capable of programming a nuclease, and the nuclease is a Cas nuclease (e.g., Cas9, Cas3, Cas13, CasX, CasY, or Cpf1 nuclease). 29. A nucleic acid vector comprising the guide RNA or DNA of any one of paragraphs 26 to 28. 30. A nucleic acid vector encoding a nuclease according to paragraph 24 or 25 and optionally a guide RNA according to paragraph 29. 31. The vector of paragraph 29 or paragraph 30, wherein the vector is a phage, phagemid, plasmid (e.g., a conjugative plasmid), or transposon.

[0319] The phage is capable of infecting a first bacterium and the phagemid is capable of producing such phage in the presence of a helper phage. 32. A pharmaceutical composition comprising a first nucleic acid vector(s) encoding a nuclease according to paragraph 24 or 25, and a second nucleic acid vector(s) encoding a guide RNA according to paragraph 29, the pharmaceutical composition further comprising a pharmaceutically acceptable diluent, excipient, or carrier. 33. A pharmaceutical composition comprising the CRISPR / Cas system of paragraph 26 and a pharmaceutically acceptable diluent, excipient, or carrier. 34. A pharmaceutical composition comprising the vector of paragraph 31 and a pharmaceutically acceptable diluent, excipient, or carrier.

[0320] Prevention of a disease or condition herein can be, for example, reducing a subject's or patient's risk of the disease or condition.

[0321] In an alternative form, the infection is caused by a first archaea instead of a first bacterium, and in this embodiment, all features and other configurations of the method of the invention relating to the killing of the first bacterium relate instead, mutatis mutandis, to the killing of the first archaea.

[0322] In embodiments, the method comprises performing a method for treating an acute microbial infection as described herein, and therefore, the features of that method as described herein are combinable with the method for treating a pathogenic bacterial infection (i.e., where the pathogenic bacterial infection is the acute microbial infection in the first method). In embodiments, the method comprises performing a method for sustained treatment of a microbial infection as described herein, and therefore, the features of that method as described herein are combinable with the method for treating a pathogenic bacterial infection (i.e., where the pathogenic bacterial infection is the microbial infection in the first method). Any optional features of the first method herein may be applied mutatis mutandis to the method for treating a pathogenic bacterial infection.

[0323] Aspects Accordingly, the present invention provides the following aspects, which are optional features of the above clauses: 1. The method of any one of paragraphs 1 to 23, wherein the infection is reduced by at least 100-fold within the first 30 minutes of performing step (b). Optionally, the infection is reduced by at least 1000-fold within the first 30 minutes of performing step (b). Optionally, the reduction in infection persists for 30 minutes immediately after the first 30 minutes of performing step (b). For example, reduction can be assessed by determining the difference in bacterial count of a first species or strain in (i) a sample (e.g., a blood sample) taken from the subject immediately before the start of the method and (ii) a sample of the same type, e.g., a blood sample, taken from the subject 30 minutes into treatment. For example, samples may be assessed for differences in colony-forming units (CFU) / ml of sample, e.g., when the samples are plated on agar in corresponding Petri dishes and incubated under identical conditions. In another example, microscopic counting of microorganisms in a sample or other routine methods known to those skilled in the art may be used. 2. The method of any one of paragraphs 1 to 23, wherein the subject's blood infection caused by the first bacterium is reduced by at least 100-fold or 1000-fold within the first 30 minutes of performing step (b). 3. Blood should be collected immediately before treatment. 5 ~10 12 (e.g., 10 7 ~10 12 3. The method of embodiment 2, wherein the cells are infected with at least 1 CFU / ml of the first bacterium. 4. The method of any one of paragraphs 1 to 23, comprising administering to the subject a nucleic acid (e.g., RNA) and a nuclease, wherein the nucleic acid complexes with the nuclease and programs the nuclease to cleave a target site in a first bacterium contained by the subject. 5. The method of embodiment 4, wherein the nuclease is administered to the subject simultaneously or sequentially with the nucleic acid. 6. The method of embodiment 4, wherein the subject comprises a nuclease before administering the nucleic acid to the subject. 7. The method of any one of aspects 4 to 6, wherein a plurality of phages are administered to the subject, each phage containing a copy of the nucleic acid, and the phage infects and delivers the nucleic acid to a first bacterium contained by the subject. 8. The method of embodiment 7, wherein the ratio of administered phage to first bacteria contained by the subject is 10 to 150. For example, the ratio is 10 to 100, i.e., a multiplicity of infection (MOI) of 10 to 100.

[0324] The ratio can be determined, for example, by using a sample (e.g., a blood sample or an intestinal sample) from a human or animal subject immediately prior to treatment and determining the number of bacteria per ml of the blood or intestinal sample. The amount of phage to be administered can then be calculated according to the determination using the sample. 9. The method of any one of paragraphs 1 to 23, wherein the infection is an infection of the lungs, brain, skin, abdomen, or urinary tract. 10. The method of any one of paragraphs 1 to 23 or any one of aspects 1 to 9, wherein the subject has undergone surgery, is under immunosuppressant medication, has a burn, has diabetes, has cancer, or has a chronic disease. 11. The method of any one of paragraphs 1 to 23 or any one of aspects 1 to 10, wherein the subject is a human over 65 years of age or a pediatric patient. 12. The method of any one of paragraphs 1 to 23 or any one of aspects 1 to 11, for treating or preventing sepsis in a subject. 13. At the start of treatment, the subject (e.g., human) must have a body temperature of <36°C or >38°C, a heart rate of >90 beats per minute, a respiratory rate of >20 breaths per minute or a PaCO2 of <4.3 kP, and a blood pressure of <4000 / mm 3 or >12,000 / mm 3 13. The method of claim 12, wherein the patient has a white blood cell count of 14. The method of paragraph 12 or 13, wherein at the start of treatment, the subject (e.g., human) exhibits the presence of two or more of: abnormal body temperature, abnormal heart rate, abnormal respiratory rate, abnormal blood gases, and abnormal white blood cell count.

[0325] Immune Checkpoint Modulation Immune checkpoints of the present invention either upregulate signals (e.g., costimulatory molecules) or downregulate signals. Inhibitory immune checkpoint molecules that can be targeted by immune checkpoint modulation in the present invention include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B- and T-lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte-associated protein (CTLA-4, also known as CD152), indoleamine 2,3 dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), and V-domain Ig suppressor of T-cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 system and / or CTLA-4.

[0326] Immune checkpoint inhibitors may be drugs such as small molecules, recombinant forms of ligands or receptors, or antibodies, such as human antibodies (e.g., WO2015016718; Pardoll, Nat Rev Cancer, 12(4):252-64, 2012, both incorporated herein by reference). Known inhibitors of immune checkpoint proteins or analogs thereof may be used, and in particular chimeric, humanized, or human forms of antibodies may be used. As will be known to those of skill in the art, alternative and / or equivalent names may be used for certain antibodies referred to in this disclosure. Such alternative and / or equivalent names may be used synonymously in the context of the present invention. For example, it is known that lambrolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

[0327] It is contemplated that any immune checkpoint inhibitor known in the art to stimulate an immune response can be used. This includes inhibitors that directly or indirectly stimulate or enhance antigen-specific T lymphocytes. Such immune checkpoint inhibitors include, but are not limited to, agents that target immune checkpoint proteins and pathways involving PD-L2, LAG3, BTLA, B7H4, and TIM3. For example, LAG3 inhibitors known in the art include soluble LAG3 (IMP321, or LAG3-Ig disclosed in WO2009044273), as well as murine or humanized antibodies that block human LAG3 (e.g., IMP701 disclosed in WO2008132601), or fully human antibodies that block human LAG3 (such as those disclosed in EP2320940). Another example is provided by the use of blocking agents against BTLA, including but not limited to antibodies that block human BTLA interaction with its ligand (such as 4C7 disclosed in WO2011014438). Yet another example is provided by the use of agents that neutralize B7H4, including but not limited to antibodies against human B7H4 (such as those disclosed in WO2013025779 and WO2013067492) or against soluble recombinant forms of B7H4 (such as those disclosed in U.S. Patent Application Publication No. 20120177645). Yet another example is provided by agents that neutralize B7-H3, including but not limited to antibodies that neutralize human B7-H3 (e.g., MGA271 and derivatives disclosed as BRCA84D in U.S. Patent Application Publication No. 20120294796). Further examples are provided by agents that target TIM3, including, but not limited to, antibodies that target human TIM3 (e.g., those disclosed in WO2013006490A2, or the anti-human TIM3 blocking antibody F38-2E2 disclosed in Jones et al., J Exp Med. 2008;205(12):2763-79).

[0328] A. PD-1 antagonists T cell dysfunction or anergy occurs concomitantly with the induction and maintenance of expression of the inhibitory receptor, programmed death 1 polypeptide (PD-1). Accordingly, provided herein is therapeutic targeting of PD-1 and other molecules that signal through interactions with PD-1, such as programmed death-ligand 1 (PD-L1) and programmed death-ligand 2 (PD-L2). PD-L1 is overexpressed in many cancers and is often associated with poor prognosis (Okazaki T et al. (et al.), Intern. Immun. 2007, 19(7):813). Accordingly, provided herein are improved methods for treating cancer by inhibiting PD-L1 / PD-1 interaction in combination with modulation of the microbiome.

[0329] For example, PD-1 axis binding antagonists include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists. Alternative names for "PD-1" include CD279 and SLEB2. Alternative names for "PD-L1" include B7-H1, B7-4, CD274, and B7-H. Alternative names for "PD-L2" include B7-DC, Btdc, and CD273. In some embodiments, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1, and PD-L2.

[0330] In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a specific aspect, the PD-1 ligand binding partner is PD-L1 and / or PD-L2. In another embodiment, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In a specific aspect, the PD-L1 binding partner is PD-1 and / or B7-1. In another embodiment, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partner. In a specific aspect, the PD-L2 binding partner is PD-1. The antagonist may be an antibody, antigen-binding fragment thereof, immunoadhensin, fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 system antagonists for use in the methods provided herein are known in the art, such as those described in U.S. Application Publication No. 20140294898, U.S. Patent Application No. 2014022021, and U.S. Patent Application Publication No. 20110008369, all of which are incorporated herein by reference.

[0331] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1-binding portion of PDL1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in WO 2006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO 2009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO 2009 / 101611. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in WO 2010 / 027827 and WO 2011 / 066342. Additional PD-1 binding antagonists include pidilizumab, also known as CT-011, MEDI0680, also known as AMP-514, and REGN2810.

[0332] In some embodiments, the immune checkpoint inhibitor is a PD-L1 antagonist, such as durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, or avelumab, also known as MSB00010118C. In certain aspects, the immune checkpoint inhibitor is a PD-L2 antagonist, such as rHIgM12B7. In some aspects, the immune checkpoint inhibitor is a LAG-3 antagonist, such as, but not limited to, IMP321 and BMS-986016. The immune checkpoint inhibitor may also be an adenosine A2a receptor (A2aR) antagonist, such as PBF-509.

[0333] In some embodiments, any antibody described herein (e.g., an anti-PD-1 antibody, anti-PD-L1 antibody, or anti-PD-L2 antibody) further comprises a human or mouse constant region. In a still further aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4. In a still further specific aspect, the human constant region is IgG1. In a still further aspect, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3. In a still further specific aspect, the antibody has reduced or minimal effector function. In a still further specific aspect, the minimal effector function results from production in a prokaryotic cell. In a still further specific aspect, the minimal effector function results from an "effector-free Fc mutation" or deglycosylation. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. Removal of glycosylation sites from the antibody is conveniently accomplished by altering the amino acid sequence so that one of the above-mentioned tripeptide sequences (for N-linked glycosylation sites) is deleted. Alterations may be made by substituting the asparagine, serine, or threonine residue within the glycosylation site with another amino acid residue (e.g., glycine, alanine, or a conservative substitution).

[0334] The antibodies or antigen-binding fragments thereof can be produced using methods known in the art, for example methods comprising culturing a host cell containing nucleic acid encoding any of the previously described anti-PD-L1, anti-PD-1, anti-PD-L2 antibodies, or antigen-binding fragments, in a form suitable for expression, under conditions suitable for the production of such antibodies or fragments, and recovering the antibodies or fragments.

[0335] B. CTLA-4 Another immune checkpoint that can be targeted using the methods provided herein is cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off switch" upon binding to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to the T-cell costimulatory protein CD28; both molecules bind to CD80 and CD86, also known as B7-1 and B7-2, respectively, on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA4 is also found on regulatory T cells and may be important for their function. T cell activation via the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules.

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

[0337] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. For example, the anti-CTLA-4 antibodies disclosed in the following documents can be used in the methods disclosed herein: U.S. Pat. No. 8,119,129; WO 01 / 14424; WO 98 / 42752; WO 00 / 37504 (CP675,206; also known as tremelimumab; formerly ticilimumab); U.S. Pat. No. 6,207,156; Hurwitz et al., 1998. The teachings of each of the foregoing publications are incorporated herein by reference. Antibodies that compete for binding to CTLA-4 with any of these art-recognized antibodies can also be used. For example, humanized CTLA-4 antibodies are described in WO 2001014424, WO 2000037504, and U.S. Patent No. 8,017,114, all of which are incorporated herein by reference.

[0338] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., WO 01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to and / or binds to the same epitope of CTLA-4 as the above-referenced antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above-referenced antibodies (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).

[0339] Other molecules for modulating CTLA-4 include soluble CTLA-4 ligands and receptors such as those described in U.S. Pat. No. 5,844,905, U.S. Pat. No. 5,885,796, and WO 1995001994 and WO 1998042752, all of which are incorporated herein by reference, and immunoadhensins such as those described in U.S. Pat. No. 8,329,867, which is incorporated herein by reference.

[0340] C. Killer Immunoglobulin-Like Receptors (KIRs) Another immune checkpoint inhibitor for use in the present invention is an anti-KIR antibody. Anti-human KIR antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art.

[0341] Instead, art-recognized anti-KIR antibodies can be used. Anti-KIR antibodies may cross-react with multiple inhibitory KIR receptors and enhance the cytotoxicity of NK cells bearing one or more of these receptors. For example, an anti-KIR antibody can bind to each of KIR2D2DL1, KIR2DL2, and KIR2DL3 and enhance NK cell activity by reducing, neutralizing, and / or reversing the inhibition of NK cytotoxicity mediated by any or all of these KIRs. In some embodiments, the anti-KIR antibody does not bind to KIR2DS4 and / or KIR2DS3. For example, monoclonal antibodies 1-7F9 (also known as IPH2101), 14F1, 1-6F1, and 1-6F5, described in International Publication WO 2006 / 003179, can be used. The teachings of these antibodies are incorporated herein by reference. Antibodies that compete for binding to KIR with any of these art-recognized antibodies can also be used. Additional art-recognized anti-KIR antibodies that can be used include, for example, those disclosed in WO 2005 / 003168, WO 2005 / 009465, WO 2006 / 072625, WO 2006 / 072626, WO 2007 / 042573, WO 2008 / 084106, WO 2010 / 065939, WO 2012 / 071411, and WO 2012 / 160448.

[0342] An exemplary anti-KIR antibody is lirilumab (also known as BMS-986015 or IPH2102). In other embodiments, the anti-KIR antibody comprises the heavy and light chain complementarity determining regions (CDRs) or variable regions (VRs) of lirilumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the heavy chain variable (VH) region of lirilumab and the CDR1, CDR2, and CDR3 domains of the light chain variable (VL) region of lirilumab. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with lirilumab.

[0343] Examples of cancers contemplated for treatment include: lung cancer, head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, cervical cancer, gastrointestinal cancer, lymphoma, preneoplastic lesions of the lung, colon cancer, melanoma, metastatic melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, Kaposi's sarcoma, keratoacanthoma, spindle cell tumor, sebaceous gland carcinoma, microcystic adnexal carcinoma, Paget's disease of the breast, atypical fibroxanthoma, leiomyosarcoma, and angiosarcoma, lentigo maligna, lentigo maligna melanoma, superficial spreading melanoma, nodular melanoma, acral lentiginous melanoma, adhesive melanoma, and bladder cancer.

[0344] In some embodiments, the subject has a cancer that is resistant (demonstrated to be resistant) to one or more anti-cancer therapies. In some embodiments, resistance to anti-cancer therapy includes recurrence of cancer or refractory cancer. Recurrence may refer to the reappearance of cancer at the original site or a new site after treatment. In some embodiments, resistance to anti-cancer therapy includes progression of cancer during treatment with an anti-cancer therapy. In some embodiments, the cancer is early stage or late stage. The subject may have a cancer that expresses (e.g., is shown to express in a diagnostic test) a PD-Ll biomarker. In some embodiments, the patient's cancer expresses a low PD-Ll biomarker. In some embodiments, the patient's cancer expresses a high PD-Ll biomarker. The PD-Ll biomarker may be detected in a sample using a method selected from FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blot, immunodetection, HPLC, surface plasmon resonance, optical spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY technology, and FISH, and combinations thereof.

[0345] In some embodiments, the cancer has low levels of T cell infiltration. In some embodiments, the cancer has no detectable T cell infiltration. In some embodiments, the cancer is a non-immunogenic cancer (e.g., non-immunogenic colorectal cancer and / or ovarian cancer).

[0346] For example, a therapeutically effective or sufficient amount of an immune checkpoint inhibitor, such as an antibody, administered to a human may range from about 0.01 to about 50 mg / kg of patient body weight, whether administered in a single dose or multiple doses. In some embodiments, the antibody used is administered, for example, at about 0.01 to about 45 mg / 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, about 0.01 to about 20 mg / kg, about 0.01 to about 15 mg / kg, about 0.01 to about 10 mg / kg, about 0.01 to about 5 mg / kg, or 0.01 to about 1 mg / kg per day. In some embodiments, the antibody is administered at 15 mg / kg. However, other dosing regimens may be useful. In one embodiment, the anti-PD-Ll antibodies described herein are administered to a human at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, or about 1400 mg on day 1 of a 21-day cycle. The dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as by infusion. The progress of this therapy is easily monitored by conventional techniques.

[0347] Anti-cancer and other therapies In some embodiments, the immune checkpoint inhibitor may be administered in combination with at least one additional therapeutic agent. The additional therapy may be a cancer therapy such as radiation therapy, surgery, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplant, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. The additional therapy may be in the form of adjuvant or neoadjuvant therapy.

[0348] In examples, the therapy for cancer (with or without administration of an immune checkpoint inhibitor) or any other disease (e.g., viral infection or autoimmune disease) may be radiation therapy, surgery, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplant, nanotherapy, or monoclonal antibody therapy. The therapy may be a combination of the foregoing. Additional therapies may also be administered.

[0349] In some embodiments, the therapy (or additional cancer treatment) is administration of a small molecule enzyme inhibitor or an anti-metastatic agent, hi some embodiments, the additional therapy is administration of a side effect limiting agent (e.g., an agent intended to reduce the occurrence and / or severity of side effects of the treatment, such as an anti-nausea agent).

[0350] In some embodiments, the therapy (or additional cancer therapy) is radiation therapy. In some embodiments, the therapy (or additional cancer therapy) is surgery. In some embodiments, the therapy (or additional cancer therapy) is a combination of radiation therapy and surgery. In some embodiments, the therapy (or additional cancer therapy) is gamma irradiation. In some embodiments, the therapy (or additional cancer therapy) is a therapy targeting the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. The therapy (or additional cancer therapy) may be one or more chemotherapeutic agents known in the art.

[0351] Administration of any compound or therapy of the present embodiments to a patient will follow general protocols for administering such compounds, taking into account the toxicity of the agents, if applicable. Thus, in some embodiments, there is a step of monitoring for toxicity resulting from the combination therapy.

[0352] The therapy may comprise or consist of administering to the subject any of the following:

[0353] 1. Chemotherapy A wide variety of chemotherapeutic agents can be used in accordance with the present embodiments. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to mean a compound or composition administered to treat cancer. These agents or drugs are classified by their mode of activity within the cell, for example, regardless of at what stage they affect the cell cycle. Alternatively, agents can be characterized based on their ability to directly crosslink and intercalate with DNA or to induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0354] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetigenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (e.g., its Adozelesin, carzelesin, and biceresin synthetic analogs); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (e.g., synthetic analogs, KW-2189 and CB1-TM1); eluterobin; pancratistatin; sarcodictyin; spongistatin; chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidase nitrogen mustards such as ciprofloxacin hydrochloride, melphalan, nobuenbiquin, fenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma II and calicheamicin omega II); dynemicins, including dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin , detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (e.g., morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycin, peplomycin, po tofilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin, and trimetrexate; fludarabine, 6-mercaptopurine, thiazolinone, and thiazolinone; Purine analogs such as mipurine and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal agents such as mitotane and trilostane; florinic acid Folic acid supplements such as aceglatone, aldophosphamide glycosides, aminolevulinic acid, eniluracil, amsacrine, bestravcil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elflornithine, elliptinium acetate, epothilone, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidainine;Maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone ;2,2',2''-Trichlorotriethylamine; Trichothecenes (e.g., T-2 toxin, veracrine A, roridin A, and anguidine); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine; Arabinoside ("AraC"); Cyclophosphamide; Taxoids, e.g., Paclitaxel and Docetaxel, Gemcitabine; 6-Thioguanine; Methionine cyclohexylcaptopurine; platinum coordination compounds such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); the topoisomerase inhibitor RFS2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl protein transferase inhibitors, transplatin, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0355] 2. Radiation therapy Other widely used agents that cause DNA damage include what are commonly known as gamma rays, X-rays, and / or directed delivery of radioisotopes to tumor cells. Other forms of DNA-damaging agents are also contemplated, such as microwaves, proton beam irradiation (U.S. Pat. Nos. 5,760,395 and 4,870,287), and ultraviolet radiation. All of these agents most likely affect a wide range of damage to DNA, the precursors of DNA, DNA replication and repair, and chromosome assembly and maintenance. X-ray dose ranges range from daily doses of 50-200 roentgens over prolonged periods (3-4 weeks) to single doses of 2000-6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by neoplastic cells.

[0356] 3. Immunotherapy Those skilled in the art will understand that immunotherapy can be used in combination with or in conjunction with the methods described herein. In the context of cancer treatment, immunotherapeutics generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is an example of an immunotherapy. The immune effector may be, for example, an antibody specific for a marker on the surface of tumor cells. The antibody may act alone as the effector of therapy or may recruit other cells that actually accomplish cell death. The antibody may also be conjugated to a drug or toxin (such as a chemotherapeutic agent, a radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve as a targeting agent. Alternatively, the effector may be a lymphocyte bearing a surface molecule that interacts either directly or indirectly with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0357] In examples, immunotherapy includes adoptive cell therapy, such as CAR-T administration, e.g., anti-CD19 or CD20 CAR-T administration.

[0358] In examples, the immunotherapeutic comprises or consists of the administration of IL-2 (eg, truncated IL-2 or pegylated IL-2 or Fc-fused IL-2).

[0359] Antibody-drug conjugates (ADCs) have emerged as a breakthrough approach for the development of cancer therapeutics. Antibody-drug conjugates (ADCs) contain a monoclonal antibody (MAb) covalently linked to a cell-killing drug. This approach combines the high specificity of MAbs for antigen targets with highly potent cytotoxic drugs, resulting in "armed" MAbs that deliver the payload (drug) to tumor cells bearing abundant levels of the antigen. Targeted delivery of the drug also minimizes contact with normal tissues, resulting in reduced toxicity and an improved therapeutic index. The FDA approval of two ADC drugs, ADCETRIS® (brentuximab vedotin) in 2011 and KADCYLA® (trastuzumab emtansine or T-DM1) in 2013, validated this approach. Currently, there are more than 30 ADC drug candidates in various stages of clinical trials for cancer treatment. As antibody engineering and linker-payload optimization become increasingly mature, the discovery and development of new ADCs increasingly relies on the identification and validation of new targets suitable for this approach and the generation of targeting MAbs. Two criteria for ADC targets are upregulated / high levels of expression in tumor cells and robust internalization.

[0360] In one aspect of immunotherapy, tumor cells must possess a marker that is amenable to targeting, i.e., that is not present on the majority of other cells. Numerous tumor markers exist, any of which may be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and pi55. An alternative aspect of immunotherapy is to combine anticancer effects with immunostimulatory effects. There are also immunostimulatory molecules, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, and gamma-IFN; chemokines such as MIP-1, MCP-1, and IL-8; and growth factors such as FLT3 ligand.

[0361] 4. Surgery Cancer or other diseases or conditions may be treated by surgery in the present invention.

[0362] Approximately 60% of individuals with cancer will undergo some type of surgery, including preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and can be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to the physical removal of at least part of a tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs surgery).

[0363] Upon partial or complete removal of cancer cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be achieved by perfusion, direct injection, or local application of the area with additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Similarly, such treatment may be at various dosages.

[0364] 5. Bacterial transplantation In embodiments, the therapy includes administering a bacterial transplant, e.g., a fecal microbial transplant containing defined bacteria, to the subject. For example, the transplant is any of the compositions disclosed in WO2018064165, the disclosure of which (particularly the compositions therein) is incorporated by reference in its entirety for possible application in the present invention. For example, the transplant is according to any of the following paragraphs (table numbers and sequence numbers refer to tables and sequences in WO2018064165, which are expressly incorporated herein for possible use in the claims): 1. A composition comprising at least one isolated or purified population of bacteria belonging to one or more of the families Ruminococcaceae, Clostridiaceae, Lachnospiraceae, Micrococcaceae, and / or Veilonellaceae. 2. A composition comprising at least two isolated or purified populations of bacteria belonging to one or more of the families Ruminococcaceae, Clostridiaceae, Lachnospiraceae, Micrococcaceae, and / or Veilonellaceae. 3. The composition of paragraph 1 or paragraph 2, wherein each of the bacterial populations is present in the composition at a concentration of at least 10^3 CFU. 4. The composition of paragraph 1 or paragraph 2, which is a live bacterial product or a live biotherapy product. 5. The composition of paragraph 1 or paragraph 2, wherein the at least one isolated or purified bacterial population or the at least two isolated or purified bacterial populations are provided as bacterial spores. 6. The composition of paragraph 1 or paragraph 2, wherein at least one bacterial population or at least two isolated or purified bacterial populations belong to Clostridiales family XII and / or Clostridiales family XIII. 7. The composition of paragraph 1 or paragraph 2, wherein at least one isolated or purified population of bacteria or at least two isolated or purified populations of bacteria belong to the family Ruminococcaceae and / or Clostridiaceae. 8. The composition of paragraph 1 or paragraph 2, wherein the population of bacteria belonging to the family Ruminococcaceae is further defined as a population of bacteria belonging to the genus Ruminococcus. 9. The composition of paragraph 8, wherein the population of bacteria belonging to the genus Ruminococcus is further specified to be a population of bacteria belonging to the species Ruminococcus bromii. 10. The composition of paragraph 1 or paragraph 2, wherein the population of bacteria belonging to the family Ruminococcaceae is further specified to be a population of bacteria belonging to the genus Faecalibacterium. 11. The composition of paragraph 10, wherein the population of bacteria belonging to the genus Faecalibacterium is further specified to be a population of bacteria belonging to the species Faecalibacterium prausnitzii. 12. The composition of paragraph 1 or paragraph 2, wherein the population of bacteria belonging to the family Micrococcaceae is further specified to be a population of bacteria belonging to the genus Rothia. 13. The composition of paragraph 1 or paragraph 2, further comprising a population of bacteria belonging to the species Porphyromonas pasteri, Clostridium hungatei, Phascolarctobacterium faecium, the genus Peptoniphilus, and / or the class Mollicutes. 14. The composition of paragraph 1 or paragraph 2, which is essentially free of a population of bacteria belonging to the order Bacteroidales. 15. The composition of paragraph 1 or paragraph 2, wherein at least one isolated or purified bacterial population or at least two isolated or purified bacterial populations belong to one or more species, subspecies, or bacterial strains selected from the group consisting of the species in Table 1 (Tables 1-29) having an enrichment index (ei) greater than 0.5. 16. The composition of paragraph 1 or paragraph 2, wherein at least one isolated or purified bacterial population or at least two isolated or purified bacterial populations are selected from the group consisting of species in Table 1 (Tables 1-29) where "ei" equals 1. 17. At least one isolated or purified bacterial population or at least two isolated or purified bacterial populations are identified by NCBI taxonomic IDs: 717959, 587, 758823, 649756, 44749, 671218, 1264, 1122135, 853, 484018, 46503, 54565, 290052, 216931, 575978, 433321, 1796646, 213810, 228924, 290054, 1509, 1462919, 29375, 337097, 1298596, 487174, 642492, 1735, 12 97424, 742766, 46680, 132925, 411467, 1318465, 1852367, 1841857, 169679, 1175296, 259063, 172901, 39488, 57172, 28118, 166486, 28133, 1529, 69 4434, 1007096, 84030, 56774, 102148, 626947, 216933, 1348613, 1472417, 100176, 824, 1471761, 1297617, 288966, 1317125, 28197, 358743, 264639, 1 265, 1335, 66219, 69473, 115117, 341220, 1732, 873513, 396504, 1796619, 45851, 2741, 105841, 86332, 1349822, 84037, 180311, 54291, 1217282, 762 984, 1185412, 154046, 663278, 1543, 398512, 69825, 1841867, 1535, 1510, 84026, 1502, 1619234, 39497, 1544, 29343, 649762, 332095, 536633, 103373 1, 574930, 742818, 177412, 1121308, 419208, 1673717, 55779, 28117, 626937, 180332, 1776382, 40519, 34062, 40518, 74426, 1216062, 293826, 850, 6 45466, 474960, 36835, 115544, 1515, 88431, 216932, 1417852, 39492, 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、1118061、357276、214851、320502、217731、246787、29371、649764、901、29374、33043、39778、682400、871665、160404、745368、408、1584、333367、47246、1096246、53342、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、109327、1531、1382、33039、311460、230143、216935、539、35519、1681、328813、214853、89014、1121115、1585974、29466、1363、292800、270498、214856、142877、133926、209880、179628、1121102、105612、1796615、39777、29353、1579、163665、53443、261299、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, 75612, 818, 371674, 338188, 88164, 588581, 676965, 546271, 1236512, 178338, 862517, 157687, 158, 51048, 1583331, 52 9, 888745, 394340, 40545, 855, 553973, 938293, 93063, 708634, 179995, 1351, 476652, 1464038, 555088, 237576, 879566, 1852371, 742727, 1377, 35830, 997353, 218538, 83771, 1605, 28111, 131109, 46609, 690567, 46206, 155615, 51616, 40542, 203, 294, 1034346, 156456, 80866, 554406, 796942, 1002367, 29347, 796944, 61592, 487175, 1050201, 762948, 137732, 1211819, 1019, 272548, 1717, 384636, 216940, 2087, 45634, 466107, 1689, 47678, 575, 979627, 840, 1660, 1236517, 617123, 546, 28135, 82171, 483, 501496, 99656, 1379, 84032, 39483, 1107316, 5 16S of a bacterium identified by an NCBI taxonomic ID selected from the group consisting of 84, 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.The composition of paragraph 1 or paragraph 2, comprising a 16S ribosomal RNA (rRNA) nucleotide sequence that is at least 90% identical (e.g., at least 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical) to an rRNA nucleotide sequence. 18. The composition of paragraph 1 or paragraph 2, wherein at least one isolated or purified bacterial population or at least two isolated or purified bacterial populations are of a species, subspecies, or bacterial strain that comprises a 16S rRNA gene sequence at least 80% identical (e.g., at least 85, 90, 95, or 98% identical) to a sequence of SEQ ID NOs: 1-876. 19. The at least one isolated or purified bacterial population or the at least two isolated or purified bacterial populations are selected from the group consisting of Bacteroides coagulans, Clostridium aldenense, Clostridium aldrichii, Clostridium alkalicellulosi, Clostridium amygdalinum, Clostridium asparagiforme, Clostridium cellulosi, Clostridium citroniae, Clostridium clariflavum DSM 19732, Clostridium clostridioforme, Clostridium colinum, Clostridium fimetarium, Clostridium hiranonis, Clostridium hungatei, Clostridium hylemonae DSM 15053, Clostridium indolis, Clostridium lactatifermentans, Clostridium leptum, Clostridium methylpentosum, Clostridium oroticum, Clostridium papyrosolvens DSM 2782, Clostridium populeti, Clostridium propionicum, Clostridium saccharolyticum, Clostridium scindens, Clostridium sporosphaeroides, Clostridium stercorarium, Clostridium straminisolvens, Clostridium sufflavum, Clostridium termitidis, Clostridium thermosuccino genes, Clostridium viride, Clostridium xylanolyticum, Desulfotomaculum guttoideum, Eubacterium rectale ATCC 33656, Eubacterium dolichum, Eubacterium eligens ATCC 27750, Eubacteriumhallii, Eubacterium infirmum, Eubacterium siraeum, Eubacterium tenue, Ruminococcus torques, Acetanaerobacterium elongatum, Acetatifactor muris, Acetivibrio cellulolyticus, Acetivibrio ethanolgignens, Acholeplasma brassicae 0502, Acholeplasma parvum, Acholeplasma vituli, Acinetobacter junii, Actinobacillus porcinus, Actinomyces bowdenii, Actinomyces dentalis, Actinomyces odontolyticus, Acutalibacter muris, Aerococcus viridans, Aeromicrobium fastidiosum, Alistipes finegoldii, Alistipes obesi, Alistipes onderdonkii, Alistipes putredinis, Alistipes shahii, Alistipes shahii WAL 8301, Alistipes timonensis JC136, Alkalibacter saccharofermentans, Alkaliphilus metalliredigens QYMF, Allisonella histaminiformans, Allobaculum stercoricanis DSM 13633, Alloprevotella rava, Alloprevotella tannerae, Anaerobacterium chartisolvens, Anaerobiospirillum thomasii, Anaerobium acetethylicum, Anaerococcus octavius ​​NCTC 9810, Anaerococcus provenciensis, Anaerococcus vaginalis ATCC 51170, Anaerocolumna jejuensis, Anaerofilum agile, Anaerofustisstercorihominis, Anaeroglobus geminatus, Anaeromassilibacillus senegalensis, Anaeroplasma abactoclasticum, Anaerorhabdus furcosa, Anaerosporobacter mobilis, Anaerostipes butyraticus, Anaerostipes caccae, Anaerostipes hadrus, Anaerotruncus colihominis, Anaerovorax odorimutans, Anoxybacillus rupiensis, Aquabacterium limnoticum, Arcobacter butzleri, Arthrospira platensis, Asaccharobacter celatus, Atopobium parvulum, Bacteroides caccae, Bacteroides caecimuris, Bacteroides cellulosilyticus, Bacteroides clarus YIT 12056, Bacteroides dorei, Bacteroides eggerthii, Bacteroides finegoldii, Bacteroides fragilis, Bacteroides gallinarum, Bacteroides massiliensis, Bacteroides oleiciplenus YIT 12058, Bacteroides plebeius DSM 17135, Bacteroides rodentium JCM 16496, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides xylanisolvens XB1A, Bacteroides xylanolyticus, Barnesiella intestinihominis, Beduini massiliensis, Bifidobacterium bifidum, Bifidobacterium dentium, Bifidobacterium longum subsp. infantis, Blautiacaecimuris, Blautia coccoides, Blautia faecis, Blautia glucerasea, Blautia hansenii DSM 20583, Blautia hydrogenotrophica, Blautia luti, Blautia luti DSM 14534, Blautia wexlerae DSM 19850, Budvicia aquatica, Butyricicoccus pullicaecorum, Butyricimonas paravirosa, Butyrivibrio crossotus, Caldicoprobacter oshimai, Caloramator coolhaasii, Caloramator proteoclasticus, Caloramator quimbayensis, Campylobacter gracilis, Campylobacter rectus, Campylobacter ureolyticus DSM 20703, Capnocytophaga gingivalis, Capnocytophaga leadbetteri, Capnocytophaga sputigena, Casaltella massiliensis, Catabacter hongkongensis, Catenibacterium mitsuokai, Christensenella minuta, Christensenella timonensis, Chryseobacterium taklimakanense, Citrobacter freundii, Cloacibacillus porcorum, Clostridioides difficile ATCC 9689 = DSM 1296, Clostridium amylolyticum, Clostridium bowmanii, Clostridium butyricum, Clostridium cadaveris, Clostridium colicanis, Clostridium gasigenes, Clostridium lentocellum DSM 5427, Clostridium oceanicum, Clostridium oryzae, Clostridiumparaputrificum, Clostridium pascui, Clostridium perfringens, Clostridium quinii, Clostridium saccharobutylicum, Clostridium sporogenes, Clostridium ventriculi, Collinsella aerofaciens, Comamonas testosteroni, Coprobacter fastidiosus NSB1, Coprococcus eutactus, Corynebacterium diphtheriae, Corynebacterium durum, Corynebacterium mycetoides, Corynebacterium pyruviciproducens ATCC BAA-1742, Corynebacterium tuberculostearicum, Culturomica massiliensis, Cuneatibacter caecimuris, Defluviitalea saccharophila, Delftia acidovorans, Desulfitobacterium chlororespirans, Desulfitobacterium metallireducens, Desulfosporosinus acididurans, Desulfotomaculum halophilum, Desulfotomaculum intricatum, Desulfotomaculum tongense, Desulfovibrio desulfuricans subsp. desulfuricans, Desulfovibrio idahonensis, Desulfovibrio litoralis, Desulfovibrio piger, Desulfovibrio simplex, Desulfovibrio zosterae, Desulfuromonas acetoxidans, Dethiobacter alkaliphilus AHT 1, Dethiosulfatibacter aminovorans, Dialister invisus, Dialister propionicifaciens, Dielmafastidiosa, Dietzia alimentaria 72, Dorea longicatena, Dysgonomonas gadei ATCC BAA-286, Dysgonomonas mossii, Eggerthella lenta, Eikenella corrodens, Eisenbergiella tayi, Emergencia timonensis, Enorma massiliensis phi, Enterococcus faecalis, Enterorhabdus muris, Ethanoligenens harbinense YUAN-3, Eubacterium coprostanoligenes, Eubacterium limosum, Eubacterium oxidoreducens, Eubacterium sulci ATCC 35585, Eubacterium uniforme, Eubacterium ventriosum, Eubacterium xylanophilum, Extibacter muris, Ezakiella peruensis, Faecalibacterium prausnitzii, Faecalicoccus acidiformans, Faecalitalea cylindroides, Filifactor villosus, Flavonifr actor plautii, Flintibacter butyricus, Frisingicoccus caecimuris, Fucophilus fucoidanolyticus, Fusicatenibacter saccharivorans, Fusobacterium mortiferum, Fusobacterium nucleatum subsp. vincentii, Fusobacterium simiae, Fusobacterium varium, Garciella nitratireducens, Gemella haemolysans, Gemmiger formicilis, Gordonibacter urolithinfaciens, Gracilibacter thermotolerans JW / YJL-S1, Granulicatellaelegans, Guggenheimella bovis, Haemophilus haemolyticus, Helicobacter typhlonius, Hespellia stercorisuis, Holdemanella biformis, Holdemania massiliensis AP2, Howardella ureilytica, Hungatella effluvii, Hungatella hathewayi, Hydrogenoanaerobacterium saccharovorans, Ihubacter massiliensis, Intestinibacter bartlettii, Intestinimonas butyriciproducens, Irregularibacter muris, Kiloniella laminariae DSM 19542, Kroppenstedtia guangzhouensis, Lachnoanaerobaculum orale, Lachnoanaerobaculum umeaense, Lachnoclostridium phytofermentans, Lactobacillus acidophilus, Lactobacillus algidus, Lactobacillus animalis, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus fornicalis, Lactobacillus iners, Lactobacillus pentosus, Lactobacillus rogosae, Lactococcus garvieae, Lactonifactor longoviformis, Leptotrichia buccalis, Leptotrichia hofstadii, Leptotrichia hongkongensis, Leptotrichia wadei, Leuconostoc inhae, Levyella massiliensis, Loriellopsis cavernicola, Lutispora thermophila, Marinilabilia salmonicolor JCM 21150, Marvinbryantiaformatexigens, Mesoplasma photuris, Methanobrevibacter smithii ATCC 35061, Methanomassiliicoccus luminyensis BIO, Methylobacterium extorquens, Mitsuokella jalaludinii, Mobilitalea sibirica, Mobiluncus curtisii, Mogibacterium pumilum, Mogibacterium timidum, Moorella glycerini, Moorella humiferrea, Moraxella nonliquefaciens, Moraxella osloensis, Morganella morganii, Moryella indoligenes, Muribaculum intestinale, Murimonas intestini, Natranaerovirga pectinivora, Neglecta timonensis, Neisseria cinerea, Neisseria oralis, Nocardioides mesophilus, Novibacillus thermophilus, Ochrobactrum anthropi, Odoribacter splanchnicus, Olsenella profusa, Olsenella uli, Oribacterium asaccharolyticum ACB7, Oribacterium sinus, Oscillibacter ruminantium GH1, Oscillibacter valericigenes, Oxobacter pfennigii, Pantoea agglomerans, Papillibacter cinnamivorans, Parabacteroides faecis, Parabacteroides goldsteinii, Parabacteroides gordonii, Parabacteroides merdae, Parasporobacterium paucivorans, Parasutterella excrementihominis, Parasutterellasecunda, Parvimonas micra, Peptococcus niger, Peptoniphilus duerdenii ATCC BAA- 1640, Peptoniphilus grossensis ph5, Peptoniphilus koenoeneniae, Peptoniphilus senegalensis JC140, Peptostreptococcus stomatis, Phascolarctobacterium succinatutens, Phocea massiliensis, Pontibacter indicus, Porphyromonas bennonis, Porphyromonas endodontalis, Porphyromonas pasteri, Prevotella bergensis, Prevotella buccae ATCC 33574, Prevotella denticola, Prevotella enoeca, Prevotella fusca JCM 17724, Prevotella loescheii, Prevotella nigrescens, Prevotella oris, Prevotella pollens ATCC 700821, Prevotella stercorea DSM 18206, P rev ote llamas silia timonensis, Propionispira arcuata, Proteus mirabilis, Providencia rettgeri, Pseudobacteroides cellulosolvens ATCC 35603 = DSM 2933、Pseudobutyrivibrio ruminis、Pseudoflavonifr actor capillosus ATCC 29799、Pseudomonas aeruginosa、Pseudomonas fluorescens、Pseudomonas mandelii、Pseudomonas nitroreducens、Pseudomonas putida、Raoultella ornithinolytica、Raoultella planticola、Raoultibactermassiliensis, Robinsoniella peoriensis, Romboutsia timonensis, Roseburia faecis, Roseburia hominis A2-183, Roseburia intestinalis, Roseburia inulinivorans DSM 16841, Rothia dentocariosa ATCC 17931, Ruminiclostridium thermocellum, Ruminococcus albus, Ruminococcus bromii, Ruminococcus callidus, Ruminococcus champanellensis 18P13 = JCM 17042, Ruminococcus faecis JCM 15917, Ruminococcus flavefaciens, Ruminococcus gauvreauii, Ruminococcus lactaris ATCC 29176, Rummeliibacillus pycnus, Saccharofermentans acetigenes, Scardovia wiggsiae, Schlegelella thermodepolymerans, Sedimentibacter hongkongensis, Selenomonas sputigena ATCC 35185, Slackia exigua ATCC 700122, Slackia piriformis YIT 12062. A composition according to paragraph 1 or paragraph 2, belonging to a species, subspecies, or bacterial strain selected from the group consisting of Staphylococcus aureus, Stenotrophomonas maltophilia, Stomatobaculum longum, Streptococcus agalactiae ATCC 13813, Streptococcus cristatus, Streptococcus equinus, Streptococcus gordonii, Streptococcus lactarius, Streptococcus parauberis, Subdoligranulum variabile, Succinivibrio dextrinosolvens, Sutterella stercoricanis, Sutterella wadsworthensis, Syntrophococcus sucromutans, Syntrophomonas zehnderi OL-4, Terrisporobacter mayombei, Thermoleophilum album, Treponema denticola, Treponema socranskii, Tyzzerella nexilis DSM 1787, Vallitalea guaymasensis, Vallitalea pronyensis, Vampirovibrio chlorellavorus, Veillonella atypica, Veillonella denticariosi, Veillonella dispar, Veillonella parvula, Victivallis vadensis, Vulcanibacillus modesticaldus, and Weissella confusa.

[0365] In an example, the implant is SER-109 or SER-262 (and optionally, the condition is C dificile infection); VE202 or SER-287 (and optionally the disease is ulcerative colitis); SER-301 (and optionally the disease is IBD); SER-401 (and optionally the condition is cancer; e.g., the therapy further comprises administration of an anti-PD-1 system antibody, e.g., an anti-PD-1 antibody); VE800 or SER-155 (and optionally the therapy comprises administration of a transplant, e.g., a hematopoietic stem cell or solid organ transplant); EDP1066 or EDP1815 (and optionally the disease is an inflammatory condition, e.g., colitis, Crohn's disease, asthma, rheumatoid arthritis (RA), psoriasis, dermatitis (e.g., atopic dermatitis), or IBD); or EDP1503 (and the disease is cancer, e.g., colorectal cancer, renal cell carcinoma, melanoma, or PD-1 recurrent cancer). In examples, the therapy includes administration of SGM-1019, SG-2-0776, or EB8018 (and optionally the disease or condition is NASH or IBD or an inflammatory condition, such as colitis, Crohn's disease, asthma, rheumatoid arthritis (RA), psoriasis, and dermatitis (e.g., atopic dermatitis)). These starting "VE" is being developed by Vadanta Biosciences, SER is being developed by Seres Therapeutics, EDP is being developed by Evelo Biosciences, SG is being developed by Second Genome, and EB is being developed by Enterome.

[0366] In examples, the disease or condition herein is an inflammatory condition, such as colitis, Crohn's disease, asthma, rheumatoid arthritis (RA), psoriasis, dermatitis (eg, atopic dermatitis), or IBD.

[0367] 6. Other Agents It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of the treatment. Such additional agents include agents that affect the upregulation of cell surface receptors and gap junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of hyperproliferative cells to apoptosis-inducing factors, or other biological agents. Increasing intercellular signaling by increasing the number of gap junctions will increase the anti-hyperproliferative effect on neighboring hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents may be used in combination with certain aspects of the present embodiments to improve the anti-hyperproliferative efficacy of the treatment. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiments. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as the antibody c225, may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy.

[0368] Diseases and Conditions Optionally, the disease or condition is selected from the following: (a) a neurodegenerative disease or condition (b) a brain disease or condition (c) a CNS disease or condition (d) Memory loss or impairment (e) heart or cardiovascular disease or condition, e.g., heart attack, stroke, or atrial fibrillation (f) liver disease or condition (g) a kidney disease or condition, e.g., chronic kidney disease (CKD) (h) pancreatic disease or condition (i) a pulmonary disease or condition, such as cystic fibrosis or COPD (j) Gastrointestinal disease or condition (k) throat or oral disease or condition (l) eye disease or condition (m) Reproductive diseases or conditions, such as diseases or conditions of the vagina, labia, penis, or scrotum (n) Sexually transmitted diseases or conditions, such as gonorrhea, HIV infection, syphilis, or chlamydia infection (o) Ear disease or condition (p) Skin disease or condition (q) Heart disease or condition (r) nasal disease or condition (s) a hematological disease or condition, such as anemia, e.g., a chronic disease of anemia or cancer (t) Viral infections (u) Pathogenic bacterial infections (v) Cancer (w) an autoimmune disease or condition, e.g., SLE (x) an inflammatory disease or condition, such as rheumatoid arthritis, psoriasis, eczema, asthma, ulcerative colitis, colitis, Crohn's disease, or IBD (y) autism (z) ADHD (aa) Bipolar disorder (bb) ALS [amyotrophic lateral sclerosis] (cc) Osteoarthritis (dd) Congenital or developmental defects or conditions (ee) miscarriage (ff) Blood coagulation status (gg) Bronchitis (hh) Dry or wet AMD (ii) angiogenesis (e.g., of tumors or eyes) (jj) cold (kk) epilepsy (ll) fibrosis, e.g., liver fibrosis or pulmonary fibrosis (mm) fungal diseases or conditions, such as thrush (nn) metabolic diseases or conditions, e.g., obesity, anorexia, diabetes, type I or type II diabetes (oo) ulcers, such as stomach ulcers or skin ulcers (pp) dry skin (qq) Sjögren's syndrome (rr) Cytokine storm (ss) hearing loss, hearing loss or impairment (tt) Slow or rapid metabolism (i.e., slower or faster than average for the subject's weight, sex, and age) (uu) Fertility disorders, e.g., infertility or subfertility (vv) Jaundice (lol) Rash (xx) Kawasaki disease (yy) Lyme disease (zz) allergies, e.g., nut, grass, pollen, house dust mite, cat or dog fur, or dander allergies (aaa) Malaria, typhoid, tuberculosis, or cholera (bbb) Depression (ccc) Mental retardation (ddd) microcephaly (eee) Malnutrition (fff) conjunctivitis (ggg) pneumonia (hhh) Pulmonary embolism (iii) Pulmonary hypertension (jjj) Bone disorders (kkk) Sepsis or septic shock (lll) Sinusitis (mmm) Stress (e.g., occupational stress) (nnn) Thalassemia, anemia, von Willebrand disease, or hemophilia (ooo) Shingles or herpes labialis (ppp) Menstruation (qqq)low sperm count

[0369] Neurodegenerative or CNS Diseases or Conditions to Treat or Prevent by the Method In examples, the neurodegenerative or CNS disease or condition is selected from the group consisting of Alzheimer's disease, geriatric psychosis, Down's syndrome, Parkinson's disease, Creutzfeldt-Jakob disease, diabetic neuropathy, Parkinsonism, Huntington's disease, Machado-Joseph disease, amyotrophic lateral sclerosis, diabetic neuropathy, and Creutzfeldt-Jakob disease. For example, the disease is Alzheimer's disease. For example, the disease is Parkinsonism.

[0370] In examples, the methods of the invention are performed on a human or animal subject to treat a CNS or neurodegenerative disease or condition, and the methods cause downregulation of Treg cells in the subject, thereby facilitating entry of systemic monocyte-derived macrophages and / or Treg cells across the choroid plexus into the subject's brain, thereby treating, preventing, or reducing the progression of the disease or condition (e.g., Alzheimer's disease). In embodiments, the methods cause an increase in IFN-gamma in the subject's CNS system (e.g., in the brain and / or CSF). In examples, the methods restore nerve fibers and / or reduce the progression of nerve fiber damage. In examples, the methods restore nerve myelin and / or reduce the progression of nerve myelin damage. In examples, the methods of the present invention treat or prevent a disease or condition disclosed in WO2015136541, and / or the methods can be used in conjunction with any of the methods disclosed in WO2015136541 (the disclosure of which is incorporated herein by reference in its entirety to provide a disclosure of potential therapeutic agents, e.g., immune checkpoint inhibitors, e.g., anti-PD-1, anti-PD-L1, anti-TIM3, or other antibodies disclosed therein, that can be administered to a subject to achieve such methods, treatment and / or prevention of diseases, conditions, CNS and neurodegenerative diseases and conditions).

[0371] Cancer to be treated or prevented by this method Cancers that can be treated include non-vascularized or substantially non-vascularized tumors, as well as vascularized tumors.Cancers may include non-solid tumors (such as hematological tumors, e.g., leukemia and lymphoma) or solid tumors.The types of cancers that can be treated with the present invention include, but are not limited to, carcinoma, blastoma, and sarcoma, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant diseases, such as sarcomas, carcinomas, and melanomas.Adult tumors / cancers and pediatric tumors / cancers are also included.

[0372] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or blood-borne) cancers include: leukemias, including acute leukemia (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myelogenous leukemia, and myeloblastic, promyeiocytic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemia (such as chronic myelocytic (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and aggressive forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0373] A solid tumor is an abnormal mass of tissue that usually does not contain cysts or fluid areas. Solid tumors may be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcoma, carcinoma, and lymphoma). Examples of solid tumors, such as sarcoma and carcinoma, include: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous eel! carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, and pheochromocytoma. Sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumors, seminoma, bladder cancer, melanoma, and CNS tumors (gliomas (including brainstem glioma and mixed glioma), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases).

[0374] In an example, the cancer is a hematological cancer. In an example, the cancer is NSCLC. In an example, the cancer is renal cell carcinoma. In an example, the cancer is urothelial carcinoma. In an example, the cancer is melanoma. Autoimmune diseases to be treated or prevented by the method Acute disseminated encephalomyelitis (ADEM) Acute necrotizing hemorrhagic leukoencephalitis Addison's disease Agammaglobulinemia Alopecia areata Amyloidosis ·Ankylosing spondylitis ·Anti-GBM / anti-TBM nephritis Antiphospholipid syndrome (APS) ·Autoimmune angioedema ·Autoimmune aplastic anemia Autoimmune autonomic neuropathy ·Autoimmune hepatitis ·Autoimmune hyperlipidemia ·Autoimmune immunodeficiency ·Autoimmune inner ear disease (AIED) Autoimmune myocarditis Autoimmune oophoritis Autoimmune pancreatitis ·Autoimmune retinopathy ·Autoimmune thrombocytopenic purpura (ATP) ·Autoimmune thyroid disease ·Autoimmune urticaria Axonal & neuronal neuropathy Barrow's disease Behçet's disease Bullous pemphigoid Cardiomyopathy Castleman's disease Celiac disease Chagas disease Chronic fatigue syndrome Chronic inflammatory demyelinating polyneuropathy (CIDP) ·Chronic recurrent multifocal osteosarcoma (CRMO) Churg-Strauss syndrome Cicatricial pemphigoid / benign mucous membrane pemphigoid Crohn's disease Cogan's syndrome ·Cold agglutinin disease Congenital cardiac conduction disorders Coxsackie's myocarditis ·CREST disease Essential mixed cryoglobulinemia Demyelinating neuropathy ·Dermatitis herpetiformis ·Dermatomyositis Devic's disease (neuromyelitis optica) Discoid lupus Dressler syndrome ·Endometriosis Eosinophilic esophagitis Eosinophilic fasciitis ·Erythema nodosum Experimental allergic encephalomyelitis Evans syndrome ·Fibromyalgia Fibrotic alveolitis ·Giant cell arteritis (temporal arteritis) Giant cell myocarditis Glomerulonephritis Goodpasture's syndrome Granulomatosis with polyangiitis (GPA) (formerly known as Wegener's granulomatosis) Graves' disease Guillain-Barré syndrome Hashimoto's encephalitis Hashimoto's thyroiditis ·Hemolytic anemia Henoch-Schönlein purpura ·Pregnancy herpes Hypogammaglobulinemia Idiopathic thrombocytopenic purpura (ITP) IgA nephropathy IgG4-related sclerosis Immunomodulatory lipoproteins Inclusion body myositis ·Interstitial cystitis Juvenile arthritis Juvenile diabetes (type 1 diabetes) Juvenile myositis Kawasaki syndrome Lambert-Eaton syndrome ·Leukocytoclastic vasculitis ·lichen planus ·Lichen sclerosus ·Ligny conjunctivitis Linear IgA disease (LAD) Lupus (SLE) Chronic Lyme disease Meniere's disease Microscopic polyangiitis ·Mixed connective tissue disease (MCTD) Mooren's ulcer Moosher-Habermann's disease Multiple sclerosis ·Myasthenia gravis Myositis Narcolepsy Neuromyelitis optica (Devic's disease) ·Neutropenia Ocular cicatricial pemphigoid ·Optic neuritis Relapsing rheumatism PANDAS (Streptococcus-associated paediatric autoimmune neuropsychiatric disorder) Paraneoplastic cerebellar degeneration Paroxysmal nocturnal hemoglobinuria (PNH) Parry-Romberg syndrome Parsonage-Turner syndrome Pars planitis (peripheral uveitis) ·Pemphigus Peripheral neuropathy Perivenous encephalomyelitis Pernicious anemia POEMS syndrome Polyarteritis nodosa Autoimmune polyendocrine syndromes types I, II, and III Rheumatic polymyalgia Polymyositis Post-myocardial infarction syndrome Postpericardiotomy syndrome Progesterone dermatitis Primary biliary cirrhosis Primary sclerosing cholangitis ·psoriasis Psoriatic arthritis Idiopathic pulmonary fibrosis Pyoderma gangrenosum Pure red cell aplasia Raynaud's phenomenon Reactive arthritis Reflex sympathetic dystrophy Reiter's syndrome Relapsing polychondritis ·Restless legs syndrome Retroperitoneal fibrosis Rheumatic fever Rheumatoid arthritis ·sarcoidosis Schmidt syndrome ·Scaryitis Scleroderma Sjögren's syndrome Sperm and testicular autoimmunity Stiff body syndrome Subacute bacterial endocarditis (SBE) Susac syndrome ·Sympathetic ophthalmia ·Takayasu arteritis ·Temporal arteritis / giant cell arteritis Thrombocytopenic purpura (TTP) Trouser-Hunt syndrome Transverse myelitis ·Type 1 diabetes ·Ulcerative colitis ·Undifferentiated connective tissue disease (UCTD) Uveitis ·Vasculitis ·Blistering skin disease ·Vitiligo Wegener's granulomatosis (now called granulomatosis with polyangiitis (GPA)) Inflammatory diseases to be treated or prevented by the method Alzheimer's disease ·Ankylosing spondylitis Arthritis (osteoarthritis, rheumatoid arthritis (RA), psoriatic arthritis) ·asthma Atherosclerosis Crohn's disease Colitis ·Dermatitis ·Diverticulitis ·Fibromyalgia ·hepatitis ·Irritable bowel syndrome (IBS) Systemic lupus erythematosus (SLE) ·Nephritis Parkinson's disease ·Ulcerative colitis

[0375] concept The present invention provides the following concept. 1. A programmable nuclease for use in a method for treating an acute microbial infection in a subject, wherein the microbial infection is caused by a first species or strain of microorganism, and the nuclease is programmable to cleave a target site contained by the genome of the microorganism infecting the subject, thereby killing or reducing the growth or proliferation of the first species or strain of microorganism, and the method of treatment comprises contacting the subject with the nuclease, wherein the nuclease is programmed to cleave the target site, thereby cleaving the genome of the microorganism contained by the subject and treating the acute microbial infection in the subject. 2. A programmable nuclease for use in a method for persistently treating a microbial (e.g., bacterial) infection in a subject, wherein the microbial infection is caused by a first species or strain of microorganism, and the nuclease is programmable to cleave a target site contained by the genome of the microorganism infecting the subject, and the first species or strain of microorganism is persistently killed or the growth or proliferation of the microorganism is reduced, the method of treatment comprising contacting the subject with the nuclease, and the nuclease being programmed to cleave the target site, thereby cleaving the genome of the microorganism contained by the subject and persistently treating the microbial infection in the subject. 3. The nuclease described in concept 2, wherein the nuclease (e.g., a programmed nuclease) and / or a nucleic acid that programs the nuclease to recognize and cleave a target site is administered to a subject at a first time point (T1) and a second time point (T2), wherein T2 is at least 1 hour after T1. 4. The nuclease of any one of concepts 1 to 3, wherein the method comprises reducing infection by at least 100-fold within the first 30 minutes of treatment (e.g., within the first 15 minutes). 5. The nuclease of any one of concepts 1 to 4, wherein the method comprises maintaining at least a 100-fold reduction in infection for at least 60 minutes (e.g., at least 120 minutes) after contacting the subject with the programmed nuclease. 6. The nuclease of any one of concepts 1 to 5, wherein the method includes a step of reducing infection such that the reduction in infection persists for 30 minutes immediately after the first 30 minutes of treatment. 7. The nuclease of any one of concepts 1 to 6, wherein the method comprises administering to the subject RNA or a nucleic acid encoding the RNA for expressing the RNA in the subject, wherein the RNA complexes with the nuclease and programs the nuclease to cleave a target site in a microorganism contained by the subject. 8. The nuclease of concept 7, wherein the nuclease is administered to the subject simultaneously or sequentially with the RNA or nucleic acid. 9. The nuclease of concept 7, wherein the subject comprises the nuclease prior to administering the RNA or nucleic acid to the subject. 10. A nuclease described in any one of paragraphs 7 to 9, wherein multiple viruses (e.g., phages) are administered to a subject, each virus containing a copy of the nucleic acid, and the viruses infect and deliver the nucleic acid to a microorganism contained by the subject. 11. The nuclease of concept 10, wherein the ratio of administered virus to microorganisms contained by the subject is 10 to 150. 12. The nuclease of any one of concepts 1 to 11, wherein the subject is a human or an animal, and optionally the subject is a human over 65 years of age or a pediatric patient. 13. The nuclease described in concept 12, wherein the infection is a pulmonary, abdominal, or urinary tract infection, or the subject has undergone surgery, is under immunosuppressant drug treatment, and / or suffers from a chronic disease. 14. The nuclease of any one of concepts 1 to 13, wherein infection is reduced by at least 90% over a period of 1 hour or longer, optionally within the first 30 minutes (e.g., within the first 15 minutes) of treatment. 15. The nuclease of any one of concepts 1 to 14, wherein the method comprises reducing infection by at least 100-fold within the first 30 minutes of treatment (e.g., within the first 15 minutes), and wherein the at least 100-fold reduction in infection is maintained for at least 60 minutes (e.g., at least 120, 145, or 180 minutes) after contacting the subject with the programmed nuclease. 16. The nuclease of any one of concepts 12 to 15, wherein the method treats or prevents sepsis and / or sepsis (e.g., septic shock) in a subject. 17. At the start of treatment, the subject (e.g., human) must have a body temperature of <36°C or >38°C, a heart rate of >90 beats per minute, a respiratory rate of >20 breaths per minute or a PaCO2 of <4.3 kP, and a blood pressure of <4000 / mm 3 or >12,000 / mm 3 17. The nuclease of claim 16, wherein the nuclease has a white blood cell count of 18. The nuclease of concept 16 or 17, wherein at the start of treatment, the subject (e.g., human) exhibits the presence of two or more of: abnormal body temperature, abnormal heart rate, abnormal respiratory rate, abnormal blood gases, and abnormal white blood cell count. 19. The nuclease of any one of concepts 1 to 18, wherein the subject is a human or animal, the microorganism is a bacterium (e.g., E. coli or C. dificile), and the subject's blood infection caused by the bacterium is reduced by at least 100-fold or 1000-fold within the first 30 minutes of treatment (e.g., within the first 15 minutes). 20. The subject's blood was collected immediately before treatment. 7 ~10 12 20. The nuclease of any one of concepts 12 to 19, wherein the nuclease infects bacteria at CFU / ml. 21. The nuclease of any one of concepts 1 to 11, wherein the subject is a plant. 22. The nuclease of any one of concepts 1 to 21, wherein the microorganism is a bacterium. 23. The nuclease of concept 22, wherein the bacterium is a gram-positive bacterium. 24. The nuclease of concept 22 or 23, wherein the bacterium is a Staphylococcus, Streptococcus, Enterococcus, Legionella, Haemophilus, Ghonnorhea, Acinetobacter, Escherichia, Klebsiella, Pseudomonas, or Stenotrophomonas bacterium (e.g., E coli (e.g., EHEC E coli), C dificile, V cholera, Staphylococcus (e.g., S aureus or MRSA), Streptococcus pyogenes, Acinetobacter baumannii, Legionella, Pseudomonas aeruginosa, Klebsiella pneumoniae bacterium). 25. The nuclease of any one of concepts 1 to 24, wherein the nuclease is a Cas nuclease (e.g., Cas3 or Cas9), a meganuclease, a TALEN (transcription activator-like effector nuclease), or a zinc finger nuclease. 26. A plurality of viruses (e.g., phages or phagemids for producing phages) for use with a nuclease described in any one of concepts 1 to 25 in a method of treatment, each virus comprising a copy of a nucleic acid defined in any one of concepts 7 to 9, and the viruses capable of infecting and delivering the nucleic acid to a microorganism contained by a subject. 27. A composition comprising a plurality of nucleic acids for programming a nuclease described in any one of concepts 1 to 25 in a method of treatment, wherein each nucleic acid is a nucleic acid defined in any one of concepts 7 to 9. 28. A CRISPR / Cas system comprising the nuclease of any one of concepts 1 to 27 for use in a method of treatment, wherein the nuclease is a Cas nuclease (e.g., Cas3 or Cas9), and the system comprises one or more guide RNAs or DNA encoding one or more guide RNAs, each guide RNA capable of programming the Cas nuclease to cleave a target site contained by the genome of the microorganism. 29. A guide RNA or DNA encoding a guide RNA for use in the system described in concept 28 for use in a method for treating an acute microbial infection in a subject, such as sepsis or sepsis. 30. A nucleic acid vector comprising the guide RNA or DNA of Concept 27 or 29. 31. The vector of concept 30, wherein the vector is a phage, phagemid, viriophage, virus, plasmid (e.g., a conjugative plasmid), or transposon. 32. An anti-sepsis or anti-sepsis composition for administration to a human or animal to treat sepsis or septicemia, comprising a plurality of vectors, each vector being as described in concept 30 or 31. 33. A method for treating an acute microbial infection in a subject, the method being as defined by any one of concepts 1 to 32. 34. Use of a nuclease, a plurality of viruses, a system, a guide RNA, a DNA, or a vector according to any one of concepts 1 to 26 and 28 to 30 in the manufacture of a composition for carrying out a method of treatment as defined by any one of concepts 1 to 33, wherein the subject is an organism other than a human or an animal. 35. Use of a nuclease, multiple viruses, systems, guide RNAs, DNA, or vectors described in any one of Concepts 1 to 26 and 28 to 30 in the manufacture of a composition for performing an ex vivo method for treating a microbial infection of a substrate, wherein the microbial infection is caused by a first species or strain of microorganism, and the nuclease is programmable to cleave a target site contained by the genome of the microorganism that has infected the substrate, thereby killing the first species or strain of microorganism or reducing its growth or proliferation, and the method of treatment comprises contacting a subject with the nuclease, wherein the nuclease is programmed to cleave the target site, thereby cleaving the genome of the microorganism contained by the subject and treating the acute microbial infection of the substrate. 36. Use of a programmable nuclease in the manufacture of a composition for performing an ex vivo method for treating a microbial infection of a substrate, wherein the microbial infection is caused by a first species or strain of microorganism, and the nuclease is programmable to cleave a target site contained by the genome of the microorganism that has infected the substrate, thereby killing the first species or strain of microorganism or reducing its growth or proliferation, and the method of treatment comprises contacting a subject with the nuclease, wherein the nuclease is programmed to cleave the target site, thereby cleaving the genome of the microorganism contained by the subject and treating the acute microbial infection of the substrate. 37. The use of any one of concepts 34, 35, or 36, wherein a nuclease (e.g., a programmed nuclease) and / or a nucleic acid that programs the nuclease to recognize and cleave a target site is administered to a subject or substrate at a first time point (T1) and a second time point (T2), wherein T2 is at least 1 hour after T1. 38. The use of any one of concepts 34 to 37, wherein infection is reduced by at least 100-fold within the first 30 minutes of treatment (e.g., within the first 15 minutes). 39. The use of any one of concepts 34 to 38, wherein the reduction in infection is maintained at least 100-fold for at least 60 minutes (e.g., at least 120 minutes) after contacting the subject with the programmed nuclease. 40. The use of any one of concepts 34 to 39, wherein the reduction in infection persists for 30 minutes immediately after the first 30 minutes of treatment. 41. The use of any one of Concepts 34 to 40, wherein the method comprises administering to a subject or substrate RNA or a nucleic acid encoding RNA for expression of RNA in or on the subject or substrate, wherein the RNA complexes with a nuclease and programs the nuclease to cleave a target site in a microorganism contained by the subject or substrate. 42. The use of concept 41, wherein the nuclease is administered to the subject or substrate simult...

Claims

1. 1. An agent for use in a method for persistently treating an E. coli bacterial infection that causes sepsis in a human or animal subject, comprising a programmable nuclease, wherein treating the infection treats or prevents sepsis in the subject, and the nuclease is programmable to cleave a target site contained by the chromosome of an E. coli bacterium that has infected the subject, thereby persistently killing the E. coli or reducing the growth or proliferation of the E. coli, the method of treatment comprising contacting the subject with the nuclease that has been programmed to cleave the target site, thereby cleaving the chromosome of the E. coli contained by the subject and persistently treating the bacterial infection in the subject; the programmable nuclease is a Cas nuclease; The method includes administering to the subject a crRNA or a nucleic acid encoding the crRNA for expressing the crRNA in the subject, wherein the crRNA complexes with the nuclease and programs the nuclease to cleave the target site in bacteria contained by the subject; the nuclease and / or crRNA or a nucleic acid encoding a crRNA is administered to the subject at a first time point (T1) and a second time point (T2), T2 being at least 1 hour after T1; The method includes reducing the infection by at least 100-fold within the first 30 minutes of the treatment; and maintaining at least a 100-fold reduction in infection for at least 60 minutes after contacting the subject with the programmed nuclease.

2. The agent for use according to claim 1, wherein T2 is at least 2 hours after T1.

3. The agent for use according to claim 2, wherein T2 is at least 24 hours after T1.

4. The agent for use according to claim 1, wherein T2 is 7 hours or less after T1.

5. The agent for use according to claim 1, wherein T2 is 24 hours or less after T1.

6. a. the nuclease is administered to the subject simultaneously or sequentially with the RNA or nucleic acid; or b. The agent for use according to any one of claims 1 to 5, wherein the subject contains the nuclease before administering the RNA or nucleic acid to the subject.

7. 7. The agent for use according to any one of claims 1 to 6, wherein a plurality of viruses are administered to the subject, each virus containing a copy of the nucleic acid, and the viruses infect the bacteria contained by the subject and deliver the nucleic acid to the microorganism.

8. The agent for use according to claim 7, wherein the ratio of administered virus to bacteria contained by the subject is 10 to 150.

9. The agent for use according to any one of claims 1 to 8, wherein the subject is a human or an animal, and the subject has undergone surgery, is under immunosuppressant drug treatment, or suffers from a chronic disease.

10. 10. The agent for use according to any one of claims 1 to 9, wherein the agent is for use in a method for treating a pathogenic E. coli bacterial infection in a subject caused by an E. coli bacterium (first bacterium), the method comprising the step of selectively killing the first bacterium contained in the subject by cleaving a target site contained in the chromosome of the first bacterium, the cleavage being carried out using the programmable nuclease that is programmed to cleave the target site, the subject suffering from a further disease or condition other than the pathogenic E. coli bacterial infection, the method comprising the step of administering a therapy to the subject to treat or prevent the further disease or condition, the nuclease treating the infection, and the therapy being effective in treating or preventing the disease or condition in the presence of the programmed nuclease.

11. 11. The agent for use according to claim 10, wherein (i) the subject is a cancer patient and the therapy comprises administration of a hematopoietic stem cell transplant, a chemotherapeutic agent, an immune checkpoint inhibitor, an immune checkpoint agonist, or an immune cell enhancer; adoptive cell therapy; radiation; or surgery, or (ii) the therapy is tissue transplantation, organ transplantation, or cell transplantation.

12. 12. The agent for use according to any one of claims 1 to 11, wherein the infection is reduced by at least 90% over a period of 1 hour or longer, optionally within the first 30 minutes (optionally within the first 15 minutes) of the treatment.

13. 13. The agent for use of any one of claims 1 to 12, wherein the method comprises reducing the infection by at least 100-fold within the first 30 minutes (optionally within the first 15 minutes) of the treatment, and wherein the at least 100-fold reduction in infection is maintained for at least 60 minutes after contacting the subject with the programmed nuclease.

14. The method of any one of claims 1 to 13, wherein the subject is a human and, at the start of treatment, the subject has a body temperature of <36°C or >38°C; a heart rate of >90 / min, a respiratory rate of >20 breaths / min or a PaCO2 of <4.3 kP; ​​and a white blood cell count of <4000 / mm3 or >12,000 / mm3.

15. 15. The method of claim 14, wherein at the start of the treatment, the subject has two or more of an abnormal body temperature, an abnormal heart rate, an abnormal respiratory rate, an abnormal blood gas, and an abnormal white blood cell count. Agents for use in

16. 16. The method of claim 1, wherein blood infection of the subject by the bacteria is reduced by at least 100 or 1000 fold within the first 30 minutes of treatment.

17. 17. A method for treating or preventing sepsis comprising administering to a subject an effective amount of a nucleic acid comprising: a) administering to said subject an effective amount of a nucleic acid comprising said nucleic acid; b) administering to said subject an effective amount of a nucleic acid comprising said nucleic acid; c) administering to said subject an effective amount of a nucleic acid comprising said nucleic acid; d) administering to said subject an effective amount of a nucleic acid comprising said nucleic acid; the nuclease and / or crRNA or a nucleic acid encoding a crRNA is administered to the subject at a first time point (T1) and a second time point (T2), T2 being at least 1 hour after T1; The method includes reducing the infection by at least 100-fold within the first 30 minutes of the treatment; and maintaining at least a 100-fold reduction in infection for at least 60 minutes after contacting the subject with the agent; the infection is an acute E. coli bacterial infection in a septic subject; the subject is a human or animal; Multiple viruses, phages, or phagemids.

18. 19. A method for treating or preventing sepsis, comprising a composition comprising a plurality of nucleic acids for programming a nuclease as defined in any one of claims 1 to 16, each nucleic acid being a nucleic acid as defined in any one of claims 1 to 17, the nuclease and / or crRNA or a nucleic acid encoding a crRNA is administered to the subject at a first time point (T1) and a second time point (T2), T2 being at least 1 hour after T1; The method includes reducing the infection by at least 100-fold within the first 30 minutes of the treatment; and maintaining at least a 100-fold reduction in infection at least 60 minutes after contacting the subject with the programmed nuclease; the infection is an acute E. coli bacterial infection in a septic subject, and the subject is a human or animal; composition.

19. 17. A CRISPR / Cas system comprising a nuclease as defined in any one of claims 1 to 16 for use in a method for the treatment or prevention of sepsis, said system comprising one or more guide RNAs or DNA encoding one or more guide RNAs, each guide RNA being capable of programming the Cas nuclease to cleave a target site comprised by the chromosome of the bacterium, The nuclease and / or guide RNA or DNA encoding the guide RNA is administered to the subject at a first time point (T1) and a second time point (T2), T2 being at least 1 hour after T1; The method includes reducing the infection by at least 100-fold within the first 30 minutes of the treatment; and maintaining at least a 100-fold reduction in infection at least 60 minutes after contacting the subject with the programmed nuclease; the infection is an acute E. coli bacterial infection in a septic subject; the subject is a human or animal; CRISPR / Cas system.

20. 1. A composition comprising a guide RNA or DNA encoding a guide RNA for use in a method for persistently treating an E. coli bacterial infection in a subject, comprising: The method includes using the guide RNA or DNA encoding the guide RNA in a system, the system further including a Cas nuclease; Each guide RNA is programmable to cleave a target site contained by the E. coli chromosome; the nuclease is programmable to cleave a target site contained by the chromosome of E. coli infected in the subject, thereby persistently killing the E. coli or reducing the growth or proliferation of the E. coli; and the method of treating infection comprises contacting the subject with the nuclease programmed to cleave the target site, thereby cleaving the chromosome of the E. coli contained by the subject and persistently treating the E. coli bacterial infection in the subject; The nuclease and / or guide RNA or DNA encoding the guide RNA is administered to the subject at a first time point (T1) and a second time point (T2), T2 being at least 1 hour after T1; the method comprises reducing the infection by at least 100-fold within the first 30 minutes of the treatment; maintaining at least a 100-fold reduction in infection at least 60 minutes after contacting the subject with the programmed nuclease; the infection is an acute E. coli bacterial infection in a septic subject, and A composition comprising a guide RNA or DNA encoding a guide RNA, wherein the subject is a human or animal.

21. 21. A nucleic acid vector comprising the guide RNA or DNA of claim 19 or 20, wherein the vector is for use in a method for treating or preventing sepsis.

22. 22. The vector for use according to claim 21, wherein the vector is a phage, phagemid, viriophage, virus, plasmid, or transposon.

23. 23. The nuclease, multiple viruses, phages, phagemids, systems, compositions or vectors for use according to claim 22, wherein T2 is at least 2 hours after T1.

24. 24. The nuclease, multiple viruses, phages, phagemids, systems, compositions or vectors for use according to claim 23, wherein T2 is at least 24 hours after T1.

25. 23. The nuclease, multiple viruses, phages, phagemids, systems, compositions or vectors for use according to claim 22, wherein T2 is 7 hours or less after T1.

26. 23. The nuclease, multiple viruses, phages, phagemids, systems, compositions or vectors for use according to claim 22, wherein T2 is 24 hours or less after T1.

27. 27. The nuclease, multiple viruses, phages, phagemids, systems, compositions or vectors for use according to any one of claims 22 to 26, wherein infection is reduced by at least 100-fold within the first 15 minutes of said treatment.

28. 28. The agent, virus, phage, phagemid, system, composition or vector for use according to any one of claims 1 to 27, wherein the method comprises oral administration of the agent, virus, phage, phagemid, guide RNA, DNA or vector.

29. The agent, virus, phage, phagemid, system, composition or vector for use according to any one of claims 1 to 28, wherein said bacterium is an EHEC E coli bacterium.

30. An agent, virus, phage, phagemid, system, composition, or vector for use according to any one of claims 1 to 29, wherein the subject is a human and the infectious disease is a pulmonary infection.

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