JP2023520583A - Methods for treating implantable device infections - Google Patents

Abstract

The present invention relates to the field of phage therapy, and in particular to providing phage-based compositions and methods for treating or preventing infections associated with implantable devices. The composition may be administered directly to the infected site of the device, optionally as a single dose and/or by other modes of administration, potentially avoiding the need for implantable device replacement. obtain. The method comprises the steps of: (a) obtaining a biological sample from the subject; (b) culturing bacteria present in the biological sample; (c) inoculating a phage that lyses the cultured bacteria. may include the step of

Description

FIELD OF THE INVENTION The present invention relates to the field of phage therapy, and in particular to providing phage-based compositions and using phage-based compositions to treat acute or chronic bacterial infections associated with implantable devices. Or to provide a method for prevention.

Discussion of Related Art The following description describes certain articles and methods for background and introductory purposes. Nothing contained herein should be construed as an "admission" of prior art. Applicants expressly reserve the right to demonstrate, where appropriate, that the articles and methods referenced herein do not constitute prior art under applicable legal provisions.

Partial or total joint replacement, most commonly in the knee and hip, is a common life-enhancing procedure that millions of patients undergo each year. In most cases, this procedure is highly successful, relieving pain and restoring the patient's function and independence. In a relatively small number of cases, prosthetic device failure (e.g., caused by wear, device breakage, malposition), or periprosthetic joint infection, an infection involving the prosthetic device and adjacent tissues Complications such as the appearance of prosthetic joint infection (PJI) can occur. Studies have revealed that the incidence of PJI in the United States is about 2% for knee replacements and up to about 1.5% for hip replacements (Tande AJ & R Patel, Clin. Microbiol. Rev. 27 (2): 302-345, 2014). However, although this number is relatively low, PJI is a very serious risk to the patient, resulting in considerable morbidity with pain and swelling, possibly requiring amputation and/or patient can lead to death. Diagnosis, treatment and management of PJI is also a significant cost burden for the health care system, projected to exceed US$1.5 billion in 2020 in the United States alone; Kurtz SM, et al. Arthroplasty 27:61-65. e61, 2012).

In general, PJI is broadly classified into three types; each is distinguished by the time when the infection appears after surgery. Thus, PJI is defined as (i) an infection that develops early within 3 months of surgery (“early onset” PJI); (ii) an infection that develops later than 3 months after surgery and before 12 or 24 months. (“late onset” PJI); and (iii) infections occurring later than 12-24 months after surgery (“late onset” PJI). PJI infections are sometimes classified as "acute" infections occurring within one month after surgery or "chronic" infections occurring more than one month after surgery. Regardless of which classification system is used, early PJI infections are primarily caused by relatively virulent microorganisms such as Staphylococcus aureus and/or aerobic Gram-negative bacteria such as Pseudomonas aeruginosa, and types of late PJI are: Coagulase-negative S. aureus (CoNS), Staphylococcus epidermidis and/or Enterococci spp. It is commonly caused by less toxic bacteria such as Both early and late forms are thought to be acquired during surgery, but late PJI infection is associated with secondary infection from another site of bacterial infection (e.g. blood flow from other foci of infection). caused by hematogenous spread of bacteria through S. aureus appears to be a common causative agent of late-onset PJI resulting from hematogenous spread (Tande & Patel, supra).

Successful treatment of PJI usually requires surgical intervention and/or medical treatment (eg, treatment with antimicrobial agents such as antibiotics) in the majority of cases. Some procedures involve debridement (i.e., surgical removal of infected/damaged tissue), treatment with antibiotics, and retention of implants (artificial joint devices) and are referred to as DAIR procedures; requires replacement of prosthetic joint devices in one- or two-stage joint replacement surgery (Tande & Patel, supra) combined with treatment with antimicrobial agents. The two-step arthroplasty procedure is generally regarded as "the most promising strategy for eradicating infection and preserving joint function", with a success rate of 87-100% for hip replacement and knee replacement. have been reported to be as high as 72-95% in . (Tande & Patel, supra). However, these two-step procedures can be complicated and expensive, requiring at least two surgeries, sometimes separated by 2-3 months or more, and prolonged treatment with antimicrobial agents. Become. This means that some patients are either unsuitable for a two-step (or even a one-step procedure) joint replacement surgery or are unwilling to undergo further surgery, in which case antibiotics There are few options other than trying to treat PJI. However, this is not recommended, and patients are generally placed on long-term or indefinite treatment with oral antimicrobials, followed by careful therapeutic monitoring (Tande & Patel, supra). ).

An additional complication to effective treatment of PJI with antibacterial agents is that the infection may be a "polymicrobial infection," meaning that more than one causative organism is present in the infection. means that This is particularly the case for early-onset PJI, where up to 35% of such infections are caused by S. cerevisiae. aureus, Enterococci spp. , and P. It is presumed to be a polymicrobial infection, usually associated with infection by aerobic Gram-negative bacteria such as C. aeruginosa (Berbari EF et al., Clin. Infect. Dis. 27:1247-1254, 1998; Chemotherap.56:2386-2391, 2012). Therefore, careful selection of antimicrobial agents is necessary if treatment is to be successful.
Although the efficacy and options for treatment of PJI have improved significantly in recent years, there are novel approaches to effectively treat and/or prevent these and other infections associated with implantable devices in joint replacement patients. Or there is a great need to identify and develop improved treatment and/or management strategies.

Tande AJ & R Patel, Clin. Microbiol. Rev. 27(2):302-345, 2014 Kurtz SM, et al. Arthroplasty 27:61-65. e61, 2012 Berbari EF et al., Clin. Infect. Dis. 27:1247-1254, 1998 Peel TN et al., Antimicrob. Agents Chemotherap. 56:2386-2391, 2012

SUMMARY OF THE INVENTION This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will become apparent from the following detailed description, including the aspects that are illustrated in any accompanying drawings and defined in the appended claims. Will.

Specifically, the disclosed invention is a method of treating an infection caused by one or more bacteria associated with a device implanted within a subject, the method comprising:
(a) identifying at least one identified phage capable of infecting the bacterium, wherein the identified phage are APT-PJI-01, APT-PJI-02, APT-PJI-03, APT-PJI -04, APT-PJI-05, APT-PJI-06, APT-PJI-07, APT-PJI-08, APT-PJI-09, APT-PJI-10, APT-PJI-11, APT-PJI-12 , APT-PJI-13, APT-PJI-14, APT-PJI-15, APT-PJI-16, APT-PJI-17, APT-PJI-18, APT-PJI-20, APT-PJI-21, APT -PJI-22, APT-PJI-23, APT-PJI-24, APT-PJI-25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI -30, APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-38 , APT-PJI-39, APT-PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45, APT-PJI-46, APT - a genomic sequence having at least 70% sequence identity to the genomic sequence of PJI-47, APT-PJI-48, APT-PJI-49 and/or APT-PJI-50, or any of the aforementioned phages; and (b) a therapeutically effective amount comprising the identified phage A method is described comprising administering a composition to a subject, wherein the composition is effective to treat and/or reduce the infection.

In another embodiment, the disclosed invention is a method of identifying a subject having, at risk of having, or suitable for treatment for a bacterial infection, the method comprising:
(a) obtaining a biological sample from a subject;
(b) culturing the bacteria present in the biological sample;
(c) cultured bacteria APT-PJI-01, APT-PJI-02, APT-PJI-03, APT-PJI-04, APT-PJI-05, APT-PJI-06, APT-PJI-07 , APT-PJI-08, APT-PJI-09, APT-PJI-10, APT-PJI-11, APT-PJI-12, APT-PJI-13, APT-PJI-14, APT-PJI-15, APT -PJI-16, APT-PJI-17, APT-PJI-18, APT-PJI-20, APT-PJI-21, APT-PJI-22, APT-PJI-23, APT-PJI-24, APT-PJI -25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, APT-PJI-31, APT-PJI-32, APT-PJI-33 , APT-PJI-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT - PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45, APT-PJI-46, APT-PJI-47, APT-PJI-48, APT-PJI-49 and/or APT - PJI-50 as well as any other lytic phage having a genomic sequence with at least 70% sequence identity to the genomic sequence of any of the aforementioned phages and capable of causing bacterial lysis. and (d) determining if the cultured bacteria are lysed by the identified phage. wherein when any of the cultured bacteria are lysed by the phage, the subject is (1) eligible for treatment with the bacteriophage for the bacterial infection; (2) suffering from the bacterial infection; and/or (3) is at risk of contracting a bacterial infection.

In further described embodiments, the composition used in any of the methods is APT-PJI-01, APT-PJI-02, APT-PJI-03, APT-PJI-04, APT-PJI-05, APT-PJI-06, APT-PJI-07, APT-PJI-08, APT-PJI-09, APT-PJI-10, APT-PJI-11, APT-PJI-12, APT-PJI-13, APT- PJI-14, APT-PJI-15, APT-PJI-16, APT-PJI-17, APT-PJI-18, APT-PJI-20, APT-PJI-21, APT-PJI-22, APT-PJI- 23, APT-PJI-24, APT-PJI-25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT- PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45, APT-PJI-46, APT-PJI-47, APT-PJI- 48, APT-PJI-49 and/or APT-PJI-50, or a genomic sequence having at least 70% sequence identity to the genomic sequence of any of the aforementioned phages and causing bacterial lysis at least one identified phage selected from the group consisting of any other lytic phage capable of

Other embodiments are described further below.

DETAILED DESCRIPTION The following definitions are provided for certain terms used in the written description that follows.

DEFINITIONS As used in this specification and claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Also, as understood by those of skill in the art, the term "phage" can be used to refer to a single phage or more than one phage.

The invention can "comprise" (open-ended) or "consist essentially of" components of the invention. As used herein, "comprising" means the listed elements, or their equivalents in structure or function, as well as any other element or elements not listed. . The terms "having" and "including" are also to be interpreted as open-ended unless the context indicates otherwise.

The terms "about" or "approximately" mean within an acceptable range of a particular value as determined by one skilled in the art, how the value is measured or determined, For example, it will depend partly on the limitations of the measurement system. For example, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, more preferably even up to 1% of a given value. Alternatively, particularly with respect to biological systems or methods, the term can mean within an order of magnitude, such as within a factor of five, or within a factor of two. Unless otherwise stated, the term "about" means within an acceptable margin of error for the specified value, such as ±1-20%, preferably ±1-10%, more preferably ±1-5%. do. In a further embodiment, "about" should be understood to mean +/-5%.

When a range of values is provided, it is understood that the invention includes each intervening value between the upper and lower limits of that range, and any other stated or intervening value within the stated range. be. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding both of those included limits are also included in the invention.

All ranges recited herein are inclusive of the endpoints, including those recited "between" two ranges of values.

Terms such as "about," "generally," "substantially," and "approximately" are not absolute and should be construed as modifying the term or value so that it is not read in the prior art. is. Such terms are defined by the context and the terms they modify as those terms are understood by those skilled in the art. It includes at least the degree of expected experimental, technique, and instrumental error for a given technique used to measure the value.

As used herein, the term "and/or" when used in a list of two or more items means that any one of the recited properties may be present or It means that any combination of two or more of the properties can be present. For example, if a composition is described as containing features A, B and/or C, the composition may include A feature only, B alone, C alone, A and B in combination, A and C in combination, B and C or combinations of A, B and C.

As understood by those skilled in the art, the term "bacteriophage" or "phage" refers to non-cellular infectious agents that replicate only in suitable host cells, ie bacterial host cells.

The term "phage therapy" refers to any disease or condition that treats a bacterial infection, or a disease or condition caused by bacteria (e.g., PJI) or that exhibits the onset or progression of symptoms or diseases/conditions associated with the presence of bacteria. refers to the treatment of Phage therapy involves administering to a patient in need of treatment one or more therapeutic phage compositions that can be used to infect, kill, or inhibit the growth of bacteria. Occasionally, the composition comprises one or more viable phage as an antimicrobial agent (eg, a composition comprising one phage type or two or more phage types in a phage "cocktail"). The one or more phage types may be obtained from stocks of phage, which may be stored as inventories. When phage therapy involves administration of two or more therapeutic phage compositions, the compositions may have different host ranges (e.g., one may have a broad host range and one may have a narrow host range). and/or one or more compositions may act synergistically with each other). Furthermore, as those skilled in the art will readily appreciate, therapeutic phage compositions used in phage therapy typically contain various conventional pharmaceutically acceptable excipients, carriers, buffers and/or agents. or a series of inactive ingredients selected from diluents.

The term "pharmaceutically acceptable" is used to refer to non-toxic substances that are compatible with biological systems such as cells, cell cultures, tissues or organisms. Examples of pharmaceutically acceptable excipients, carriers, buffers and/or diluents are well known to those skilled in the art, see, for example, Remington's Pharmaceutical Sciences (latest edition), Mack Publishing Company, Easton, can be found in Pa. For example, pharmaceutically acceptable excipients include wetting or emulsifying agents, pH buffering agents, binders, stabilizers, preservatives, bulking agents, adsorbents, disinfectants, detergents, sugar alcohols, gelling agents. or thickening agents, flavoring agents and coloring agents. Pharmaceutically acceptable carriers include macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, trehalose, lipid aggregates (such as oil droplets or liposomes), and inert virus particles. are mentioned. Pharmaceutically acceptable diluents include, but are not limited to, water and saline.

The present invention relates to providing phage-based compositions and methods for treating infections associated with implantable devices, such as prosthetic joint infections (“PJI”). The compositions and methods may offer considerable simplicity, as the treatment may involve administering the composition directly to the infected area (e.g., a joint) as a single dose as needed, For example, phage therapy could potentially avoid the need for replacement of implantable devices (eg, prosthetic joint devices for infected joints).

Accordingly, in a first aspect, the present invention provides a pharmaceutical composition comprising at least two different bacteriophages capable of lysing infections associated with implantable devices, such as prosthetic joint infections (PJI) in patients. wherein the pharmaceutical composition is formulated for administration to the patient directly to the site of infection by IV and/or orally.

In one embodiment, at least two different bacteriophages lyse a single bacterial species or two or more bacterial species (i.e., in the case of a polymicrobial infection) that may be present in an implantable device infection ( phage types selected for their ability to kill, ie, kill).

Selection of phage type for therapeutic indication can be based on the results of tests to determine the type of bacteria (i.e., the causative organism) present in the implantable device infection; Bacteria present in the culture were identified by 16S rRNA gene sequencing (as described, for example, in Whelan FJ et al., Ann. Am. Thorac. Soc. 11:513-521, 2014). can be readily determined by standard techniques well known to those skilled in the art, including Biological samples may also be used in assays to assess bacteria for susceptibility to individual phages, which can be very informative in selecting phage types for inclusion in compositions.

The choice of phage type can also be based on predictions or predictions of which causative organisms are present in implantable device infections. Similarly, phage type selection can also be based on predictions or predictions of which causative organisms are present in a particular geographic location.

For example, the phage selected for inclusion in the composition is the bacterium commonly responsible for delayed onset device infections, eg, delayed onset PJI, ie, S. cerevisiae. aureus (eg, coagulase-negative S. aureus) and Enterococcus spp. (eg, E. fecalis and Enterococcus fecium). Such compositions are generally described in S.M. It may also be effective in treating late-onset PJI caused by C. aureus. Alternatively, the selected phage can be used to treat acute or chronic infections. Other bacteria that can be targeted by including appropriately selected phage types include other coagulase-negative Staphylococcus species reported to cause PJI, such as S. cerevisiae. simulans (Razonable RR et al., Mayo Clin. Proc. 76:1067-1070, 2001); caprae (Allignet J et al., Microbiology 145 (Part 8):2033-2042, 1999) and S. lugdunensis (Sampathkumar P et al., Mayo Clin. Proc. 75:511-512, 2000); Lancefield A, B, C and G groups (Meehan AM et al., Clin. Infect. Dis. 36:845-849, 2003; Zeller V et al., Presse Med. 38:1577-1584, 2009; and Kleshinski J et al., South. Med. agalactiae and S. Various Streptococcus spp. , aerobic Gram-negative bacteria such as Escherichia coli (Jaen N et al., Rev. Esp. Quimioter. 25:194-198, 2012), other Enterobacteriaceae (Hsieh PH et al., Clin. Infect. Dis. 49:1036-1043, 2009), eg, Enterobacter clocae; and others, eg, Clostridium spp. , Actinomyces spp. , Peptostreptococcus spp. , Cutibacterium acnes (formerly Propionibacterium acnes), Klebsiella pneumoniae, P. aeruginosa and Bacteroides fragilis (Tande & Patel, supra).

It is further contemplated that the identified phage included in the composition can be matched phage compositions based on the prevalent infections in a particular geographic location. Examples of such "geomatched" phage are described in USSN 62/956,729, filed Jan. 3, 2020, which is hereby incorporated by reference in its entirety. .

Accordingly, one aspect of the invention is a method of treating an infection caused by one or more bacteria associated with a device implanted within a subject, the method comprising:
(a) identifying at least one identified phage capable of infecting the bacterium, the identified phage being APT-PJI-01, APT-PJI-02, APT-PJI listed in Table 1; -03, APT-PJI-04, APT-PJI-05, APT-PJI-06, APT-PJI-07, APT-PJI-08, APT-PJI-09, APT-PJI-10, APT-PJI-11 , APT-PJI-12, APT-PJI-13, APT-PJI-14, APT-PJI-15, APT-PJI-16, APT-PJI-17, APT-PJI-18, APT-PJI-20, APT -PJI-21, APT-PJI-22, APT-PJI-23, APT-PJI-24, APT-PJI-25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI -29, APT-PJI-30, APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI-34, APT-PJI-35, APT-PJI-36, APT-PJI-37 , APT-PJI-38, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45, APT - at least 70% sequence identity to the genomic sequence of PJI-46, APT-PJI-47, APT-PJI-48, APT-PJI-49 and/or APT-PJI-50, or any of the aforementioned phages (b) an identified phage selected from a library comprising at least one phage selected from any other lytic phage that has a genomic sequence with a cytotoxicity and is capable of causing bacterial lysis; and (b) an identified phage administering to the subject a therapeutically effective amount of a composition comprising: wherein the composition is effective to treat and/or reduce the infection.

Table 1 lists published phages (e.g., APT-PJI-01, APT-PJI-02, APT-PJI-03, APT-PJI-04, APT-PJI-05, APT-PJI-06, APT-PJI -07, APT-PJI-08, APT-PJI-09, APT-PJI-10, APT-PJI-11, APT-PJI-12, APT-PJI-13, APT-PJI-14, APT-PJI-15 , APT-PJI-16, APT-PJI-17 and APT-PJI-18) and proprietary phages that are not publicly available (e.g., APT-PJI-20, APT-PJI-21, APT-PJI-22, APT-PJI-23, APT-PJI-24, APT-PJI-25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, APT- PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI- 39, APT-PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45, APT-PJI-46, APT-PJI-47, APT-PJI-48, APT-PJI-49 and/or APT-PJI-50).

In preferred embodiments, the compositions and methods are performed using any combination of proprietary phage. Specifically, APT-PJI-20, APT-PJI-21, APT-PJI-22, APT-PJI-23, APT-PJI-24, APT-PJI-25, APT-PJI-26, APT-PJI -27, APT-PJI-28, APT-PJI-29, APT-PJI-30, APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI-34, APT-PJI-35 , APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT - at least selected from the group consisting of PJI-44, APT-PJI-45, APT-PJI-46, APT-PJI-47, APT-PJI-48, APT-PJI-49 and/or APT-PJI-50 One, at least two, at least three, at least four and/or at least five phage are used to treat a patient suffering from a PJI infection as described herein.

In another aspect, the disclosed invention is a method of identifying a subject having, at risk of having, and/or amenable to phage treatment for a bacterial infection, the method comprising:
(a) obtaining a biological sample from a subject;
(b) culturing the bacteria present in the biological sample;
(c) cultured bacteria APT-PJI-01, APT-PJI-02, APT-PJI-03, APT-PJI-04, APT-PJI-05, APT-PJI-06, APT-PJI-07 , APT-PJI-08, APT-PJI-09, APT-PJI-10, APT-PJI-11, APT-PJI-12, APT-PJI-13, APT-PJI-14, APT-PJI-15, APT -PJI-16, APT-PJI-17, APT-PJI-18, APT-PJI-20, APT-PJI-21, APT-PJI-22, APT-PJI-23, APT-PJI-24, APT-PJI -25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, APT-PJI-31, APT-PJI-32, APT-PJI-33 , APT-PJI-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT - PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45, APT-PJI-46, APT-PJI-47, APT-PJI-48, APT-PJI-49 and/or APT - PJI-50 as well as any other lytic phage having a genomic sequence with at least 70% sequence identity to the genomic sequence of any of the aforementioned phages and capable of causing bacterial lysis. and (d) determining if the cultured bacteria are lysed by the identified phage. wherein when any of the cultured bacteria are lysed by the phage, the subject is (1) eligible for treatment with the bacteriophage for the bacterial infection; (2) suffering from the bacterial infection; and/or (3) is at risk of contracting a bacterial infection.

In preferred embodiments, the composition comprises APT-PJI-01, APT-PJI-02, APT-PJI-03, APT-PJI-04, APT-PJI-05, APT-PJI-06, APT-PJI-07 , APT-PJI-08, APT-PJI-09, APT-PJI-10, APT-PJI-11, APT-PJI-12, APT-PJI-13, APT-PJI-14, APT-PJI-15, APT -PJI-16, APT-PJI-17, APT-PJI-18, APT-PJI-20, APT-PJI-21, APT-PJI-22, APT-PJI-23, APT-PJI-24, APT-PJI -25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, APT-PJI-31, APT-PJI-32, APT-PJI-33 , APT-PJI-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT - PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45, APT-PJI-46, APT-PJI-47, APT-PJI-48, APT-PJI-49 and/or APT - consisting of PJI-50 or any other lytic phage having a genomic sequence with at least 70% sequence identity to the genomic sequence of any of the aforementioned phages and capable of causing bacterial lysis comprising at least one identified phage selected from the group;

In even more preferred embodiments, the composition comprises APT-PJI-01, APT-PJI-02, APT-PJI-03, APT-PJI-04, APT-PJI-05, APT-PJI-06, APT-PJI -07, APT-PJI-08, APT-PJI-09, APT-PJI-10, APT-PJI-11, APT-PJI-12, APT-PJI-13, APT-PJI-14, APT-PJI-15 , APT-PJI-16, APT-PJI-17, APT-PJI-18, APT-PJI-20, APT-PJI-21, APT-PJI-22, APT-PJI-23, APT-PJI-24, APT -PJI-25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, APT-PJI-31, APT-PJI-32, APT-PJI -33, APT-PJI-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT-PJI-40, APT-PJI-41 , APT-PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45, APT-PJI-46, APT-PJI-47, APT-PJI-48, APT-PJI-49 and/or or APT-PJI-50 or any other lytic phage having a genomic sequence with at least 70% sequence identity to the genomic sequence of any of the aforementioned phages and capable of causing bacterial lysis at least two identified phage selected from the group consisting of

In further embodiments, the at least two identified phages (a) cause lysis in the same bacterial strain; (b) cause lysis in different bacterial strains.

In another further aspect, one of the two identified phages is (a) APT-PJI-01, APT-PJI-02, APT-PJI-03, APT-PJI-04, APT-PJI- 05, APT-PJI-06, APT-PJI-07, APT-PJI-08, APT-PJI-09, APT-PJI-10, APT-PJI-11, APT-PJI-12, APT-PJI-22, APT-PJI-23, APT-PJI-24, APT-PJI-25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, APT- PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI- 39, APT-PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45 and APT-PJI-46 and in (a) (b) APT-PJI-13, APT-PJI-14 and any other lytic phage having a genomic sequence with at least 70% sequence identity to the genomic sequence of any of the aforementioned phages in (b). (c) at least 70 against the genomic sequence of APT-PJI-15, APT-PJI-16, APT-PJI-17, APT-PJI-18 and any of the foregoing phages in (c); selected from any other lytic phage having a genomic sequence with % sequence identity; (d) APT-PJI-20 or APT-PJI-21 and (d) or (e) any other lytic phage having a genomic sequence having at least 70% sequence identity to the genomic sequence of any of the aforementioned phages in APT-PJI -47, APT-PJI-48, APT-PJI-49 or APT-PJI-50 and a genomic sequence having at least 70% sequence identity to the genomic sequence of any of the aforementioned phages in (e) selected from the group consisting of any other lytic phage having

In a further aspect, the two identified phage are selected from each of (a) and (b); selected from each of (a) and (c) from the phages described in the preceding paragraph selected from each of (a) and (d); selected from each of (a) and (e); and/or selected from each of (b) and (c), (b) and selected from each of (d); selected from each of (b) and (e); and/or selected from each of (c) and (d); each of (c) and (e) and/or selected from each of (d) and (e). In a further aspect, the composition comprises at least three identified phages, at least one of which is from the phages described in the preceding paragraphs (a), (b), (c), selected from each of (d) and/or (e);

In other further embodiments, one of the two identified phages is (a) (a) APT-PJI-22, APT-PJI-23, APT-PJI-24, APT-PJI-25, APT -PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI -34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT-PJI-42 , APT-PJI-43, APT-PJI-44, APT-PJI-45 and APT-PJI-46 and at least 70% sequence identity to the genomic sequence of any of the aforementioned phages in (a) (b) APT-PJI-20 or APT-PJI-21 and any of the foregoing phages in (b) or (c) APT-PJI-47, APT-PJI-48, or (c) APT-PJI-47, APT-PJI-48, APT-PJI-49 or APT-PJI-50 and any other lytic phage having a genomic sequence with at least 70% sequence identity to the genomic sequence of any of the aforementioned phages in (c) selected from the group consisting of

In a further embodiment, the two identified phage are selected from each of (a) and (b); selected from each of (a) and (c) from the phages described in the preceding paragraph and/or selected from each of (b) and (c). In a further aspect, the composition comprises at least three identified phages, wherein at least one of the identified phages is derived from the phages of (a), (b) and (c) described in the preceding paragraph. selected from each.

In a further aspect, the composition is in the range of 10 ⁵ to 10 ¹³ pfu, preferably 10 ⁶ to 10 ¹² pfu; 10 ⁷ to ^{10 11 pfu} ; 10 ⁸ to ^{10 11 pfu} ; providing a dose of each phage within the range of ^{9-10 10} pfu or a dose of approximately 10 ⁶ pfu, 10 ⁷ pfu, 10 ⁸ pfu, 10 ⁹ pfu, 10 ¹⁰ pfu, 10 ¹¹ pfu, 10 ¹² pfu or 10 ¹³ pfu do. In a most preferred embodiment, the dose of each phage in the composition is approximately 10 ⁹ pfu.

Accordingly, the composition can be formulated such that a suitable dose of each phage type is included in the composition. By way of example only, suitable doses of phage are in the range of 10 ⁵ -10 ¹³ pfu, more preferably 10 ⁹ -10 ¹² pfu. Most preferably, the compositions are formulated so that there is a dose of about 10 ⁹ pfu, 10 ¹⁰ pfu or 10 ¹¹ pfu of each phage type.

In a preferred embodiment, the bacterium is S. cerevisiae. aureus, S. epidermidis, E. Enterococcus spp. including Enterococcus fecalis and Enterococcus fecium. , S. simulans, S. caprae and S. Other coagulase-negative Staphylococcus species, including Lancefield A, B, C and G groups, including S. lugdunensis. agalactiae and S. Streptococcus spp., including Streptococcus pneumoniae. , aerobic Gram-negative bacteria including E. coli, other Enterobacteriaceae including Enterobacter clocae, Clostridium spp. , Actinomyces spp. , Peptostreptococcus spp. , Cutibacterium acnes, Klebsiella pneumoniae, P. aeruginosa and at least one of Bacteroides fragilis.

In another preferred embodiment, the bacterium is S. cerevisiae. aureus, S. epidermidis, E. Enterococcus spp. including Enterococcus fecalis and Enterococcus fecium. , S. simulans, S. caprae and S. Other coagulase-negative Staphylococcus species, including Lancefield A, B, C and G groups, including S. lugdunensis. agalactiae and S. Streptococcus spp., including Streptococcus pneumoniae. , aerobic Gram-negative bacteria including E. coli, other Enterobacteriaceae including Enterobacter clocae, Clostridium spp. , Actinomyces spp. , Peptostreptococcus spp. , Cutibacterium acnes, Klebsiella pneumoniae, P. aeruginosa and Bacteroides fragilis from two or more different bacterial strains.

In an even more preferred embodiment, the bacterium is S. cerevisiae. aureus, S. epidermidis and/or E. fecalis. In order to treat such infections, the composition may contain S. aureus, S. epidermidis and/or E. at least two phages with different lytic specificities that are capable of infecting at least two of the S. fecalis. In other aspects, the composition comprises, for example, at least three phage with different lytic specificities, each phage being associated with S. cerevisiae. aureus, S. epidermidis and E. fecalis.

Preferably, the lytic phage is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% relative to the genomic sequence of any of the aforementioned phages. , have genomic sequences with 97%, 98% or 99% sequence identity.

Percentage sequence identities referred to herein are calculated by comparing two polynucleotide sequences using the BLAST algorithm from the National Center for Biotechnology Information database (NCBI; Bethesda, MD, United States of America). It will be understood that

The terms "percent (%) sequence similarity", "percent (%) sequence identity", etc., generally refer to different nucleic acid molecules that may or may not share a common evolutionary origin. Refers to the degree of identity or correspondence between nucleotide sequences or between different amino acid sequences of polypeptides (see Reeck et al., supra). Sequence identity may be determined using any of several publicly available sequence comparison algorithms such as BLAST, FASTA, DNA Strider, GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin). and so on.

To determine the percent identity between two amino acid sequences or between two nucleic acid molecules, the sequences are aligned for optimal comparison purposes. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical positions/total number of positions (e.g., overlapping positions) x 100 ). In one embodiment, the two sequences are the same length or about the same length. The percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent sequence identity, typically exact matches are counted.

The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized to compare two sequences is the algorithm described by Karlin and Altschul, Proc. Natl. Acad. Sci. USA 1993, 90:5873-5877, modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 1990, 87:2264 algorithm. Such algorithms are described by Altschul et al. Mol. Biol. 1990;215:403, incorporated in the NBLAST and XBLAST programs. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the sequences of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein sequences of the invention. To obtain gapped alignments for comparison purposes, Altschul et al., Nucleic Acids Res. 1997, 25:3389 can be utilized. Alternatively, PSI-Blast can be used to perform an iterative search which detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used. ncbi.com on the world wide web. nlm. nih. See gov/BLAST/.

Another non-limiting example of a mathematical algorithm utilized for comparing sequences is the algorithm of Myers and Miller, CABIOS 1988;4:1 1-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weighted residue table, a gap length penalty of 12 and a gap penalty of 4 can be used.

In a preferred embodiment, the percent identity between two amino acid sequences is calculated using the method of Needleman and Wunsch incorporated into the GAP program in the GCG software package (Accelrys, Burlington, Mass.; available on the world wide web at acceleratorrys.com). Using the algorithm (J. Mol. Biol. 1970, 48:444-453), a Blossom62 matrix or a PAM250 matrix, gap weights of 16, 14, 12, 10, 8, 6 or 4, and 1, 2, 3, Determined using length weights of 4, 5 or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, NWSgapdna. Determined using the CMP matrix, gap weights of 40, 50, 60, 70 or 80, and length weights of 1, 2, 3, 4, 5 or 6. A particularly preferred set of parameters (and one that can be used when the practitioner is unsure what parameters to apply to determine whether a molecule is the limit of sequence identity or sequence homology of the present invention) uses the Blossom62 scoring matrix with a gap open penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

Another non-limiting example of how percent identity can be determined can be found in Current Protocols In Molecular Biology (FM Ausubel et al., eds., 1987) Supplement 30, section 7.7.18, Table 7.7.1. Using a software program such as the software program described. Preferably, default parameters are used for the alignment. A preferred alignment program is BLAST, using default parameters. In particular, preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic Code=Standard; Filter=None; Strand=Both; Cutoff=60; Expect=10; Matrix=BLOSUM62; sort = by high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet address: http://www. ncbi. nlm. nih. gov/cgi-bin/BLAST.

Statistical analysis of the properties described herein can be performed by standard tests such as t-test, ANOVA or chi-square test. Typically statistical significance is at the level of p=0.05 (5%), more preferably p=0.01, p=0.001, p=0.0001, p=0.000001 measured.

In preferred aspects, the methods described herein involve a device, wherein the device is (a) permanently implanted in the subject; (b) temporarily implanted in the subject; and/or (d) selected from artificial joints, left ventricular assist devices (LVADs), stents, metal rods, indwelling catheters, spinal devices, and/or bone devices.

In a further aspect, the infection is prosthetic joint infection (PJI), chronic bacterial infection, acute bacterial infection, refractory infection, biofilm associated infection, and/or implantable device associated infection. is selected from

In a further aspect, the methods described herein relate to a composition, the composition comprising, for example,
(a) by IV injection; (b) by direct injection to the site of infection; (c) prophylactically; (d) prior to surgery; (e) alternatively to surgery; ) once (ie, as a “single shot”), and/or (h) as a course of treatment of 2 weeks or more.

In further aspects, lysis of bacteria by phage can be achieved by, but not limited to: (a) growth inhibition; (b) optical density; (c) metabolic output; (d) photometry (e.g. fluorescence, absorption and transmission assays); and/or (e) can be measured using assays known in the art, such as plaque formation.

In preferred embodiments, photometric assays used to measure lysis employ additives that cause and/or enhance detection of photometric signals. Examples of such additives include, but are not limited to, tetrazolium dyes.

In other preferred embodiments, the biological sample is (a) synovial fluid; (b) the area surrounding the implantable device; (c) the site of infection; (d) a sample during surgery; (e) a swab of the device; (f) biofilms; (g) fistulae; and/or (h) aspirates of infected sites.

In a further aspect, the bacterial infection is (a) multidrug resistant; (b) clinically refractory to antimicrobial treatment; (c) refractory to antimicrobial treatment due to biofilm formation. and/or (d) clinically refractory due to the subject's inability to tolerate the antimicrobial due to adverse reactions.

As understood herein, the terms “multidrug resistance,” “multi drug resistant,” “multi drug resistance,” “MDR,” etc. are used herein to and are known to those skilled in the art, i.e., multidrug-resistant bacteria are resistant to multiple different antimicrobial agents, e.g., antibiotics; more particularly, multiple different classes of It is an organism that exhibits resistance to antibiotics. Bacterial infections to be treated are understood herein to include bacteria within biofilms and/or planktonic growth modes.

Examples of typical MDR bacteria that may be treated include the "ESKAPE" pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia), which are often nosocomial in nature and can cause severe local and systemic infections. , Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter sp). Specifically, these include, for example, methicillin-resistant Staphylococcus aureus (MRSA); vancomycin-resistant Enterococcus faecium (VRE); carbapenem-resistant Klebsiella pneumonia (NDM-1); MDR-Pseudomonas aeruginosa; bacter baumannii .

Among the ESKAPE pathogens, A. baumannii is a Gram-negative, encapsulated opportunistic pathogen that spreads readily in hospital intensive care units. For example, A. baumannii infections are typically found in the respiratory tract, urinary tract and wounds. many A. baumannii clinical isolates are also MDRs, which severely limits available treatment options, and untreatable infections in trauma lead to prolonged healing times, the need for extensive surgical debridement, and in some cases It often results in further amputation or complete amputation of the limb. Because blast-related injuries, especially in military populations, are associated with significant tissue destruction and concomitant massive blood loss, these injuries are at high risk for infectious complications. A. baumannii and other MDR ESKAPE pathogens can colonize and survive in many environmental settings, those skilled in the art will appreciate the urgent need for new therapeutics against these pathogens.

In a further aspect, the subject has a device-associated infection.

In preferred embodiments, the infection is prosthetic joint infection (PJI). For example, compositions of the invention may be useful in treating PJI of any type. Thus, by appropriate selection of two or more phages, the present compositions will be beneficial in treating early-onset, late-onset and late-onset PJI, as well as PJI that is the result of polymicrobial infection. obtain. Similarly, in other preferred embodiments, the compositions described herein can be used to treat acute and/or chronic infections, preferably PJI infections. The compositions of the invention may be particularly useful in treating PJI associated with hip, knee, shoulder and elbow joint replacement surgery.

In some embodiments, the libraries used in the methods described herein comprise phage prescreened to exclude phage containing undesirable and/or virulence characteristics. Examples of such undesirable and/or virulence characteristics are toxin genes or other bacterial virulence factors, phages with lysogenic properties and/or carrying lysogenic genes, bacterial virulence factor genes or Phage carrying antibiotic resistance genes, phage carrying any antibiotic resistance gene or capable of conferring antibiotic resistance on bacterial strains, and eliciting inappropriate immune responses and/or strong allergic responses in mammalian systems. are selected from phages that provoke Examples of making such prescreened phage libraries are described, for example, in US 10,357,522, the disclosure of which is incorporated herein by reference in its entirety.

Possible consequences of using bacteriophages to treat bacterial infections are known to be antibiotic resistance, toxin formation and unwanted innate immune responses in mammals (Quiros et al., Antimicrob Agents Chemother 58:606-609, 2014; de Vale et al., Front. Microbiol. 7:42, 2016). In some aspects, phage that contain undesirable and/or virulence characteristics can be initially excluded from the library. It is well understood that it is also within the skill in the art to genetically alter phage to eliminate and/or reduce undesirable and/or virulence phenotypes. The deleterious phenotype can be traced to genes located on the single-stranded DNA of the phage. To eliminate these genes and to prevent phages from horizontally transmitting these features to bacteria associated with chronic infections within implantable devices, CRISPRs (clustered and regularly arranged short palindromic sequences) were used. Systems such as repeats) can be used to edit genetic material. Phage with DNA or RNA genomes can be engineered using the CRISPR system.
Gene editing using CRISPR/Cas9

CRISPR and CRISPR-associated protein 9 (Cas9), referred to as the CRISPR/Cas9 system, can be used to effect gene editing. Gene editing includes, but is not limited to, gene insertions, gene replacements, gene deletions, frameshifts, single nucleotide changes, nonsense mutations, missense mutations, and the like. Gene editing can also involve editing regulatory sequences to regulate gene expression. Based on these techniques, genes can be mutated, repaired or regulated in a variety of cell lines and germline sequences. A review of the CRISPR/Cas9 system can be found in Hsu et al., "Development and Applications of CRISPR-Cas9 for Genome Engineering" Cell (2014) vol. 157:1262. Guidance for use of the CRISPR/Cas9 system may be found in Addgene (addgene.org/CRISPR/guide).

Using the CRISPR/Cas9 system to "knock out" and "knock in" specific genes and selectively activate or repress specific genes, specific regions of DNA , imaging DNA in living cells using fluorescence microscopy, as well as many other applications.

According to embodiments of the present invention, the CRISPR/Cas9 system can be used to edit genes in phage to eliminate and/or reduce unwanted and/or toxic phenotypes. By reducing or eliminating the virulence phenotype, the undesirable effects of bacteriophage therapy for treating bacterial infections, including antibiotic resistance, toxin formation and unwanted innate immune responses in mammals are reduced or eliminated. As such, these phages are expected to be useful in the treatment of infectious diseases.

According to other embodiments of the invention, phage edited by the CRISPR/Cas9 system can be administered to a patient. In still further embodiments of the invention, phage edited by the CRISPR/Cas9 system may be used in combination with one or more additional agents described herein.

Phage genome editing, which has traditionally been a time-consuming and difficult process, can be efficiently performed using the CRISPR/Cas9 system. In some cases, editing by the CRISPR/Cas9 system can occur within 24 hours of delivery of the Cas9 gene, guide RNA, and, if applicable, nucleotide replacement sequences.
Components of the CRISPR/Cas9 system

In general, CRISPR/Cas9 systems consist of at least two components: (i) a Cas9 protein, which is a nuclease capable of cleaving both strands of a DNA duplex; and (ii) a specific target for a gene of interest (genomic target). Include at least one RNA sequence (eg, a guide RNA (gRNA) sequence, eg, a single-stranded guide RNA (sgRNA) sequence) designed to target Cas9 to a location or locus. The gRNA contains a targeting sequence homologous to the genomic target, as well as a scaffolding domain that binds Cas9, in order to recruit Cas9 to the genomic target.
non-homologous end joining

In some embodiments, the CRISPR/Cas9 system induces double-stranded DNA breaks in genomic targets. This cleavage is thought to stimulate cellular repair mechanisms that employ non-homologous end joining (NHEJ). Non-homologous end joining, which can lead to gene disruption by random insertion or deletion, is believed to be the primary mechanism for repairing double-strand breaks (DSBs). NHEJ-mediated DSB repair can disrupt genomic target open reading frames (ORFs), resulting in non-functional proteins.

For the NHEJ method, Cas9 and gRNA are introduced into host cells, eg, as pre-assembled complexes or inserted into one or more expression vectors.
Homology Directed Repair

In other embodiments, the CRISPR/Cas9 system is utilized to repair, replace or insert one or more nucleotides in a genomic target. In this method, a replacement nucleotide sequence is introduced into the cell. The replacement nucleotide sequence now contains the desired editing and homology regions upstream and downstream of the genomic target. Homologous recombination repair (HDR) requires Cas9, e.g., recombinant Cas9 from Streptococcus pyogenes, complexed with a gRNA, e.g., in vitro transcribed sgRNA, and a replacement nucleotide sequence.

Homologous recombination repair (HDR) can be used to perform gene editing ranging from single nucleotide base changes to large nucleotide sequence insertions. To utilize HDR for gene editing, replacement nucleotide sequences are delivered into cells along with gRNA and Cas9. The replacement nucleotide sequence contains the desired genome editing and additional homologous sequences immediately upstream and downstream of the genomic target sequence (referred to as left and right homology arms). Replacement nucleotide sequences can be single-stranded oligonucleotides, double-stranded oligonucleotides, double-stranded DNA plasmids, etc., depending on the particular application. Generally, the replacement nucleotide sequence does not contain protospacer adjacent motif (PAM) sequences so as not to be targeted for Cas9 cleavage.
gRNA

As discussed above, gRNAs contain a targeting sequence and a scaffolding domain. When expressed, Cas9 and gRNA form a riboprotein complex through interaction of the gRNA with the scaffold domain. Upon gRNA binding, Cas9 undergoes a conformational change to a DNA-bound form while allowing the targeting sequence to freely interact with the genomic target DNA. For Cas9 to cleave genomic target DNA, the targeting sequence should show high homology to the genomic target sequence. Generally, the target sequence contains 20 or about 20 nucleotides and can be synthetic RNA.

Generally, "target sequence" or "targeting sequence" refers to the sequence of the gRNA (eg, the portion that does not associate with the scaffold domain). "Genomic target" or "genomic target sequence" refers to a sequence or locus in the genome that is targeted for editing.

The gRNA uses the targeting sequence to focus the nuclease activity of Cas9 on the genomic target. Once the target sequence binds or hybridizes to the genomic target, Cas9 cleaves the genomic DNA at or near that site. The genomic target of Cas9 can be altered by altering the target sequence.

In some embodiments, gRNAs can be synthesized using commercially available kits, such as the Guide-it sgRNA In Vitro Transcription Kit. In vitro transcription may be used to generate gRNA that can be purified using techniques known in the art.
genomic target

A genomic target sequence should be unique relative to the rest of the genome of the organism or cell to avoid off-target effects.

Furthermore, the genomic target sequence should be immediately upstream of the genomic protospacer adjacent motif (PAM) sequence. A PAM sequence is required for Cas9 binding, and the exact PAM sequence depends on the species of Cas9 used. Cas9 from Streptococcus pyogenes is widely used in genome engineering. In general, Cas9 binds to the PAM sequence resulting in a DNA break approximately 3 base pairs upstream of the PAM sequence.

An example of a PAM sequence is 5'-NGG-3'. A genomic target sequence can be present on either strand of the genomic DNA.

Several online tools (e.g., http://crispr.mit.edu/ or https://chopchop.rc.fas.harvard.edu/) are available for selecting PAM sequences and A list of potential genomic target sequences within a genomic locus or genomic location (eg, those encoding proteins such as CXCR4, PD-1, etc.) is also provided. These tools also predict off-target effects to allow selection of genomic target sequences that minimize cleavage of genomic DNA at other locations.
Vectors and host cells

Construction of various types of vectors, including vectors for CRISPR/Cas9 systems, can be found in US Patent Application No. 2014/0273226, incorporated herein by reference.

Generally, polynucleotides, e.g., polynucleotides encoding Cas9, polynucleotides encoding gRNA sequences, polynucleotides encoding replacement nucleotide sequences, etc., can be incorporated into any desired DNA- or RNA-based vector without limitation. For example, expression vectors, subcloning vectors, shuttle vectors, vectors designed for use in in vitro transcription reactions, cosmids, phagemids, and retroviruses (e.g., lentiviruses), adenoviruses, adeno-associated viruses (AAV), and others. Polynucleotides can be cloned into vectors derived from mammalian viruses such as Epstein-Barr virus, as well as episomal EBNA-based vectors of Epstein-Barr virus origin.

In some embodiments, vectors may be in circular or linear form. Linear forms can be used in subcloning steps, such as cloning a gRNA target sequence into a host vector.

Those skilled in the art will appreciate that a wide variety of expression vectors for expressing the gene product are within the scope of embodiments of the present invention. An expression vector can be optimally designed to express a protein, eg, Cas9, variant Cas9, etc., in a host cell. For example, a vector containing a nucleotide sequence encoding a protein (eg, Cas9 open reading frame (ORF)) and appropriate regulatory elements can be delivered to host cells by any suitable method, such as transfection, transduction. Any type and amount of regulatory element involved in the regulation of transcription or translation may be incorporated into the expression vector and placed upstream or downstream of the ORF. Once inside the host cell, the host cell's own machinery, such as an endogenous RNA polymerase, can be used to synthesize mRNA, which is translated into protein.

In other embodiments, the expression vector is optimally designed to express a functional RNA molecule, eg, a guide RNA for tethering Cas9 to a genomic target sequence.

In some embodiments, Cas9 mRNA and gRNA are expressed from the same expression vector. In other embodiments, Cas9 mRNA and gRNA are expressed from different expression vectors. The Cas9 gene inserted into the expression vector can be, for example, a native bacterial Cas9 gene derived from Streptococcus pyogenes, Staphylococcus aureus, and the like. In some embodiments, the expression vector may be a mammalian expression vector, eg, a human expression vector, and may contain one or more promoter elements, eg, a bacteriophage promoter element such as the T7 promoter.

In further embodiments, the gene encoding Cas9 can be operably linked to one or more genes encoding nuclear localization signals, thereby targeting the expressed Cas9/gRNA to the nucleus of the host cell. be done.

In still other embodiments, the gene encoding Cas9 is optimized to reflect preferred codon usage for the organism in which gene editing is performed. For example, when gene editing is performed in phage, the gene encoding Cas9 within the expression vector construct is optimized to reflect preferred codon usage.

In some embodiments, phage can be isolated and pre-assembled gRNA/Cas9 complexes can be electroporated into host cells with optional nucleotide replacement sequences. A gRNA sequence can be designed to have homology to exon coding regions of one or more genes.

In other embodiments, the CRISPR/Cas9 system allows targeting of multiple loci (genomic targets) by cloning multiple gRNAs into a single vector.

The embodiments also provide compositions and methods for in vivo gene replacement, gene mutation and gene repair using the CRISPR/Cas9 systems described herein. This system is useful for manipulating cellular genomes in a highly specific manner. In some embodiments, engineered cell lines generated by the CRISPR/Cas9 system are useful in therapeutic transplantation to treat human disease. Human diseases resulting from bacterial infections can be treated with the compositions and methods of the invention, or with edited cell lines produced by the compositions and methods of the invention.

According to embodiments of the present invention, a method for specific genomic modification of a host cell or phage is provided, comprising (i) a first polynucleotide encoding a Cas9 protein or variant thereof and a gRNA encoding wherein the gRNA comprises a target sequence homologous to the genomic locus of interest; (ii) comprising the genomic locus of interest; providing a host cell, (iii) delivering the expression vector construct into the host cell, and (iv) expressing the first and second polynucleotides within the host cell. In some embodiments, the method may further comprise visualizing, identifying or selecting host cells with gene editing at the genomic target.

According to another embodiment of the present invention, a method for specific genomic modification of a host cell is provided, comprising (i) a first providing a first expression vector comprising a polynucleotide and a second expression vector comprising a second polynucleotide encoding a gRNA, wherein the gRNA comprises a target sequence homologous to a genomic target (ii) providing a host cell containing the genomic target, (iii) delivering the two expression vector constructs into the host cell (e.g., via transfection), and (iv) the host This includes expressing the first and second polynucleotides within the cell. In some embodiments, the method may comprise visualizing, identifying or selecting host cells that have gene editing at the genomic target. Expression can be modulated, eg, repressed or activated, by targeting regulatory regions, such as promoter regions, that control expression of the gene of interest.

According to yet another embodiment of the present invention, a method for specific genomic modification of a host cell is provided, the method comprising (i) providing a Cas9 protein complexed with a gRNA; , wherein the gRNA comprises a target sequence homologous to the genomic target, (ii) electroporating the Cas9 protein/gRNA complex into a host cell containing the genomic target, (iii) the genomic target (genomic Selecting a host cell based on the level of expression of the protein encoded by the gene locus). In some embodiments, the method comprises staining host cells for the presence of a cell surface protein encoded by a genomic target, and using flow cytometry to determine if the host expressing low levels of the cell surface protein It can further comprise selecting the cells.

In still other embodiments of the invention, more than one genomic target (locus of interest) is targeted for gene editing simultaneously. Thus, an expression vector may contain multiple nucleotide sequences, each nucleotide sequence encoding a different gRNA with a different targeting sequence, each targeting sequence corresponding to a genomic target. Alternatively, multiple expression vectors can be used, each expression vector containing one or more targeting sequences, each targeting sequence corresponding to a genomic target.

Those skilled in the art will appreciate that embodiments of the present invention are not limited to the polynucleotide or polypeptide sequences referred to herein, and also encompass variant polypeptide sequences and variant polynucleotide sequences. Variant polypeptide sequences include polypeptides with conservative amino acid substitutions in their amino acid sequence. Variant polynucleotide sequences include polynucleotides that have been modified to have alterations in their nucleotide sequences that, when expressed, have essentially the same function or activity as a polypeptide expressed by an unmodified nucleotide sequence. Polynucleotides that result in a polypeptide having

In some embodiments, the CRISPR-CAS9 system is used to edit phage. In some other embodiments, the Type IE CRISPR-Cas system is used to edit phage. In still other embodiments, a Type III CRISPR-CAS10 system is used to edit phage.

For example, protocols described in recent publications may be modified to delete toxic and/or unwanted phenotypes in phages of the families Myoviridae, Siphoviridae and Podoviridae (Bari et al., Synth Biol. 2017; 6(12):2316-2325; Box et al., Journal of Bacteriology Jan 2016, 198(3)578-590, Tao et al., ACS Synth Biol. .2017;7:42458). Bari et al., with and without the native CRISPR-Cas system, demonstrated that S . describes a type III-A CRISPR-Cas10 system for editing phages targeting S. aureus strains. This system provides a mechanism for selecting phage-derived sequences, such as those with point mutations at multiple loci, and for restoring recombination of phage that have acquired the desired mutations. When using RNA sequences as maps, Cas enzymes cleave targeted genes with deleterious and/or toxic characteristics. Another publication (Yosef et al., Proceedings of the National Academy of Sciences Jun 2015, 112(23) 7267-7272) describes lysogenic phage-based CRISPR-Cas delivery to the genome of antibiotic-resistant bacteria. ing.

In some embodiments, phages or phagemids are used to deliver DNA to bacterial cells.

In some embodiments, the CRISPR system can be used to eliminate specific bacterial strains by removing them from the microbiome or for specific treatments.

In a further preferred embodiment, the method involves the detection of unwanted and/or virulence genes, preferably bacterial virulence genes, such as bacterial endogenous genes, single nucleotide polymorphisms (SNPs), epichromosomal genes, antibiotics. Substance resistance genes, genes encoding one or more virulence factors, genes encoding one or more toxins, genes highly conserved between species, genera or phyla, and/or host physiology. CRISPR systems, such as the CRISPR/Cas9 system, are relied upon to edit genes that encode enzymes involved in biochemical pathways with products that can regulate .

In a preferred embodiment, the CRISPR system is as described in Akarsu et al. (2019) PLoS Comput Biol 15(4): e1006946, Xie et al., Nucleic Acids Res. 2018;46(D1):D749-D753, Harms et al., Mol Cell. 2018;70(5):768-784 and do Vale et al., 2016, and toxin loci involved in important biological functions including plasmid maintenance, phage protection, persistence and virulence Targets the antitoxin locus.

Examples of bacterial pathogens include Escherichia coli, Shigella dysenteriae, Yersinia pestis, Francisella tularensis, Bacillus anthracis, Staphylococcus aureus, Streptococcus pyogenes, Vibrio cholerae, Pseudom These include onas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii and Salmonella enterica Typhi. In an even more preferred embodiment, the bacterium is S. cerevisiae. aureus, S. epidermidis and/or E. fecalis.

Examples of toxins include pertussis toxin and adenylate cyclase toxin (ACT) secreted by Bordetella pertussis, anthrax toxin from Bacillus anthracis and leukotoxin of Staphylococcus aureus, AIP5 from Photobacterium damselaepiscicida (Phdp) 6, mycolactone, produced by Mycobacteriumulcerans polyketide molecules, other bacterial secretion products not officially called toxins, such as S. aureus superantigen-like protein (SSL) and phenol-soluble modulin (PSM), Clostridium C3 toxin, Shiga toxin, cholera toxin, hemolysin, leukocidin, fimbrial and afimbrial adhesins, proteases, lipases, endonucleases, Endotoxin and exotoxin cytotoxic agents, microcin and colicin. In some embodiments, the toxin is S . It is encoded by a superantigen enterotoxin gene such as the Sek gene of S. aureus. In some embodiments, the toxin is an exotoxin such as toxic shock syndrome toxin-1 (TSST-1); an enterotoxin such as SEA, SEB, SECn, SED, SEE, SEG, SEH and SEI, and ETA and ETB exfoliating toxins such as

Examples of genes that confer virulence traits to E. coli (e.g., O157:H7) include stx1 and stx2 (encoding Shiga-like toxins) and espA (induction of enterocyte effacement (LEE) A/E lesions). involved), but are not limited to: Other examples of genes that confer virulence traits to E. coli include fimA (pilus large subunit), csgD (curli regulator) and csgA. An example of a gene that confers virulence traits to Yersinia pestis is yscF (plasmid-carried (pCD1) T3SS external needle subunit). An example of a gene that confers a virulence trait to Francisella tularensis is fslA. An example of a gene that confers a virulence trait to Bacillus anthracis is pag (anthrax toxin, a cell-binding protective antigen). Examples of genes that confer virulence traits to Vibrio cholerae include, but are not limited to, ctxA and ctxB (cholera toxin), tcpA (toxin coregulatory pili) and toxT (master virulence regulator). Examples of genes that confer virulence traits to Pseudomonas aeruginosa include genes encoding the production of the siderophore pioverdin (e.g., sigma factor pvdS, biosynthetic genes pvdL, pvdl, pvdJ, pvdH, pvdA, pvdF, pvdQ, pvdN , pvdM, pvdO, pvdP, the transporter genes pvdL, pvdR, pvdT and opmQ), genes encoding the production of the siderophore piochelin (e.g. pchD, pchC, pchB, pchA, pchE, pchF and pchG), and toxins. Examples include, but are not limited to, genes that do (eg, exoU, exoS and exoT). Examples of genes that confer virulence traits to Klebsiella pneumoniae include, but are not limited to, fimA (adherent, type I fimbrial large subunit) and cps (capsular polysaccharide). Examples of genes that confer virulence traits to Acinetobacter baumannii include, but are not limited to, ptk (capsular polymerization) and epsA (assembly). Examples of genes that confer virulence traits to Salmonella enterica Typhi include hilA (regulator of entry, SPI-1), ssrB (regulator of SPI-2), and bile resistance, including the efflux pump genes acrA, acrB and tolC. Related genes include, but are not limited to.

Examples of antibiotic resistance genes or various methods for identifying them are known in the art. Antimicrobial susceptibility testing (WGS-AST) as described in Su et al., Journal of Clinical Microbiology Feb 2019, 57(3) e01405-18 and Ruppe et al., Nat Microbiol. Genomic methods to identify resistance phenotypes in strains, such as whole genome sequencing for antibiotic resistance determinants (ARDs) identified from the gut microbiota in 2019;4(1):112-123, are known in the art. explained in the field. In some embodiments, the resistance gene confers resistance to narrow-spectrum beta-lactam antibiotics of the penicillin class of antibiotics. In other embodiments, the resistance gene confers resistance to methicillin (eg, methicillin or oxacillin), or flucloxacillin, or dicloxacillin, or some or all of these antibiotics. In selected embodiments, the CRISPR system comprises methicillin-resistant S. cerevisiae. aureus (MRSA) and/or vancomycin-resistant S. aureus (MRSA). aureus (VRSA) selectively targeted. In certain embodiments, the resistance gene may confer resistance to linezolid, daptomycin, quinupristin/dalfopristin. In some embodiments, the resistance gene is fosfomycin resistance gene fosB, tetracycline resistance gene tetM, kanamycin nucleotidyl transferase aadD, bifunctional aminoglycoside modifying enzyme gene aacA-aphD, chloramphenicol acetyltransferase cat, mupirocin resistance gene ileS2 , vancomycin resistance genes vanX, vanR, vanH, vraE, vraD, methicillin resistance factors femA, fmtA, mecl, streptomycin adenylyltransferase spcl, spc2, antl, ant2, pectinomycin adenyltransferase spd, ant9, aadA2 and Selected from any other resistance gene.

In a preferred embodiment, the CRISPR system comprises a broad-spectrum beta-lactamase resistance factor (ESBL factor), CTX-M-15, beta-lactamase, New Delhi metallo-beta-lactamase (NDM)-1,2,5,6 and tetracycline Target one or more of the antibiotic resistance genes selected from A (tetA).

Examples of genes that confer resistance to aminoglycosides include, but are not limited to, aph, aac and aad variants and other genes encoding aminoglycoside modifying enzymes. Examples of genes containing SNPs that confer aminoglycoside resistance include, but are not limited to, rpsL, rrnA and rrnB. Examples of genes that confer beta-lactam resistance include genes encoding beta-lactamase (bla) (e.g., TEM, SHV, CTX-M, OXA, AmpC, IMP, VIM, KPC, NDM-1, beta- lactamase family) and mecA. Examples of genes containing SNPs that confer daptomycin resistance include, but are not limited to, mprF, yycFG, rpoB and rpoC. Examples of genes that confer macrolide-lincosamide-streptogramin B resistance include, but are not limited to, ermA, ermB and ermC. Examples of genes that confer quinolone resistance include, but are not limited to, qnrA, qnrS, qnrB, qnrC and qnrD. Examples of genes containing SNPs that confer quinolone resistance include, but are not limited to, gyrA and parC. Examples of genes containing SNPs that confer trimethoprim/sulfonamide resistance include, but are not limited to, the dihydrofolate reductase (DHFR) gene and the dihydropteroate synthase (DHPS) gene. Examples of genes that confer vancomycin resistance include, but are not limited to, the vanA (eg, vanRS and vanHAX), vanB and vanC operons.

In some embodiments, the CRISPR system can be used to target SNPs that cause overexpression of genes encoding multidrug efflux pumps, such as acrAB, mexAB, mexXY, mexCD, mefA, msrA and tetL. .

As examples of highly conserved genes (referred to herein as "remodeling" genes) that can typically represent a microbial species, genus or phylum for the purpose of reconstructing complex microbial communities include, but are not limited to, the ribosomal components rrnA, rpsL, rpsJ, rplO, rpsM, rplC, rpsH, rplP and rpsK, the transcription initiation factor infB, and the tRNA synthetase pheS.

Examples of genes encoding enzymes involved in biochemical pathways with products capable of modulating host physiology (referred to herein as "regulatory" genes) include: (1) (2) the enzyme involved in the production of polysaccharide A by Bacteroides fragilis, which leads to T regulatory cell (TREG) development, IL-10 response and increased TH1 cell numbers; (3) genes encoding enzymes involved in butyrate production leading to the secretion of inducible antimicrobial peptides; (4) short chains leading to increased energy recovery, obesity, modulation of inflammation and healing of gastrointestinal wounds. (5) genes encoding enzymes involved in the conversion of choline to methylamine, which can disrupt glucose homeostasis leading to non-alcoholic fatty liver disease and cardiovascular disease; (6) genes encoding enzymes involved in the production of neuromodulatory compounds such as y-aminobutyric acid, noradrenaline, 5-HT, dopamine and acetylcholine; and (7) involved in the formation of lactate and propionate associated with anxiety. Genes encoding enzymes include, but are not limited to.

In addition to the CRISPR/Cas9 system, other genome editing tools can be used to modify and/or eliminate toxic and/or undesirable characteristics. Examples of such phage genome editing tools include other CRISPR-based systems, engineered TALEN (transcription activator-like effector nuclease) variants, engineered zinc finger nuclease (ZFN) variants, and homologous recombination-based technique, bacteriophage recombineering (BRED) of electroporated DNA, a system described in Chen et al. Other systems include, but are not limited to, such as

As understood herein, terms such as "effective amount" and "therapeutically effective amount" of the pharmaceutical composition of the invention are used to, for example, eradicate and/or survive bacterial pathogens in a subject. by altering the virulence or antibiotic susceptibility of phage-resistant bacterial pathogens and/or providing additional benefits when the compositions are co-administered with effective and/or ineffective antibiotics. It refers to the amount of composition suitable to elicit a therapeutically beneficial response. Such responses can include, for example, preventing, ameliorating, treating, inhibiting and/or reducing one or more pathological symptoms associated with bacterial infections. Those skilled in the art will appreciate that the initial amount of the composition described herein should be sufficient to control the bacterial population before reaching the lethal threshold. Animal models suggest that 10 ⁹ -10 ¹¹ pfu/ml phage particles per dose would likely be the maximum tolerated dose, based on the protein load acutely presented to the liver in adults. (this would be scaled down in the pediatric population). It is speculated that this is an acute bolus sufficient to reduce the bacterial load sufficient to enhance the immune response. In particular, phage "viremia" can be measured in blood after administration. Given the host's immune response and sequestration in the reticuloendothelial system (liver and spleen), animal models suggest that viremia is fairly transient.

Appropriate effective amounts of the compositions of the present invention can be readily determined by those skilled in the art, and may depend on the age, weight, species (if non-human) and medical condition of the subject being treated. Furthermore, those skilled in the art will appreciate that the type of infection (e.g., systemic or localized) and the availability of treatment for that infection can also affect dosages deemed effective. . Those skilled in the art will appreciate that initial information can be gathered in laboratory experiments, and that effective amounts of the compositions described herein for humans can then be determined by dosing trials and routine experimentation. will understand

The compositions of the present invention can be administered systemically, parenterally (e.g., by intracisternal injection and intracisternal infusion techniques), intradermal, transmembrane, transdermal (including topical), intramuscular, intraperitoneal, intravenous It is still further contemplated that the subject may be administered by a variety of routes according to conventional methods, including, but not limited to, intraarterial, intralesional, subcutaneous, oral and intranasal (e.g., inhalation) routes of administration. . Administration can also be by continuous infusion or by bolus injection.

Additionally, the compositions of the present invention can be administered in various dosage forms. These include, for example, liquid preparations and suspensions, including preparations for parenteral, subcutaneous, intradermal, intramuscular, intraperitoneal or intravenous administration (eg, injectable administration). For example, sterile isotonic aqueous solutions, suspensions, emulsions or viscous compositions which can be buffered to a selected pH. In certain embodiments, the compositions of the invention are targeted as injectable compositions, including but not limited to injectable compositions for delivery by intramuscular, intravenous, subcutaneous or transdermal injection. It is contemplated herein to be administered to. Such compositions can be formulated using various pharmaceutical excipients, carriers or diluents well known to those skilled in the art.

In another specific embodiment, the compositions of the invention, and/or pharmaceutical agents administered therewith, such as antibiotics, may be administered orally. Oral formulations for administration according to the methods of the present invention include various dosage forms such as solutions, powders, suspensions, tablets, pills, capsules, caplets, sustained release formulations, or sustained release formulations. preparations with solid or liquid fills, such as liquids coated with gelatin, which dissolves in the stomach and is delivered to the intestine. Such formulations include various pharmaceutically acceptable excipients as described herein including, but not limited to, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose and magnesium carbonate. obtain.

It is contemplated herein that, in certain embodiments, compositions for oral administration may be in liquid formulations or may be present as part of a dissolvable paper that rests on the tongue. Such formulations have a pharmaceutically acceptable thickener that can produce high viscosity compositions that facilitate mucosal delivery of active agents, for example, by providing prolonged contact with the lining of the stomach. agent. Such viscous compositions can be made by those skilled in the art using conventional methods and using pharmaceutical excipients and reagents such as methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose and carbomer.

Other dosage forms suitable for nasal or respiratory (mucosal) administration are contemplated herein, for example dosage forms in the form of squeeze spray dispensers, pump dispensers or aerosol dispensers. Dosage forms suitable for rectal or vaginal delivery are also contemplated herein. Where appropriate, compositions for use with the methods of the invention may be lyophilized and delivered to the subject with or without rehydration using conventional methods.

Thus, as can be appreciated, the methods of the present invention can be administered according to various regimens, i.e., in amounts and in a manner sufficient, and for a sufficient period of time, to provide a clinically meaningful benefit to the subject. , including administering a composition of the invention to a subject. Dosing regimens suitable for use in the present invention can be determined by those skilled in the art according to conventional methods. For example, it is herein stated that an effective amount can be administered to a subject as a single dose, a series of repeated doses administered over several days, or a single dose followed by one or more additional "boosting" doses. is contemplated. The terms "dose" or "dosage" as used herein refer to physically discrete units suitable for administration to a subject, each dose calculated to provide the desired response. containing a predetermined amount of an active pharmaceutical ingredient.

The administrative regimen, such as amount administered, number of treatments, and effective amount per unit dose, etc., depends on the judgment of the practitioner and is peculiar to each subject. Factors to be considered in this regard include the physical and clinical condition of the subject, route of administration, intended treatment goal, and efficacy, stability and toxicity of the particular composition. As understood by those skilled in the art, a "boosting dose" can include the same dose or a different dose than the initial dose. Indeed, when a series of doses are administered to produce the desired response in a subject, those skilled in the art will understand that in that case an "effective amount" can encompass two or more doses.

In preferred embodiments, the compositions described herein can be administered both locally and systemically, for example, via IV, intra-articular, IM and/or direct injection at the site of infection. For example, for PJI, the compositions described herein can be injected directly into the infected joint. Accordingly, the compositions may be formulated as injectable fluid, semisolid, or depot-type preparations, as will be readily apparent to those skilled in the art. However, PJI can also be treated by IV injection and/or a combination of both direct injection to the site of infection and administration by IV, intra-articular and/or IM.

When an infected joint is also treated by debridement and/or replacement of a prosthetic joint device in one-stage or two-stage joint replacement surgery, the composition may be administered directly to the joint during surgery. For example, the composition may be administered once (i.e., a "single shot") or multiple times as may be required (the "single shot" may be administered to the site of infection. (including both direct injection or IV). However, it is believed that the composition could potentially be administered as a single shot, further avoiding the need to replace prosthetic joint devices in infected joints.

In some embodiments, the composition comprises an additional active agent or preparation, such as one or more antibiotics (eg, rifampin and/or fluoroquinolones, such as ciprofloxacin), one or more It may further include more antiseptic agents and/or one or more other therapeutic molecules, eg, small molecules or biologics with antiseptic activity. These additional agents can be administered as part of the composition, or can be administered simultaneously with, but separately from, the phage composition.

In a further aspect, the invention provides a method of treating a patient having or at risk of developing a prosthetic joint infection (PJI), comprising administering a pharmaceutical composition according to the first aspect. including administering.

In some embodiments, the patient is S. aureus, S. epidermidis, E. fecalis, S. caprae and/or S. have PJI caused by a bacterium selected from S. lugdunensis.

In other embodiments, the patient is S. aureus, S. epidermidis, E. fecalis, S. caprae and/or S. lugdunensis determined to be caused by a bacterium selected from PJI. Said determination can be made, for example, by culturing synovial fluid obtained by aspiration from the affected joint and determining the identity of the bacteria in the culture, for example, by 16S rRNA gene sequence analysis.

In other embodiments, the patient is S. aureus, S. epidermidis, E. fecalis, S. caprae and/or S. There is a risk of developing PJI caused by bacteria selected from S. lugdunensis.

The methods of the invention may be useful in treating or preventing PJI of any type. Thus, by appropriately selecting two or more phages for inclusion in the present compositions, the present methods are beneficial in treating early, late and late onset forms of PJI, as well as PJI that is the result of polymicrobial infections. can be

The method comprises lysis (i.e., , death) and/or in an amount sufficient to prevent the development of a bacterial infection. An effective amount can be administered in one or more administrations. Typically, the effective amount is sufficient to treat the disease or condition or otherwise attenuate, reverse, stabilize, reverse, slow the progression or onset of PJI, Sufficient to delay or prevent.

Infections to be treated include not only bacterial infections that are already present in the patient, but also prevention of future infections that may or may not occur in patients with implantable devices, including but not limited to: Not limited. For example, a bacterial infection (e.g., a geographically matched composition) of a particular strain of bacteria known to occur frequently in a particular geographic location may be used prophylactically to prevent future bacterial infections. The modalities can be treated with the compositions of the present disclosure.

In further detail, the method may comprise administering the composition directly to an infected joint or to an artificial joint at risk of developing PJI. In the latter case, the method can be performed intraoperatively (ie, the composition can be administered at the time the joint replacement is being performed). When the method is used to treat infected joints, the method may avoid the need for replacement of prosthetic joint devices. That is, the method can be performed in lieu of surgery. In other cases where infected joints are also treated by debridement and/or replacement of prosthetic joint devices in one- or two-stage joint replacement surgery, the method can be conveniently performed intraoperatively (i.e. The composition may be administered to the joint during surgery). The composition may be administered once (ie, a "single shot") or multiple times as may be required.

In some embodiments, the methods of the invention may be practiced as combination therapy. For example, a composition comprising at least two different phages may be added to an additional active agent or preparation, such as one or more antibiotics (eg, rifampin and/or fluoroquinolones, such as ciprofloxacin),1 or more microbicides, and/or as a combination therapy comprising administration of one or more other therapeutic molecules, eg, small molecules or biologics with microbicide activity.

When administered as a combination therapy with an additional active agent or preparation, can the composition comprise at least two different phages with that additional active agent or preparation (i.e., a single composition as), or otherwise a separate composition may be used. When administered in separate compositions, the therapeutic phage composition and the additional active agent or preparation may be administered simultaneously or sequentially in any order (eg, within seconds or minutes (eg, 5 to 10 minutes)). within 60 minutes) or within hours (eg, within 2-48 hours).

Although the invention has been described herein with reference to embodiments, it is understood that these embodiments and the examples provided herein are merely illustrative of the principles and applications of the invention. should. Thus, numerous modifications may be made to the illustrative embodiments and examples, and other arrangements may be devised without departing from the spirit and scope of the invention as defined by the appended claims. should be understood. All patent applications, patents, publications and references cited herein are hereby incorporated by reference in their entirety.

The invention will now be further illustrated with reference to the following examples. It will be understood that the following is exemplary only and that modifications may be made in detail while remaining within the scope of the invention.
Example 1: Selection of phages for inclusion in PJI compositions

S. aureus (MRSA and/or MSSA), S. epidermidis, E. fecalis, S. caprae and/or S. A composition is designed to treat PJI that can be caused by one or more of S. lugdunensis. Candidate phage for inclusion in the composition include phage publicly available from the Felix d'Herelle Reference Center for Bacterial Viruses of the University Laval (Quebec City, Quebec, Canada; www.phage.ulaval.ca) and table 1 (above) from the proprietary phages listed.
Formulation of injectable compositions

by combining about 10 ¹¹ pfu of each of the following phages APT-PJI-01, APT-PJI-13 and APT-PJI-15 in a pharmaceutically acceptable diluent (e.g. isotonic saline) In one particular example, an injectable fluid composition is prepared. The composition may be provided as a pre-filled syringe.
Example 2: Selection of phages for inclusion in PJI compositions

実施例３：机上のケーススタディ The composition of Example 1 is effective against other pathogenic bacteria (e.g., S. lugdunensis, E. fecium, S. agalactiae, P. aeruginosa, K. pneumoniae, E. coli and E. coli) of implantable device infections (e.g., PJI). One or more phage targeted for lysis of S. clocae) can be readily modified by adding to the composition or replacing one or more phage in the composition. Examples of some candidate phage for inclusion in such compositions are available from the Felix d'Herelle Reference Center for Bacterial Viruses of the University Laval (Quebec City, Quebec, Canada; www.phage.ulaval.ca). and are listed in Table 2 below.

Example 3: Desktop Case Study

Two months after undergoing a ceramic-on-polyethylene hip replacement, a 55-year-old male patient reported to the orthopedic surgeon that the joint was in considerable pain. The patient is referred for a CT scan of the affected joint, which confirms joint swelling and suggests periprosthetic tissue infection. Blood tests also provide additional evidence of PJI. A synovial fluid sample is then taken from the hip joint by joint aspiration (athrocentesis) and the bacteria present in the synovial fluid are cultured in liquid medium. The 16S rRNA sequence database is then used to identify the bacterial species present by subjecting the extracted bacterial DNA to sequence analysis of the V1-V3 regions of the 16S rRNA gene. The patient's PJI was S. aureus and E. fecalis is the cause.

The patient was given a single dose containing approximately 10 ⁹ pfu of each of the APT-PJI-01 (targeting S. aureus) and APT-PJI-15 (targeting E. fecalis) phages in pharmaceutical saline. A single dose of therapeutic phage composition is administered by direct injection into the affected joint. The patient is then treated with intravenous (IV) phage (APT-PJI-01 and/or APT-PJ-15) for 10 days. After treatment, patients are closely monitored for clinical response. After 4 weeks, patients report little or no pain remaining. The joint may be aspirated to determine if an infection remains. Additional CT scans may be performed, if necessary, to determine that the joint is no longer infected.

The invention is not limited to the embodiments herein described above, which may vary in arrangement and detail without departing from the spirit of the invention. The entire teachings of any patent, patent application or other publication referenced herein are hereby incorporated by reference as if fully set forth herein.

Claims (25)

A method of treating an infection caused by one or more bacteria associated with a device implanted in a subject, said method comprising:
(a) identifying at least one identified phage capable of infecting said bacterium, said identified phage being APT-PJI-01, APT-PJI-02, APT-PJI-03, APT -PJI-04, APT-PJI-05, APT-PJI-06, APT-PJI-07, APT-PJI-08, APT-PJI-09, APT-PJI-10, APT-PJI-11, APT-PJI -12, APT-PJI-13, APT-PJI-14, APT-PJI-15, APT-PJI-16, APT-PJI-17, APT-PJI-18, APT-PJI-20, APT-PJI-21 , APT-PJI-22, APT-PJI-23, APT-PJI-24, APT-PJI-25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT -PJI-30, APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI -38, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45, APT-PJI-46 , APT-PJI-47, APT-PJI-48, APT-PJI-49 and/or APT-PJI-50, or a genome having at least 70% sequence identity to the genomic sequence of any of the aforementioned phages selected from a library comprising at least one phage selected from any other lytic phage having a sequence and capable of causing lysis of said bacterium; and (b) comprising said identified phage A method comprising administering to said subject a therapeutically effective amount of a composition, said composition being effective in treating and/or reducing said infection. A method of identifying a subject having or at risk of having a bacterial infection, said method comprising:
(a) obtaining a biological sample from said subject;
(b) culturing the bacteria present in said biological sample;
(c) APT-PJI-01, APT-PJI-02, APT-PJI-03, APT-PJI-04, APT-PJI-05, APT-PJI-06, APT-PJI- 07, APT-PJI-08, APT-PJI-09, APT-PJI-10, APT-PJI-11, APT-PJI-12, APT-PJI-13, APT-PJI-14, APT-PJI-15, APT-PJI-16, APT-PJI-17, APT-PJI-18, APT-PJI-20, APT-PJI-21, APT-PJI-22, APT-PJI-23, APT-PJI-24, APT- PJI-25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, APT-PJI-31, APT-PJI-32, APT-PJI- 33, APT-PJI-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45, APT-PJI-46, APT-PJI-47, APT-PJI-48, APT-PJI-49 and/or APT-PJI-50 as well as any other lytic phage having a genomic sequence with at least 70% sequence identity to the genomic sequence of any of the aforementioned phages and capable of causing lysis of said bacteria. and (d) said cultured bacteria are lysed by said identified phage; wherein, when any of the cultured bacteria are lysed by the phage, the subject is (1) eligible for treatment with a bacteriophage for the bacterial infection, ( 2) having a bacterial infection; and/or (3) being at risk of having a bacterial infection. The composition is APT-PJI-01, APT-PJI-02, APT-PJI-03, APT-PJI-04, APT-PJI-05, APT-PJI-06, APT-PJI-07, APT-PJI -08, APT-PJI-09, APT-PJI-10, APT-PJI-11, APT-PJI-12, APT-PJI-13, APT-PJI-14, APT-PJI-15, APT-PJI-16 , APT-PJI-17, APT-PJI-18, APT-PJI-20, APT-PJI-21, APT-PJI-22, APT-PJI-23, APT-PJI-24, APT-PJI-25, APT -PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI -34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT-PJI-42 , APT-PJI-43, APT-PJI-44, APT-PJI-45, APT-PJI-46, APT-PJI-47, APT-PJI-48, APT-PJI-49 and/or APT-PJI-50 or from the group consisting of any other lytic phage having a genomic sequence having at least 70% sequence identity to the genomic sequence of any of the aforementioned phages and capable of causing lysis of said bacteria 3. The method of claim 1 or 2, comprising at least one identified phage selected. The composition is APT-PJI-01, APT-PJI-02, APT-PJI-03, APT-PJI-04, APT-PJI-05, APT-PJI-06, APT-PJI-07, APT-PJI -08, APT-PJI-09, APT-PJI-10, APT-PJI-11, APT-PJI-12, APT-PJI-13, APT-PJI-14, APT-PJI-15, APT-PJI-16 , APT-PJI-17, APT-PJI-18, APT-PJI-20, APT-PJI-21, APT-PJI-22, APT-PJI-23, APT-PJI-24, APT-PJI-25, APT -PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI -34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT-PJI-42 , APT-PJI-43, APT-PJI-44, APT-PJI-45, APT-PJI-46, APT-PJI-47, APT-PJI-48, APT-PJI-49 and/or APT-PJI-50 or from the group consisting of any other lytic phage having a genomic sequence having at least 70% sequence identity to the genomic sequence of any of the aforementioned phages and capable of causing lysis of said bacteria 4. A method according to any preceding claim comprising at least two identified phages selected. wherein the at least two identified phage are
(a) said identified phage causes lysis in the same bacterial strain;
(b) said identified phage causes lysis in different bacterial strains;
(c) at least one of said identified phage is APT-PJI-01, APT-PJI-02, APT-PJI-03, APT-PJI-04, APT-PJI-05, APT-PJI-06, APT- PJI-07, APT-PJI-08, APT-PJI-09, APT-PJI-10, APT-PJI-11, APT-PJI-12, APT-PJI-22, APT-PJI-23, APT-PJI- 24, APT-PJI-25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI-34, APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT-PJI-40, APT- at least 70% relative to the genomic sequence of PJI-41, APT-PJI-42, APT-PJI-43, APT-PJI-44, APT-PJI-45 and APT-PJI-46 and any of the aforementioned phages selected from the group consisting of any other lytic phage having a genomic sequence with sequence identity;
(d) at least one of said identified phage has a genomic sequence having at least 70% sequence identity to the genomic sequence of APT-PJI-13, APT-PJI-14 and any of said phage; selected from the group consisting of any lytic phage of
(e) at least one of said identified phage is at least 70 relative to the genomic sequence of APT-PJI-15, APT-PJI-16, APT-PJI-17 and APT-PJI-18 and any of said phages; selected from any other lytic phage having a genomic sequence with % sequence identity;
(f) at least one of said identified phage has a genomic sequence having at least 70% sequence identity to the genomic sequence of APT-PJI-20 and APT-PJI-21 and any of said phage; selected from the group consisting of any lytic phage of
(g) at least one of said identified phage is APT-PT-PJI-22, APT-PJI-23, APT-PJI-24, APT-PJI-25, APT-PJI-26, APT-PJI-27; APT-PJI-28, APT-PJI-29, APT-PJI-30, APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI-34, APT-PJI-35, APT- PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT-PJI- 44, APT-PJI-45 or APT-PJI-46 and any other lytic phage having a genomic sequence with at least 70% sequence identity to the genomic sequence of any of the aforementioned phages selected;
(h) at least one of said identified phage is at least 70 relative to the genomic sequence of APT-PJI-47, APT-PJI-48, APT-PJI-49 or APT-PJI-50 and any of said phages; selected from the group consisting of any other lytic phage having a genomic sequence with % sequence identity,
5. The method of claim 4, characterized in that (i) at least one of said identified phage is selected from any of the combinations of (a)-(h). Said composition provides a dose of each phage within the range of 10 ⁵ -10 ¹³ pfu, preferably said dose of ^each phage is 10 ^{6 -10 12 pfu ;} 10 ¹¹ pfu; 10 ⁹ to 10 ¹¹ pfu; within the range of 10 ⁹ to 10 ¹⁰ pfu;
or a dose of approximately 10 ⁶ pfu, 10 ⁷ pfu, 10 ⁸ pfu, 10 ⁹ pfu, 10 ¹⁰ pfu, 10 ¹¹ pfu, 10 ¹² pfu or 10 ¹³ pfu,
A method according to any one of the preceding claims. The bacterium is S. cerevisiae. aureus, S. epidermidis, E. Enterococcus spp. including Enterococcus fecalis and Enterococcus fecium. , S. simulans, S. caprae and S. Other coagulase-negative Staphylococcus species, including Lancefield A, B, C and G groups, including S. lugdunensis. agalactiae and S. Streptococcus spp., including Streptococcus pneumoniae. , aerobic Gram-negative bacteria including E. coli, other Enterobacteriaceae including Enterobacter clocae, Clostridium spp. , Actinomyces spp. , Peptostreptococcus spp. , Cutibacterium acnes, Klebsiella pneumoniae, P. 10. A method according to any preceding claim selected from at least one of Bacteroides fragilis and Bacteroides fragilis. The bacterium is S. cerevisiae. aureus, S. epidermidis, E. Enterococcus spp. including Enterococcus fecalis and Enterococcus fecium. , S. simulans, S. caprae and S. Other coagulase-negative Staphylococcus species, including Lancefield A, B, C and G groups, including S. lugdunensis. agalactiae and S. Streptococcus spp., including Streptococcus pneumoniae. , aerobic Gram-negative bacteria including E. coli, other Enterobacteriaceae including Enterobacter clocae, Clostridium spp. , Actinomyces spp. , Peptostreptococcus spp. , Cutibacterium acnes, Klebsiella pneumoniae, P. The method of any preceding claim selected from two or more different bacterial strains selected from aeruginosa and Bacteroides fragilis. The bacterium is S. cerevisiae. aureus, S. epidermidis, E. fecalis, S. caprae and/or S. lugdunensis, according to any one of the preceding claims. Said composition is obtained from S.I. aureus, S. epidermidis, E. fecalis, S. caprae and/or S. lugdunensis, comprising at least two phage with different lytic specificities capable of infecting at least two of the preceding claims. Said composition comprises at least three phages with different lytic specificities, each phage being associated with S. cerevisiae. aureus, S. epidermidis, E. fecalis, S. caprae and/or S. lugdunensis, according to the preceding claim, capable of being infected with at least one of S. lugdunensis. said other lytic phage is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% relative to the genomic sequence of any of said phages , 97%, 98% or 99% sequence identity. the composition comprising:
(a) an identified phage matched to a bacterial strain known to exist in a geographic location;
(b) APT-PJI-20, APT-PJI-21, APT-PJI-22, APT-PJI-23, APT-PJI-24, APT-PJI-25, APT-PJI-26, APT-PJI-27 , APT-PJI-28, APT-PJI-29, APT-PJI-30, APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI-34, APT-PJI-35, APT -PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT-PJI -44, APT-PJI-45, APT-PJI-46, APT-PJI-47, APT-PJI-48, APT-PJI-49 and/or APT-PJI-50. A method according to any one of the preceding claims. the device is
(a) permanently implanted in said subject;
(b) is temporarily implanted in said subject;
(c) is removable; and/or (d) an artificial joint, left ventricular assist device (LVAD), stent, metal rod, indwelling catheter, spinal device and/or spinal device, and/or bone device and/or bone selected from fixtures,
A method according to any one of the preceding claims. said infection is selected from prosthetic joint infection (PJI), chronic bacterial infection, acute bacterial infection, refractory infection, biofilm associated infection, implantable device associated infection, A method according to any one of the preceding claims. the composition comprising:
(a) by IV injection;
(b) by direct injection into the site of infection;
(c) by intra-articular injection;
(d) by IM injection;
(e) prophylactically;
(f) prior to surgery;
(g) instead of surgery;
(h) during surgery;
(i) at one time (i.e., as a "single shot") and/or (j) as a course of treatment for two weeks or more,
10. The method of any one of the preceding claims, administered. Dissolution
(a) growth inhibition;
(b) optical density;
(c) metabolic output;
4. A method according to any one of the preceding claims, measured by (d) photometric (eg fluorescence, absorption and transmission assays); and/or (e) changes in plaque formation. The method of any preceding claim, wherein the photometric change is measured using an additive that causes and/or enhances detection of a photometric signal, preferably the additive is a tetrazolium dye. wherein the biological sample is
(a) synovial fluid;
(b) the area surrounding the implantable device;
(c) infection site;
(d) intraoperative samples;
(e) swabbing the device;
(f) biofilms;
4. A method according to any one of the preceding claims, obtained from (g) a fistula; and/or (h) an aspirate of an infected site. the bacterial infection is
(a) is multi-drug resistant;
(b) clinically refractory to antimicrobial treatment;
(c) clinically refractory to antimicrobial treatment due to biofilm formation; and/or (d) due to the subject's inability to tolerate antimicrobials due to adverse reactions. clinically refractory,
A method according to any one of the preceding claims. 4. The method of any one of the preceding claims, wherein the subject is suffering from a device-associated infection. 4. The method of any of the preceding claims, wherein the subject is suffering from prosthetic joint infection (PJI). 4. The method of any one of the preceding claims, wherein the library comprises phage prescreened to exclude phage containing undesirable and/or virulence characteristics. said excluded phage containing undesirable and/or virulence characteristics are toxin genes or other bacterial virulence factors, phage with lysogenic properties and/or carrying lysogenic genes, bacterial virulence factors phages that carry genes or antibiotic resistance genes, phages that carry any antibiotic resistance gene or that can confer antibiotic resistance to bacterial strains, and elicit inappropriate immune responses and/or are strong in mammalian systems; A method according to any preceding claim, selected from phages that elicit an allergic response. 25. The method of claim 23 or 24, wherein said unwanted and/or toxic features are eliminated by genome editing.