Note: Descriptions are shown in the official language in which they were submitted.
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METHOD FOR TREATING IMPLANTABLE DEVICE INFECTIONS
BACKGROUND OF THE INVENTION
Field of the Invention
[001] The invention relates to the field of phage therapy and is
particularly
directed at providing a phage-based composition and method for treating or
preventing acute or chronic bacterial infections associated with an
implantable device
using phage-based compositions.
Discussion of the Related Art
[002] In the following discussion, certain articles and methods will be
described
for background and introductory purposes. Nothing contained herein is to be
construed
as an "admission" of prior art. The Applicant expressly reserves the right to
demonstrate,
where appropriate, that the articles and methods referenced herein do not
constitute prior
art under the applicable statutory provisions.
[003] Partial or total joint replacement surgery, most commonly of knees
and hips,
is a common, life-enhancing procedure undertaken by millions of patients each
year. In
most cases, the procedure is highly successful, bringing pain relief and
restoration of
function and independence to patients. In a relatively small number of cases,
complications can be experienced such as prosthetic device failure (e.g.
caused by wear,
device fracture or malpositioning), or the appearance of prosthetic joint
infection (PJI),
also known as periprosthetic joint infection, which is an infection involving
the joint
prosthetic device and adjacent tissues. Studies have determined that the
incidence of PJI
in the United States is in the order of 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, while this number is relatively low, PJI represents a very serious
risk to the
patient bringing with it considerable morbidity involving pain and swelling,
and in some
cases, can necessitate amputation and/or lead to patient death. The diagnosis,
treatment
and management of PJI is also a very significant cost burden on the health
care system,
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and has been predicted to be greater than USD1.5 billion in the United States
alone in
2020; Kurtz SM et al., J. Arthroplasty 27:61-65.e61, 2012).
[004] Generally, doctors have categorized PJI into three major kinds of
PJI; each
distinguished by the period of time, after surgery, that the infection
appears. Thus, PJI
includes infections that: (i) occur early, within 3 months of surgery ("early
onset" PJI);
(ii) are delayed, appearing after 3 months but before 12 or 24 months of the
surgery
("delayed onset" PJI); and (iii) infections that occur later than 12 to 24
months following
the surgery ("late onset" PJI). Others have categorized PJI infections as
"acute"
(infections occurring within 1 month after surgery or "chronic" which occur
after 1 month
from surgery. Regardless of which classification system is used, early PJI
infections are
mostly caused by relatively virulent microorganisms such as Staphylococcus
aureus
and/or aerobic gram-negative bacteria such as Pseudomonas aeruginosa, whereas
the
delayed PJI types are commonly due to less virulent bacteria such as coagulase-
negative
S. aureus (CoNS), Staphylococcus epidermidis and/or Enterococci spp. Both the
early
and delayed kinds are believed to be acquired during the surgery, whereas late
PJI
infections may frequently eventuate as a secondary infection from another site
or sites of
bacterial infection (e.g. arising from hematogenous spread of bacteria from
other
infectious foci through the blood). S. aureus appears to be the common
causative
microorganism for late onset PJI arising from hematogenous spread (Tande &
Patel,
supra).
[005] The successful treatment of PJI typically requires surgical
intervention
and/or medical therapy (e.g. with antimicrobial agent(s) such as antibiotics)
in the
majority of cases. Some treatments involve a debridement (i.e. surgical
removal of
infected/damaged tissue), antibiotic treatment, and implant (prosthetic
device)
retention, and are referred to as a DAIR procedure, while in other cases, the
treatment
requires the replacement of the prosthetic device in a one- or two-stage
arthroplasty
exchange (Tande & Patel, supra) in combination with treatment with an
antimicrobial
agent(s). Two-stage arthroplasty exchange procedures, are typically regarded
to be the
"most definitive strategy in terms of infection eradication and preservation
of joint
function" with success rates reported as high as 87-100% for hip replacements
and 72-
95% for knee replacements (Tande & Patel, supra). However, these two-stage
procedures
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can be complex and costly, requiring at least two surgeries, sometimes
separated by a
period of 2-3 months or more, and extensive periods of treatment with an
antimicrobial
agent(s). This means that some patients are not suitable for a two-stage
arthroplasty
exchange (or even a one-stage procedure) or are unwilling to undertake further
surgery,
in which case there is little option other than to attempt to treat the PJI
with an
antimicrobial agent(s). However, this is not recommended and commonly leads to
the
patient being placed on prolonged or indefinite treatment with oral
antimicrobial agent(s)
and careful ongoing therapeutic monitoring (Tande Sr Patel, supra).
[006] An added complication to the effective treatment of PJI with
antimicrobial
agent(s) is that the infection can be "polymicrobial", meaning that there is
more than one
causative microorganism present in the infection. This is particularly the
case with early
onset PJI, where it has been estimated that up to 35% of such infections are
polymicrobial
commonly involving infection by S. aureus, Enterococci spp. and aerobic gram-
negative
bacteria such as P. aeruginosa (Berbari EF et al., Clin. Infect. Dis. 27:1247-
1254, 1998;
Peel TN et al., Antimicrob. Agents Chemotherap. 56:2386-2391, 2012).
Consequently, a
careful selection of antimicrobial agent(s) is needed if the treatment is to
be successful.
[007] While the efficacy and options for treatments of PJI has
significantly
improved in recent years, there is a great ongoing need to identify and
develop new or
improved treatments and/or management strategies for effectively treating
and/or
preventing these infections in joint replacement patients, as well as other
infections
associated with implantable devices.
SUMMARY OF THE INVENTION
[008] 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 be apparent
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from the following written Detailed Description including those aspects
illustrated in any
accompanying drawings and defined in the appended claims.
[009] Specifically, the disclosed invention describes a method
of treating an
infection caused by one or more bacterium associated with a device implanted
within a
subject, wherein said method comprises: (a) identifying at least one
identified phage
capable of infecting the bacterium, wherein said identified phage is selected
from a library
comprising at least one phage selected from APT-PJI-01, APT-PM-02, APT-PJI-03,
APT-
PM-04, APT-PM-05, APT-PJI-06, APT-PM-07, APT-PJI-08, APT-PM-09, APT-PM-io,
APT-PJI-12, APT-PM-13, APT-PM-14, APT-PM-15, APT-PM-16, APT-PJI-17,
APT-PM-18, APT-PJI-20, APT-PM-21, APT-PJI-22, APT-PJI-23, APT-PM-24, APT-PJI-
25, APT-PM-26, APT-PM-27, APT-PM-28, APT-PM-29, APT-PJI-30, APT-PJI-31, APT-
PJI-32, APT-PJI-33, APT-PM-34, APT-PJI-35, APT-PJI-36, APT-PM-37, APT-PM-38,
APT-PM-39, APT-PM-4o, APT-PM-41, APT-PM-42, APT-PM-43, APT-PM-44, APT-PM-
45, APT-PM-46, APT-PM-47, APT-PM-48, APT-PM-49, and/or APT-PJI-50, or any
other
lytic phage that has a genomic sequence with at least 70% sequence identity to
the
genomic sequence of any of the foregoing phage and which is capable of causing
lysis of
the bacterium; and (b) administering to said subject a therapeutically
effective amount of
a composition comprising said identified phage, wherein said composition is
effective in
treating and/or reducing said infection.
[ow] In other embodiments, the disclosed invention describes a
method of
identifying a subject suffering, at risk of suffering, or eligible for
receiving treatment for a
bacterial infection, comprising the steps of: (a) obtaining a biological
sample from the
subject; (b) culturing a bacterium present in the biological sample; (c)
inoculating the
cultured bacterium with an identified phage selected from a library comprising
at least
one phage selected from one or more of APT-PJI-oi, APT-PM-02, APT-PM-03, APT-
PJI-
04, APT-PM-05, APT-PM-06, APT-PM-07, APT-PJI-o8, APT-PM-09, APT-PM-io, APT-
PJI-ii, APT-PJI-12, APT-PJI-13, APT-PM-14, APT-PM-15, APT-PJI-16, APT-PJI-17,
APT-PM-18, APT-PJI-2o, APT-PM-21, APT-PJI-22, APT-PJI-23, APT-PM-24, APT-PJI-
25, APT-PM-26, APT-PM-27, APT-PM-28, APT-PM-29, APT-PJI-30, APT-PJI-31, APT-
PJI-32, APT-PJI-33, APT-PM-34, APT-PJI-35, APT-PJI-36, APT-PM-37, APT-PM-38,
APT-PM-39, APT-PM-4o, APT-PM-41, APT-PM-42, APT-PM-43, APT-PM-44, APT-PJI-
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45, APT-PM-46, APT-PM-47, APT-PM-48, APT-PM-49, and/or APT-PJI-5o, and any
other lytic phage that has a genomic sequence with at least 70% sequence
identity to the
genomic sequence of any of the foregoing phage and which is capable of causing
lysis of
the bacterium; and (d) determining whether the cultured bacterium are lysed by
the
identified phage, wherein when any of the cultured bacterium are lysed by the
phage, the
subject is determined (1) to be eligible for treatment by bacteriophage for
said bacterial
infection (2) to be suffering from a bacterial infection; and/or (3) at risk
of suffering from
a bacterial infection.
[on] In further described embodiments, the composition used in
either method
comprises at least one identified phage selected from the group consisting of
APT-PJI-oi,
APT-PM-02, APT-PJI-03, APT-PM-04, APT-PM-05, APT-PJI-o6, APT-PM-07, APT-PJI-
o8, APT-PM-09, APT-PM-io, APT-PJI-11, APT-PJI-12, APT-PJI-13, APT-PM-14, APT-
PJI-15, APT-PM-16, APT-PM-17, APT-PM-18, APT-PM-20, APT-PJI-21, APT-PJI-22,
APT-PJI-23, APT-PM-24, APT-PJI-25, APT-PJI-26, APT-PJI-27, APT-PJI-28, APT-PJI-
29, APT-PJI-30, APT-PM-31, APT-PJI-32, APT-PJI-33, APT-PM-34, APT-PJI-35, APT-
PM-36, APT-PM-37, APT-PJI-38, APT-PM-39, APT-PJI-40, APT-PM-41, APT-PM-42,
APT-PM-43, APT-PM-44, APT-PM-45, APT-PM-46, APT-PM-47, APT-PM-48, APT-PJI-
49, and/or APT-PM-50, or any other lytic phage that has a genomic sequence
with at least
70% sequence identity to the genomic sequence of any of the foregoing phage
and which
is capable of causing lysis of the bacterium.
[01.2] Other embodiments are further described below.
DETAILED DESCRIPTION
[013] The following definitions are provided for specific terms which are
used in
the following written description.
Definitions
[014] As used in the specification and claims, the singular form "a", "an"
and "the"
include plural references unless the context clearly dictates otherwise. Also,
as
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understood by one of ordinary skill in the art, the term "phage" can be used
to refer to a
single phage or more than one phage.
[015] The present invention can "comprise" (open ended) or "consist
essentially
of' the components of the present invention. As used herein, "comprising"
means the
elements recited, or their equivalent in structure or function, plus any other
element or
elements which are not recited. The terms "having" and "including" are also to
be
construed as open ended unless the context suggests otherwise.
[016] The term "about" or "approximately" means within an acceptable range
for
the particular value as determined by one of ordinary skill in the art, which
will depend
in part on how the value is measured or determined, e.g., the limitations of
the
measurement system. For example, "about" can mean a range of up to 20%,
preferably
up to io%, more preferably up to 5%, and more preferably still up to 1% of a
given value.
Alternatively, particularly with respect to biological systems or processes,
the term can
mean within an order of magnitude, preferably within 5 fold, and more
preferably within
2 fold, of a value. Unless otherwise stated, the term "about" means within an
acceptable
error range for the particular value, such as 1-20%, preferably i-io% and
more
preferably 1-5%. In even further embodiments, "about" should be understood to
mean-q-5%.
[017] Where a range of values is provided, it is understood that each
intervening
value, between the upper and lower limit of that range and any other stated or
intervening
value in that stated range is encompassed within the invention. The upper and
lower
limits of these smaller ranges may independently be included in the smaller
ranges, and
are also 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 either both of those included limits are also included in the
invention.
[o18] All ranges recited herein include the endpoints, including
those that recite a
range "between" two values.
[01.9] Terms such as "about," "generally," "substantially,"
"approximately" and the
like are to be construed as modifying a term or value such that it is not an
absolute, but
does not read on the prior art. Such terms will be defined by the
circumstances and the
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terms that they modify as those terms are understood by one of ordinary skill
in the art.
This includes, at very least, the degree of expected experimental error,
technique error
and instrument error for a given technique used to measure a value.
[020] Where used herein, the term "and/or" when used in a list of two or
more
items means that any one of the listed characteristics can be present, or any
combination
of two or more of the listed characteristics can be present. For example, if a
composition
is described as containing characteristics A, B, and/or C, the composition can
contain A
feature alone; B alone; C alone; A and B in combination; A and C in
combination; B and
C in combination; or A, B, and C in combination.
[021] The term "bacteriophage" or "phage", as understood by one of ordinary
skill
in the art, refers to a non-cellular infective agent that reproduces only in a
suitable host
cell, that is, a bacterial host cell.
[022] The term "phage therapy" refers to any therapy to treat a bacterial
infection
or a disease or condition that is caused by bacteria (e.g. PJI) or which shows
symptoms
or disease/condition development or progress associated with the presence of
bacteria.
Phage therapy may involve the administration to a patient requiring treatment
of one or
more therapeutic phage composition that can be used to infect, kill or inhibit
growth of a
bacterium, which comprises one or more viable phage as an antibacterial agent
(e.g. a
composition comprising one phage type or two or more phage type in a phage
"cocktail").
The one or more phage types may be obtained from stocks of the phage, which
may be
held in storage in an inventory. AAThere a phage therapy involves the
administration of
more than one therapeutic phage composition, then the compositions may have a
different host range (e.g. one may have a broad host range and one may have a
narrow
host range, and/or one or more of the compositions may act synergistically
with one
another). Further, as will be readily understood by one of ordinary skill in
the art, the
therapeutic phage composition(s) used in a phage therapy will also typically
comprise a
range of inactive ingredients selected from a variety of conventional
pharmaceutically
acceptable excipients, carriers, buffers, and/or diluents.
[023] The term "pharmaceutically acceptable" is used to refer to a non-
toxic
material that is compatible with a biological system such as a cell, cell
culture, tissue, or
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organism. Examples of pharmaceutically acceptable excipients, carriers,
buffers, and/or
diluents are familiar to one of ordinary skill in the art and can be found,
e.g. in
Remington's Pharmaceutical Sciences (latest edition), Mack Publishing Company,
Easton, Pa. For example, pharmaceutically acceptable excipients include, but
are not
limited to, wetting or emulsifying agents, pH buffering substances, binders,
stabilizers,
preservatives, bulking agents, adsorbents, disinfectants, detergents, sugar
alcohols,
gelling or viscosity enhancing additives, flavoring agents, and colors.
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 inactive virus particles.
Pharmaceutically acceptable diluents include, but are not limited to, water
and saline.
[024] The present invention is directed at providing a phage-based
composition
and method for treating an infection associated with an implantable device,
such as for
example, a prosthetic joint infection ("PJI"). The composition and method may
offer
considerable simplicity inasmuch as the treatment may comprise administering
the
composition directly to the infected area (e.g., joint), optionally as a
single dose, such that,
for example, the phage therapy may potentially avoid any requirement for
replacing the
implantable device (e.g., the prosthetic device of the infected joint).
[025] Thus, in a first aspect, the invention provides a pharmaceutical
composition
comprising at least two different bacteriophage that are capable of lysis of
an infection
associated with an implantable device (e.g., a prosthetic joint infection
(PJI) in a patient),
wherein the pharmaceutical composition is formulated for administration to the
patient
either directly to the location of the infection, by IV, and/or orally.
[026] In one embodiment, the at least two different bacteriophage may be
phage
types selected for their capability of causing lysis (i.e. killing) of a
single bacterial species
or two or more bacterial species (i.e. in the case of a polymicrobial
infection) that may be
present in the implantable device infection.
[027] The selection of the phage types for therapeutic indications may be
based
upon the results of testing to determine the type(s) of bacteria present in
the implantable
device infection (i.e. the causative microorganisms); for example, by synovial
fluid
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aspiration and subsequent culture of the fluid on solid or in liquid media,
bacteria present
in the culture can be readily determined by standard techniques well known to
one of
ordinary skill in the art including 16S rRNA gene sequence analysis (e.g. as
described in
Whelan FJ et al., Ann. Am. Thorac. Soc. 11:513-521, 2014). The biological
sample may
also be used in assays to assess the bacteria for their susceptibility to
individual phage,
which can be invaluable in making the selection of the phage types for
inclusion in the
composition.
[028] The selection of the phage types may also be based upon a prediction
or
expectation of which causative microorganisms are present in the implantable
device
infection. Similarly, the selection of phage types may also be based upon a
prediction or
expectation of which causative microorganisms are present at a particular
geographical
location.
[029] For example, the phage selected for inclusion in the composition may
include types that are capable of lysis of bacteria commonly causative of
delayed onset a
device infection, such as for example, a delayed onset PJI, namely S. aureus
(e.g.
coagulase-negative S. aureus) and Enterococcus spp. (e.g. E. fecalis and
Enterococcus
fecium). Such a composition might also be effective for treating late onset
PJI, which is
commonly caused by S. aureus. Alternatively, the phage selected can be used to
treat an
acute infection or a chronic infection. Other bacteria that may be targeted by
inclusion of
appropriately selected phage type(s) include: other coagulase-negative
Staphylococcus
species that have been reported to cause PJI, such as S. simulans (Razonable
RR et al.,
Mayo CHn. Proc. 76:1067-1070, 2001), S. 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); various Streptococcus spp. that have been associated with
PJI,
including Lancefield groups A, B, C and G (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. J. 93:1217-1220, 2000), S. agalactiae and S. pneumoniae (Raad J et al.,
Semin.
Arthritis Rheum. 34:559-569, 2004), aerobic gram-negative bacteria such as E.
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) such as Enterobacter clocae; and
others such
as Clostridium spp., Actinomyces spp., Peptostreptococcus spp., Cutibacterium
acnes
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(formerly Propionibacterium acnes), Klebsiella pneumoniae, P. aeruginosa and
Bacteroides fragilis (Tande & Patel, supra).
[030] Moreover, it is also contemplated that the identified
phage to be included in
a composition could be a matched phage composition based on the infections
commonly
experienced in a certain geographic location. Examples of such -geomatched"
phage are
described in USSN 62/956,729, filed on January 3, 2020, and which is hereby
incorporated by reference in its entirely.
[031] Thus, one aspect of the invention is a method of treating
an infection caused
by one or more bacterium associated with a device implanted within a subject,
wherein
said method comprises:
(a) identifying at least one identified phage capable of infecting the
bacterium,
wherein said identified phage is selected from a library comprising at least
one phage
selected from APT-PJI-oi, APT-PJI-02, APT-PJI-03, APT-PJI-04, APT-PJI-o5, APT-
PJI-06, APT-PM-07, APT-PJI-08, APT-PM-09, APT-PJI-io, APT-PM-11, APT-PJI-
12, APT-PJI-13, APT-PJI-14, APT-Pa-15, APT-PJI-16, APT-PM-17, APT-PJI-18, APT-
PJI-20, APT-PJI-21, APT-PJI-22, APT-PJI-23, APT-PM-24, APT-PJI-25, APT-PJI-
26, APT-PM-27, APT-PJI-28, APT-PJI-29, APT-PJI-30, APT-PJI-31, APT-PJI-32,
APT-PM-33, APT-PM-34, APT-PM-35, APT-PM-36, APT-PM-37, APT-PM-38, APT-
PJI-39, APT-PJI-40, APT-PA-41, APT-PM-42, APT-PA-43, APT-PM-44, APT-PJI-
45, APT-PM-46, APT-PM-47, APT-PM-48, APT-PM-49, and/or APT-PM-50 as
described in Table 1 or any other lytic phage that has a genomic sequence with
at least
70% sequence identity to the genomic sequence of any of the foregoing phage
and
which is capable of causing lysis of the bacterium; and
(b) administering to said subject a therapeutically effective amount of a
composition comprising said identified phage, wherein said composition is
effective
in treating and/or reducing said infection.
[032] Table 1 provides relevant information for the public phage
(e.g, APT-PJI-oi,
APT-PJI-02, APT-PJI-o3, APT-PJI-04, APT-PJI-o5, APT-PJI-o6, APT-PJI-o7, APT-
PJI-
o8, APT-PJI-09, APT-PJI-io, APT-PJI-n, APT-PJI-12, APT-PJI-13, APT-PJI-14, APT-
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APT-PJI-16, APT-PJI-17, and APT-PJI-18) as well as the proprietary, not
publicly
available phage (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-3o, 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-PM-39, APT-PJI-40, APT-PJI-41, APT-PJI-42, APT-PJI-43, APT-PM-44,
APT-PJI-45, APT-PJI-46, APT-PJI-47, APT-PJI-48, APT-PJI-49, and/or APT-PJI-
50).
[033] In preferred embodiments, the compositions and methods are
performed
with any combination of the proprietary phage. Specifically, at least one, at
least two, at
least three, at least four and/or at least five phage selected from the group
consisting of
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-PM-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 are used to treat a
patient
suffering from a PJI infection as described herein.
11
CA 03174729 2022- 10- 5
,-,
),
E'
APT-14-PCT Final
'.'
Table 1
Phage Bacterial
GenBank
Phage ID
SEQ ID
Bacteria Name host Comments
Accession
code
NO:i
(HER#)* HER#*
NO.
Podoviridae, species
APT-PJI-oi P68 (49) 1049
44AHJD; also lyses NC 004679 2
HERnoi
Podoviridae, species
44AHJD NC_oo4678
APT-PJI-02 1101 44AHJD;
also lyses 1
(ioi) HER1:49
NC _007053
APT-PJI-03 3A (225) 1225
Siphoviridae, species 3A 6
APT-PJI-04 77(226) 1226 Siphoviridae,
species 77 NC 005356 3
species 11
hoviridae,
APT-PJI-o5 71 (238) 1238
SipNC _007059 7
(P11-M15)
IV
APT-PJI-06 187 (239) 1239
Siphoviridae, species 187 NC_007047 4
S. auretts 2638A
APT-PJI-o7 1283
Siphoviridae NC _007051 5
(283)
APT-PJI-o8 CSi (466) 1466
Siphoviridae
APT-PJI-o9 DW2 (467) 1466
Siphoviridae NC 024391 10
APT-PJI-io K (474) 1474
Myoviridae NC_005880 9
APT-PJI-ii 812 (475) 1475
Myoviridae NC_029080 12 XI
m
-a
Myoviridae,
Remus
APT-PJI-12 1528
Staphylococcus species NC0
031031 13
(528)
m
Remus
E
m
Z
APT-PJI-o 2 2
16 ¨I
_______________________________________________________________________________
_____________________________________ Cl,
I
M
M
¨I
9
`,7
APT-14-PCT Final
'.'
Tablet
Phage Bacterial
GenBank
Phage ID
SEQ ID
Bacteria Name host Comments
Accession
code
NO:t
(HER#)* HER#*
NO.
APT-PJI-023
17
APT-PJI-024
18
APT-PJI-025
19
APT-PJI-026
20
APT-PJI-027
21
APT-PJI-028
22
APT-PJI-029
23 i-
(A)
APT-PJI-030
24
APT-PJI-031
25
APT-PJI-032
26
APT-PJI-033
27
APT-PJI-034
28 xj
M
APT-PJI-035
29
C.)
m
APT-PJI-036
30 E
m
z
¨i
APT-PJI-03 7
31 0)
I
m
m
-1
,-,
),
E'
APT-14-PCT Final
'.'
Table 1
Phage Bacterial
GenBank
Phage ID SEQ ID
Bacteria Name host Comments
Accession
NO:i
code
(HER#)* HER#*
NO.
APT-PJI-o38
32
APT-Pa-039
33
APT-PJI-040
34
APT-PJI-041
35
APT-PJI-042
36
APT-Pa-043
37
APT-Pa-044
38
4.
APT-PR-045
39
APT-PJI-o46
40
APT-PJI-13 392 (292) 1292 Siphoviridae
S. epidermidis
APT-PJI-14 6ec (555) 1555 Siphoviridae
K12935213 11
APT-PJI-15 VD13 ( Siphoviridae, species
44) 1044 VD13
NC 024212.1 8 x
m
APT-PJI-16 182 (8o) io8o Podoviridae species 182
E. fecalis
0m
VD1884
E
APT-PJI-17 1323 Siphoviridae
m
(323)
z
-1
APT-PJI-18 1 (339) 1339
Myoviridae, species 1 (1)
_______________________________________________________________________________
___________________________________ I
M
M
-I
9
8
-
,1 APT-14-PCT Final
.
2
õ
. Table 1
Phage Bacterial
GenBank
Phage ID
SEQ ID
Bacteria Name host Comments
Accession
code
NO:i
(HER#)* HER#*
NO.
APT-PJI-20
14
S. caprae
APT-PM-021
15
S. lug dunensis APT-PJI-047 41
APT-PJI-048 42
APT-PM-049
43
APT-PM-050
44
* d'Herelle (HER) numbers as indicated at www.phage.ulaval.ca
i-
u-.
x
m
-a
0
m
E
m
z
-1
Cl,
mx
m
-1
WO 2021/207082
PCT/US2021/025794
[034]
In another aspect, the invention disclosed is a method of identifying
a subject
suffering, at risk of suffering, and/or eligible for phage treatment for a
bacterial infection,
comprising the steps of:
(a) obtaining a biological sample from the subject;
(b) culturing a bacterium present in the biological sample;
(c) inoculating the cultured bacterium with an identified phage selected
from a
library comprising at least one phage selected from one or more of APT-PM-oi,
APT-
PM-02, APT-PM-03, APT-PM-04, APT-PM-o5, APT-PM-o6, APT-PM-07, APT-PJI-
o8, APT-PM-09, APT-PM-io, APT-PJI-n, APT-PM-12, APT-PM-13, APT-PM-14,
APT-PM-15, APT-PM-16, APT-PM-17, APT-PM-18, APT-PJI-20, APT-PM-21, APT-
PM-22, APT-PM-23, APT-PM-24, APT-PM-25, APT-PM-26, APT-PM-27, APT-PM-
28, APT-PJI-29, APT-PJI-3o, APT-PJI-31, APT-PJI-32, APT-PJI-33, APT-PJI-34,
APT-PM-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PM-39, APT-PM-40, APT-
PJI-41, APT-PM-42, APT-PM-43, APT-PM-44, APT-PM-45, APT-PM-46, APT-PJI-
47, APT-PM-48, APT-PM-49, and/or APT-PJI-5o, and any other lytic phage that
has
a genomic sequence with at least 70% sequence identity to the genomic sequence
of
any of the foregoing phage and which is capable of causing lysis of the
bacterium; and
(d) determining whether the cultured bacterium are lysed by the identified
phage,
wherein when any of the cultured bacterium are lysed by the phage, the subject
is
determined (1) to be eligible for treatment by bacteriophage for said
bacterial infection
(2) to be suffering from a bacterial infection; and/or (3) at risk of
suffering from a
bacterial infection.
[035]
In preferred embodiments, the composition comprises at least one
identified phage selected from the group consisting of APT-PJI-oi, APT-Pa-02,
APT-
PM-03, APT-PM-04, APT-PM-05, APT-PM-06, APT-PM-07, APT-PM-o8, APT-PM-09,
APT-PM-io, APT-PM-11, APT-PM-12, APT-PM-13, APT-PM-14, APT-PM-15, APT-PM-16,
APT-PM-17, APT-PM-18, APT-PM-20, APT-PM-21, APT-PJI-22, APT-PM-23, APT-PJI-
24, APT-PM-25, APT-PM-26, APT-PM-27, APT-PM-28, APT-PM-29, APT-PM-30, APT-
PM-31, APT-p,11-32, APT-PM-33, APT-PM-34, APT-PM-35, APT-PJI-36, APT-PJI-37,
APT-PJI-38, APT-PJI-39, APT-PJI-40, APT-PJI-41, APT-Pa-42, APT-PM-43, APT-PJI-
16
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44, APT-PM-45, APT-PM-46, APT-PM-47, APT-PM-48, APT-PM-49, and/or APT-PJI-
50, or any other lytic phage that has a genomic sequence with at least 70%
sequence
identity to the genomic sequence of any of the foregoing phage and which is
capable of
causing lysis of the bacterium.
[036] In even further preferred embodiments, the composition comprises at
least
two identified phage selected from the group consisting of APT-PM-oi, APT-PJI-
02, APT-
PJI-o3, APT-PJI-o4, APT-PJI-o5, APT-PJI-o6, APT-PJI-o7, APT-PJI-o8, APT-PJI-
o9,
APT-PM-10, APT-PM-11, APT-PJI-12, APT-PM-13, APT-PM-14, APT-PJI-15, APT-PJI-
16,
APT-PM-17, APT-PM-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-M-32, APT-PM-33, APT-PM-34, APT-PJI-35, APT-PJI-36, APT-PM-37,
APT-PJI-38, APT-PM-39, APT-PM-40, APT-PM-41, APT-PM-42, APT-PM-43, APT-RII-
44, APT-PM-45, APT-PM-46, APT-PM-47, APT-PM-48, APT-PM-49, and/or APT-PJI-
50, and any other lytic phage that has a genomic sequence with at least 70%
sequence
identity to the genomic sequence of any of the foregoing phage and which is
capable of
causing lysis of the bacterium.
[037] In further aspects, the at least two identified phage: (a) cause
lysis in the
same strain of bacterium; (b) cause lysis in different strains of bacterium.
[038] In other further aspects, one of the two identified phage: (a) is
selected from
the group consisting of APT-PJI-01, APT-PJI-02, APT-PM-03, APT-PM-04, APT-PM-
05,
APT-PJI-o6, APT-PM-07, APT-PJI-08, APT-PM-09, APT-PM-io, APT-PM-n, APT-PJI-
12, APT-PJI-22, APT-PJI-23, APT-PM-24, APT-PJI-25, APT-PJI-26, APT-PM-27, APT-
PJI-28, APT-PM-29, APT-PJI-30, APT-PM-31, APT-PM-32, APT-PJI-33, APT-PM-34,
APT-PJI-35, APT-PJI-36, APT-PJI-37, APT-PJI-38, APT-PJI-39, APT-PM-4o, APT-PM-
41, APT-PM-42, APT-PM-43, APT-PM-44, APT-PM-45, and APT-PM-46, and any other
lytic phage that has a genomic sequence with at least 70% sequence identity to
the
genomic sequence of any of the foregoing phage in (a); (b) is selected from
the group
consisting of APT-PJI-13, APT-PM-14 and any other lytic phage that has a
genomic
sequence with at least 70% sequence identity to the genomic sequence of any of
the
foregoing phage in (b); (c) is selected from the group consisting of selected
from APT-PJI-
15, APT-PM-16, APT-PM-17, APT-PM-18 and any other lytic phage that has a
genomic
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sequence with at least 70% sequence identity to the genomic sequence of any of
the
foregoing phage in (c); (d) is selected from the group consisting of APT-PJI-
20 or APT-
PJI-21 and any other lytic phage that has a genomic sequence with at least 70%
sequence
identity to the genomic sequence of any of the foregoing phage in (d); or (e)
is selected
from the group consisting of APT-PJI-47, APT-PJI-48, APT-PJI-49, or APT-PJI-
5o, and
any other lytic phage that has a genomic sequence with at least 70% sequence
identity to
the genomic sequence of any of the foregoing phage in (e).
[039] In further aspects, the two identified phage are: selected from each
of (a)
and (b); selected from each of (a) and (c); selected from each of (a) and (d);
selected from
each of (a) and (e); and/or selected from each of (b) and (c), selected from
each of (b) and
(d); selected from each of (b) and (e); and/or selected from each of (c) and
(d); selected
from each of (c) and (e); and/or selected from each of (d) and (e) from the
phage
described in the previous paragraph. In further aspects, the composition
comprises at
least three identified phage, where at least one of the identified phage is
selected from
each of (a), (b), (c), (d) and/or (e) from the phage described in the previous
paragraph.
[040] In other further aspects, one of the two identified phage: (a) is
selected from
the group consisting of (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 any other lytic phage that has a genomic sequence with at least
70% sequence
identity to the genomic sequence of any of the foregoing phage in (a); (b) is
selected from
the group consisting of APT-PJI-20 or APT-PJI-21 and any other lytic phage
that has a
genomic sequence with at least 70% sequence identity to the genomic sequence
of any of
the foregoing phage in (b); or (c) is selected from the group consisting of
APT-PJI-47,
APT-PJI-48, APT-PJI-49, or APT-PJI-50, and any other lytic phage that has a
genomic
sequence with at least 70% sequence identity to the genomic sequence of any of
the
foregoing phage in (c).
[041] In further aspects, the two identified phage are: selected from each
of (a)
and (b); selected from each of (a) and (c); and/or selected from each of (b)
and (c) from
the phage described in the previous paragraph. In further aspects, the
composition
18
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comprises at least three identified phage, where at least one of the
identified phage is
selected from each of (a), (b), and (c) from the phage described in the
previous paragraph.
[042] In further aspects, the composition provides a dose of each phage in
the
range of 105 to 1013 pfu, and preferably, at a range of lob to 1012 pfu; 107
to loll pfu; 108 to
1011 pfu; 109 to 1011 pfu; 109 to 1010 pfu; or at a dose of approximately 106
pfu, 107 pfu, 108
pfu, 1o9 pfu, 1010 pfu, io pfu, 1012 pfu, or 1013 pfu. In most preferred
embodiments, the
dose of each phage in the composition is approximately 109 pfu.
[043] Thus, the composition may be formulated so as to provide a suitable
dose
of each phage type included in the composition. By way of example only, a
suitable dose
of the phage(s) may be in the range of 105 to 1013 pfu, and more preferably
109 to 1012 pfu.
Most preferably, the composition is formulated to provide a dose of each phage
type
present of about 109 pfu, 1010 pfu, or 1011 pfu.
[044] In preferred embodiments, the bacterium is selected from at least one
of S.
aureus, S. epidermidis, Enterococcus spp., including E. fecalis and
Enterococcus fecium,
other coagulase-negative Staphylococcus species, including S. simulans, S.
caprae and S.
lugdunensis, Streptococcus spp., including Lancefield groups A, B, C and G, S.
agalactiae
and S. pneumoniae, aerobic gram-negative bacteria including E. coil, other
Enterobacteriaceae including Enterobacter clocae, Clostridium spp.,
Actinomyces spp.,
Peptostreptococcus spp., Cutibacterium acnes, Klebsiella pneumoniae, P.
aeruginosa
and Bacteroides fragilis.
[045] In other preferred embodiments, the bacterium is selected from two or
more
different strains of bacterium selected from S. aureus, S. epidermidis,
Enterococcus spp.,
including E. fecalis and Enterococcus fecium, other coagulase-negative
Staphylococcus
species, including S. simulans, S. caprae and S. lugdunensis, Streptococcus
spp.,
including Lancefield groups A, B, C and G, S. agalactiae and S. pneumoniae,
aerobic
gram-negative bacteria including E. coil, other Enterobacteriaceae including
Enterobacter clocae, Clostridium spp., Actinomyces spp., Pep tostreptococcus
spp.,
Cuti bacterium acnes, Klebsiella pneumoniae, P. aeruginosa and Bacteroides
fragilis.
[046] In even further preferred embodiments, the bacterium is selected from
S.
aureus, S. epidermidis and/or E. fecalis. To treat such an infection, the
composition
19
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could comprise at least two phage having different lytic specificities capable
of infecting
at least two of S. aureus, S. epidermidis and/or E. fecalis. In other aspects,
the
composition comprises, for example, at least three phage having different
lytic
specificities, each phage capable of infecting at least one of S. aureus, S.
epidermidis and
E. fecalis.
[047] Preferably, the lytic phage has a genomic sequence with at least 75%,
80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to
the
genomic sequence of any of the foregoing phage.
[048] Sequence identity percentages referred to herein are to be understood
as
having been 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).
[049] The terms "percent (%) sequence similarity", "percent (%) sequence
identity",
and the like, generally refer to the degree of identity or correspondence
between different
nucleotide sequences of nucleic acid molecules or amino acid sequences of
polypeptides
that may or may not share a common evolutionary origin (see Reeck et al.,
supra).
Sequence identity can be determined using any of a number of publicly
available sequence
comparison algorithms, such as BLAST, PASTA, DNA Strider, GCG (Genetics
Computer
Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin),
etc.
[050] To determine the percent identity between two amino acid sequences or
two
nucleic acid molecules, the sequences are aligned for optimal comparison
purposes. The
percent identity between the 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, or are about, of 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.
[051] The determination of percent identity between two sequences can be
accomplished using a mathematical algorithm. A non-limiting example of a
CA 03174729 2022- 10- 5
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mathematical algorithm utilized for the comparison of two sequences is the
algorithm of
Karlin and Altschul, Proc. Natl. Acad. Sci. USA 1990, 87: 2 2 64, modified as
in Karlin and
Altschul, Proc. Natl. Acad. Sci. USA 1993, 90:5873-5877. Such an algorithm is
incorporated into the NBLAST and XBLAST programs of Altschul et al, J. Mol.
Biol. 1990;
215: 403. BLAST nucleotide searches can be performed with the NBLAST program,
score
= loo, wordlength = 12, to obtain nucleotide sequences homologous to 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 protein
sequences of
the invention. To obtain gapped alignments for comparison purposes, Gapped
BLAST
can be utilized as described in Altschul et al, Nucleic Acids Res. 1997,
25:3389.
Alternatively, PSI-Blast can be used to perform an iterated search that
detects distant
relationship between molecules. See Altschul et al. (1997) supra. When
utilizing BLAST,
Gapped BLAST, and PSI-Blast programs, the default parameters of the respective
programs (e.g., XBLAST and NBLAST) can be used. See ncbi.nlm.nih.gov/BLAST/ on
the
WorldWideWeb.
[052] Another non-limiting example of a mathematical algorithm utilized for
the
comparison of 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 PAM12o weight residue table, a gap length
penalty of
12, and a gap penalty of 4 can be used.
[053] In a preferred embodiment, the percent identity between two amino
acid
sequences is determined using the algorithm of Needleman and Wunsch (J. Mol.
Biol.
1970, 48:444-453), which has been incorporated into the GAP program in the GCG
software package (Accelrys, Burlington, MA; available at accelrys.corn on the
WorldWideWeb), using either a Blossum 62 matrix or a PAM25o matrix, a gap
weight of
16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 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 using a NWSgapdna.CMP matrix, a
gap
weight of 40, 50, 6o, 70, or 80, and a length weight of 1, 2, 3, 4, 5, or 6. A
particularly
preferred set of parameters (and the one that can be used if the practitioner
is uncertain
21
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about what parameters should be applied to determine if a molecule is a
sequence identity
or homology limitation of the invention) is using a Blossum 62 scoring matrix
with a gap
open penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of
5.
[054] Another non-limiting example of how percent identity can be
determined is by
using software programs such as those described in Current Protocols In
Molecular
Biology (F. M. Ausubel et al., eds., 1987) Supplement 30, section 7.7.18,
Table 7.7.1.
Preferably, default parameters are used for 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=6o; expect=io; Matrix=BLOSUM62; Descriptions =50
sequences;
sort by=HIGH SCORE;
Databases=non-redundant,
GenBank+EMBL+DDBJ+PDB+GenBank
CDS
translations+SwissProtein+SPupdate+PIR. Details of these programs can be found
at the
following Internet address: http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST.
[055] Statistical analysis of the properties described herein may be
carried out by
standard tests, for example, t-tests, ANOVA, or Chi squared tests. Typically,
statistical
significance will be measured to a level of p=o.05 (5%), more preferably
p=o.oi, p=o.00l,
p=o.000l, p=o.00000i
[056] In preferred aspects, the methods described herein relate to devices,
wherein
said device is: (a) permanently implanted in the subject; (b) temporarily
implanted in
the subject; (c) removable; and/or (d) is selected from a prosthetic joint, a
left-ventricular
assist device (LVAD), a stent, a metal rod, an in-dwelling catheter, spinal
hardware,
and/or bone hardware.
[057] In further aspects, the infection is selected from: a prosthetic
joint infection
(PJI), a chronic bacterial infection, an acute bacterial infection, a
refractory infection, an
infection associated with a biofilm, and/or an infection associated with an
implantable
device.
[058] In further aspects, the methods described herein relate to
compositions,
wherein said composition is administered, for example, (a) by IV injection;
(b) by direct
injection to the site of infection; (c) prophylactically; (d) prior to
surgery; (e) in lieu of
22
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surgery; (g) during surgery; (h) on a single occasion (i.e., as a "single
shot"); and/or (i) as
a therapeutic course over 2 weeks or more.
[059] In preferred aspects, lysis of a bacterium by a phage can be
measured using
assays known in the art, such as 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) plaque formation.
[o6o] In preferred aspects, the photometric assay used to measure
lysis utilizes an
additive that causes and/or enhances the photometric signal detection. An
example of
such an additive, include, but is not limited to tetrazolium dye.
[061] In other preferred aspects, the biological sample is obtained from:
(a) synovial
fluid; (b) an area surrounding an implantable device; (c) a site of infection;
(d) an intra-
operative sample; (e) a swab of the device; (f) a biofilm; (g) a fistula;
and/or (h) an
aspiration of a site of infection.
[062] In further aspects, the bacterial infection is (a) multi-drug
resistant; (b)
clinically refractory to antimicrobial treatment; (c) clinically refractory to
antimicrobial
treatment due to biofilm production; and/or (d) clinically refractory due to
the subject's
inability to tolerate antimicrobials due to adverse reactions.
[063] As understood herein, the terms, "multidrug resistant", "multi drug
resistant", "multi drug resistance", "MDR" and like terms may be used
interchangeably
herein, and are familiar to one of skill in the art, i.e., a multidrug
resistant bacteria is an
organism that demonstrates resistance to multiple different antibacterial
drugs, e.g.,
antibiotics; and more specifically, resistance to multiple different classes
of antibiotics.
It is understood herein that bacterial infections to be treated comprise
bacteria in biofilm
and/or planktonic growth modes.
[064] Examples of typically MDR bacteria that maybe treated include, but
are not
limited to the "ESKAPE" pathogens (Enterococcus faecium, Staphylococcus
aureus,
Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and
Enterobacter sp), which are often nosocomial in nature and can cause severe
local and
systemic infections. Specifically, these include, e.g., methicillin-resistant
Staphylococcus
aureus (MRSA); vancomycin-resistant Enterococcus faecium (VRE); carbapenem-
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resistant Klebsiella pneumonia (NDM-1); MDR-Pseudomonas aeruginosa; and MDR-
Acinetobacter baumannii.
[065] Among the ESKAPE pathogens, A. baumannii is a Gram-negative,
encapsulated, opportunistic pathogen that is easily spread 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 MDR, which
severely
restricts the available treatment options, with untreatable infections in
traumatic wounds
often resulting in prolonged healing times, the need for extensive surgical
debridement,
and in some cases the further or complete amputation of limbs. Notably, blast-
related
injuries in military populations are associated with significant tissue
destruction with
concomitant extensive blood loss and therefore these injuries are at high risk
for
infectious complications. One of skill in the art will appreciate that given
the ability for
A. baumannii and other MDR ESKAPE pathogens to colonize and survive in a host
of
environmental settings, there is an urgent need for new therapeutics against
these
pathogens.
[o66] In further aspects, the subject is suffering from a hardware
related infection.
[067] In preferred embodiments, the infection is a prosthetic
joint infection (PJI).
For example, the composition of the invention may be useful for treating PJI,
regardless
of type. That is, by appropriately selecting the two or more phage, the
composition may
be beneficial in treating early, delayed and late onset types of PJI, as well
as PJI that are
the result of a polymicrobial infection. Similarly, in other preferred
embodiments, the
compositions described herein can be used to treat an acute and/or chronic
infection, and
preferably a PJI infection. The composition of the invention may be useful for
treating
PJI associated particularly with prosthetic hip, knee, shoulder and elbow
replacements.
[o68] In some aspects, the library used in the methods described
herein comprises
phage pre-screened to exclude phage comprising undesirable and/or toxic
characteristics.
Examples of such undesirable and/or toxic characteristics are selected from
toxin genes
or other bacterial virulence factors, phages which possess lysogenic
properties and/or
carry lysogeny genes, phages which transduce bacterial virulence factor genes
or
antibiotic resistance genes, phages which carry any antibiotic-resistance
genes or can
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confer antibiotic resistance to bacterial strains, and phages which elicit an
inappropriate
immune response and/or provoke a strong allergenic response in a mammalian
system.
Examples of producing such pre-screened libraries of phage are described in,
for example,
US 10,357,522 (the disclosure of which is hereby incorporated by reference in
its entirety).
[069] It is known that antibiotic resistance, toxin formation, and unwanted
mammalian innate immune responses are possible outcomes from the use of
bacteriophages to treat bacterial infections (Quiros et. al., Antimicrob
Agents
Chemother 58: 606-609, 2014; do Vale et. al., Front. Microbiol. 7:42, 2016).
In some
respects, phages comprising undesirable and/or toxic characteristics can be
eliminated
initially from the library. It is also well understood within the skill in the
art to genetically
alter the phage to eliminate and/or reduce undesirable and/or toxic phenotype.
The
detrimental phenotypes can be traced back to genes located on the phage's
single-
stranded DNA. To remove these genes and prevent phages from horizontally
transferring
these characteristics to bacteria associated with chronic infections within
implantable
devices, systems such as CRISPR (Clustered Regularly Interspaced Short
Palindromic
Repeats) can he used to edit the genetic material. Phages with DNA or RNA
genom es may
be engineered using CRISPR systems.
Gene Editing Using CRISPR/Cas9
[070] CRISPR and CRISPR-associated protein 9 (Cas9), referred to as the
CRISPR/Cas9 system, may be used to perform gene editing. Gene editing
includes, but
is not limited to, gene insertions, gene replacements, gene deletions, frame
shifts, single
nucleotide changes, nonsense mutations, missense mutations, etc. Gene editing
may also
include editing of regulatory sequences to modulate gene expression. Based on
these
techniques, genes may be mutated, repaired, or even modulated in a variety of
cell lines
and germline sequences. A review of the CRISPR/Cas9 system may be found in
Hsu, et
al., "Development and Applications of CRISPR-Cas9 for Genome Engineering" Cell
(2014) vol. 157:1262. A guide to using the CRISPR/Cas9 system may be found at
Addgene
(add.gene.org/CRiSPR/gu.ide).
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[071] The CRISPR/Cas9 system can be employed to "knock-out" and "knock-in"
specific genes in various cell types and organisms as well as selectively
activate or repress
specific genes, purify specific regions of DNA, image DNA in live cells using
fluorescence
microscopy, as well as many other uses.
[072] According to embodiments of the present invention, the CRISPR/Cas9
system
can be used to edit the gene in phages to eliminate and/or reduce undesirable
and/or toxic
phenotype. By reducing or eliminating toxic phenotype, the undesirable effects
of
bacteriophage therapy to treat bacterial infections including antibiotic
resistance, toxin
formation, and unwanted mammalian innate immune responses be reduced or
eliminated, and it is expected that the phages are useful in treatment of
infection.
[073] According to other embodiments of the present invention, the phages
that have
been edited by the CRISPR/Cas9 system may be administered to the patient. In
still
further embodiments of the invention, the phages that have been edited by the
CRISPR/Cas9 system may be combined with one or more additional agents as
described
herein.
[074] Genome editing of phages, traditionally a challenging and time-
consuming
process, may be efficiently performed using the CRISPR/Cas9 system. In some
cases,
editing by the CRISPR/Cas9 system may occur within 24 hours of delivery of the
Cas9
gene, the guide RNA, and as applicable, the nucleotide replacement sequence.
Components of the CRISPR/Cas9 system
[075] In general, the CRISPR/Cas9 system comprises at least two components:
(i) a
Cas9 protein, which is a nuclease capable of cutting both stands of a DNA
double helix;
and (ii) at least one RNA sequence (e.g., a guide RNA (gRNA) sequence, such as
a single-
guide RNA (sgRNA) sequence) that is designed to target Cas9 to a specific
location or
locus of a gene of interest (a genomic target). The gRNA comprises a targeting
sequence
homologous to the genomic target, as well as a scaffolding domain that binds
to Cas9, in
order to recruit Cas9 to the genomic target.
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Non-Homologous End Joining
[076] In some embodiments, the CRISPR/Cas9 system induces double stranded
DNA breaks in the genomic target, which are thought to stimulate cell repair
mechanisms
using non-homologous end joining (NHEJ). Non-homologous end joining, which can
lead to gene disruption by random insertions or deletions, is thought to be a
primary
mechanism for repairing double strand breaks (DSBs). NHEJ-mediated DSB repair
can
result in disruption of the open reading frame (ORF) of the genomic target,
leading to a
non-functional protein.
[077] For NHEJ methods, Cas9 as well as a gRNA is introduced into the host
cell,
e.g., either as a preassembled complex or inserted into one or more expression
vectors.'
Homology Directed Repair
[078] In other embodiments, the CRISPR/Cas9 system is utilized to repair,
replace
or insert one or more nucleotides at the genomic target. In this method, a
replacement
nucleotide sequence is introduced into a cell, wherein the replacement
nucleotide
sequence contains the desired edits as well as regions of homology upstream
and
downstream of the genomic target. To perform homology directed repair (HDR),
Cas9,
e.g., a recombinant Cas9 from Streptococcus pyogenes, complexed with a gRNA,
e.g., an
in vitro transcribed sgRNA, and a replacement nucleotide sequence is needed.
[079] Homology Directed Repair (HDR) may be used to generate gene edits
ranging
from a single nucleotide base change to large nucleotide sequence insertions.
To utilize
HDR for gene editing, a replacement nucleotide sequence is delivered into the
cell along
with the gRNA and Cas9. The replacement nucleotide sequence contains the
desired
genomic edit(s) as well as additional homologous sequences immediately
upstream and
downstream of the genomic target sequence (referred to as a left homology arm
and a
right homology arm). The replacement nucleotide sequence can be a single
stranded
oligonucleotide, a double-stranded oligonucleotide, a double-stranded DNA
plasmid
depending on the specific application, and so forth. Generally, the
replacement nucleotide
sequence will not contain a Protospacer Adjacent Motif (PAM) sequence so as
not to
become a target for Cas9 cleavage.
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gRNA
[080] As discussed previously, gRNA comprises a targeting sequence and a
scaffolding domain. Once expressed, Cas9 and the gRNA form a riboprotein
complex
through interactions between the gRNA scaffolding domain. Upon gRNA binding,
Cas9
undergoes a conformational change into a form that binds DNA, while allowing
the
targeting sequence to remain free to interact with genomic target DNA. For
Cas9 to cleave
genomic target DNA, the targeting sequence should exhibit high homology to the
genomic
target sequence. In general, the target sequence comprises 20 nucleotides, or
about 20
nucleotides, and may be a synthetic RNA.
[081] In general, "target sequence" or "targeting sequence" refers to a
sequence (e.g.,
the portion that is not associated with the scaffolding domain) of the gRNA.
"Genomic
target" or "Genomic target sequence" refers to a sequence or locus of the
genome targeted
for editing.
[082] The gRNA focuses the nuclease activity of Cas9 to the genomic target
using the
target sequence. Once the target sequence binds or hybridizes to the genomic
target, Cas9
cleaves the genomic DNA at or near that site. By changing the target sequence,
the
genomic target of Cas9 can be modified.
[083] In some embodiments, gRNA may be synthesized using commercially
available kits, e.g., a Guide-it sgRNA In Vitro Transcription Kit. In vitro
transcription may
be used to produce gRNAs that may be purified using techniques known in the
art.
Genomic target
[084] The genomic target sequence should be unique as compared to the
remainder
of the genome of the organism or cell in order to avoid off target effects.
[085] Additionally, the genomic target sequence should be present
immediately
upstream of a genomic Protospacer Adjacent Motif (PAM) sequence. The PAM
sequence
is needed for Cas9 binding, with the exact PAM sequence being dependent upon
the
species of Cas9 used. Cas9 from Streptococcus pyo genes is widely used in
genomic
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engineering. In general, Cas9 binds to the PAM sequence, and DNA cleavage
occurs
approximately 3 base pairs upstream of the PAM sequence.
[086] An example of a PAM sequence is 5'-NGG-3". Genomic target sequences
may
reside on either strand of genomic DNA.
[087] Several online tools
(e.g., http://crispr.mit.edu/ or
https://chopchop.rc.fas.harvard.edu/) are available for selecting PAM
sequences, and
also provide a list of potential genomic target sequences within a genomic
locus or
location of interest (e.g., within a genomic location encoding for a protein
such as CXCR4,
PD-1, etc.). These tools also predict off target effects in order to allow
selection of a
genomic target sequence that minimizes cleavage of genomic DNA at other
locations.
Vectors and Host Cells
[088] Construction of various types of vectors, including vectors for the
CRISPR/Cas9 system, maybe found in U.S. Patent Application No. 2014/0273226,
which
is incorporated herein by reference.
[089] In general, polynucleotides, e.g., polynucleotides encoding Cas9,
polynucleotides encoding a gRNA sequence, polynucleotides encoding a
replacement
nucleotide sequence, etc., can be incorporated into any desired DNA or RNA
based vector,
without limitation. For example, a polynucleotide may be cloned into an
expression
vector, a subcloning vector, a shuttle vector, a vector designed for use with
in vitro
transcription reactions, cosmids, phagemids, and vectors derived from
mammalian
viruses, including retroviruses (for example, lentiviruses), adenoviruses,
adenoassociated
viruses (AAV), and episomal EBNA-based vectors of Epstein-Barr virus origin.
[090] In some embodiments, vectors may be in circular form or in linearized
form.
The linearized form may be used in subcloning steps, e.g., cloning the gRNA
target
sequence into a host vector.
[091] One of skill in the art will understand that a wide variety of
expression vectors
for expression of gene products are within the scope of present invention
embodiments.
An expression vector can be optimally designed to express a protein, e.g.,
Cas9, a variant
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Cas9, etc., in a host cell. For example, a vector comprising the nucleotide
sequence
encoding a protein (e.g., a Cas9 open reading frame (ORF)) and suitable
regulatory
elements can be delivered into the host cell by any suitable method of
transfection,
transduction, etc. Any type and any quantity of regulatory elements, involved
in
regulation of transcription or translation, may be incorporated into the
expression vector
and may be located upstream or downstream of the ORF. Once in the host cell,
the host
cell's own machinery, e.g., endogenous RNA polymerases, etc., may be employed
to
synthesize mRNA, which is translated to produce the protein.
[092] In other embodiments, expression vectors are optimally designed to
express a
functional RNA molecule, e.g., a guide RNA for tethering Cas9 to a genomic
target
sequence.
[093] In some embodiments, the Cas9 mRNA and the gRNA are expressed from
the
same expression vector. In other embodiments, the Cas9 mRNA and the gRNA are
expressed from different expression vectors. The Cas9 gene inserted into the
expression
vector may be a native bacterial Cas9 gene from, e.g., Streptococcus pyo
genes,
Staphylococcus aureus, etc. In some embodiments, the expression vectors may be
mammalian expression vectors, e.g., such as human expression vectors, and may
contain
one or more promoter elements, e.g., a bacteriophage promoter element such as
a T7
promoter.
[094] In further embodiments, the gene encoding for Cas9 may be operably
linked
with one or more genes encoding nuclear localization signals, resulting in
targeting of the
expressed Cas9/gRNA to the host cell nucleus.
[095] In still other embodiments, the gene encoding for Cas9 is optimized
to reflect
preferred codon usage for the organism in which gene editing is being
performed. For
example, if gene editing is being performed in phages, then the gene encoding
for Cas9 in
the expression vector construct, is optimized to reflect preferred codon
utilization.
[096] In some embodiments, phages may be isolated, and a preassembled
gRNA/Cas9 complex along with an optional nucleotide replacement sequence may
be
electroporated into host cells. The gRNA sequences may be designed to have
homology
with exonic coding regions of one or more genes.
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[097] In other embodiments, CRISPR/Cas9 systems allow targeting of multiple
genetic loci (genomic targets) by cloning multiple gRNAs into a single vector.
[098] Present embodiments also provide compositions and methods for in vivo
gene
replacement, gene mutation, and gene repair using the CRISPR/Cas9 system
described
herein. This system is useful for engineering cell genomes in a highly
specific manner. In
some embodiments, engineered cell lines produced by the CRISPR/Cas9 system are
useful for therapeutic transplantation to treat human diseases. Human diseases
arising
from bacterial infections can be treated with compositions and methods of the
invention,
or with edited cell lines produced by the compositions and methods of the
invention.
[099] According to embodiments of the invention, methods are provided for
specific
genomic modification of host cells or phages, including: (i) providing an
expression
vector construct comprising a first polynucleotide encoding a Cas9 protein or
a variant
thereof, and a second polynucleotide encoding a gRNA, wherein the gRNA
comprises a
target sequence homologous to a genomic locus of interest, (ii) providing a
host cell
comprising the genomic locus of interest, (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 for host cells having gene edits at the genomic target.
[loo] According to other embodiments of the invention, methods are
provided for
specific genomic modifications of host cells, including: (i) providing a first
expression
vector comprising a first polynucleotide encoding a Cas9 protein and having a
transcriptional regulatory domain, or a variant thereof, 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
comprising the
genomic target, (iii) delivering the two expression vector constructs into the
host cell (e.g.,
via transfection), and (iv) expressing the first and second polynucleotides
within the host
cell. In some embodiments, the method may comprise visualizing, identifying or
selecting
for host cells having gene edits at the genomic target. By targeting a
regulatory region
such as a promoter region, controlling expression of a gene of interest,
expression may be
modulated, e.g., repressed or activated.
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[101] According to still other embodiments of the invention, methods are
provided
for specific genomic modification of host cells, including: (i) providing a
Cas9 protein
complexed with a gRNA, wherein the gRNA comprises a target sequence homologous
to
a genomic target, (ii) electroporating the Cas9 protein/gRNA complex into a
host cell
comprising the genomic target, (iii) selecting host cells based upon
expression levels of
the protein encoded by the genomic target (genomic locus). In some
embodiments, the
method may further comprise staining the host cell for the presence of a cell
surface
protein encoded by the genomic target, and selecting host cells expressing low
levels of
the cell surface protein using flow cytometry.
[102] In still other embodiments of the invention, more than one genomic
target (loci
of interest) is targeted for gene editing at the same time. Accordingly, the
expression
vector may comprise multiple nucleotide sequences, each encoding a different
gRNA with
a different targeting sequence, each targeting sequence corresponding to a
genomic
target. Alternatively, multiple expression vectors may be used, each
expression vector
comprising one or more_targeting sequences, each targeting sequence
corresponding to a
genomic target.
[103] One of skill in the art will appreciate that present invention
embodiments are
not limited to the polynucleotide or polypeptide sequences referred to herein,
and will
also encompass variant polypeptide and polynucleotide sequences. Variant
polypeptide
sequences include polypeptides having conservative amino acid substitutions in
their
amino acid sequences. Variant polynucleotide sequences include polynucleotides
that are
modified to have a change in a nucleotide sequence, and upon expression,
result in a
polypeptide having essentially the same function or activity as the
polypeptide expressed
by the unmodified nucleotide sequence.
[104] In some embodiments, CRISPR-CAS9 system is used for editing phages.
In
some other embodiments, a type I-E CRISPR-Cas system is used for editing
phages. In
yet other embodiments, a type III CRISPR-CASio system is used for editing
phages.
[105] For example, the protocols described in recent publications may be
modified
to delete a toxic and/or undesirable phenotype in phages of Myoviridae,
Siphoviridae,
and Podoviridae families (Ban et. al., Synth Biol. 2017;6(12):2316-2325; Box
et. al,
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Journal of Bacteriology Jan 2016, 198 (3) 578-590, Tao et. At, ACS Synth Biol.
2017;6(10):1952-1961; and Park et. al., Sci Rep. 2017;7:42458). Bari et. al.,
describes a
Type III-A CRISPR-Casio system for editing phages targeting S. aureus strain
with or
without a natural CRISPR-Cas system. The system provides a mechanism to select
phage-
derived sequences such as those harboring point mutations at multiple genetic
loci and
recover phage recombinants that have acquired desired mutations. Using RNA
sequences
as a map, the Cas enzyme cleaves the targeted gene carrying the deleterious
and/or toxic
characteristic. Another publication (Yosef et. al., Proceedings of the
National Academy of
Sciences Jun 2015, 112 (23) 7267-7272) describes temperate phage based CRISPR-
Cas
delivery into the genome of antibiotic-resistant bacteria.
[106] In some embodiments, a phage or phagemid is used to deliver DNA to
the
bacterial cell.
[107] In some embodiments, a CRISPR system can be used to eliminate
specific
bacterial strains by removing them from the microbiome or for specific
therapy.
[108] In further preferred embodiments, the method relies on the CRISPR
system
such as the CRISPR/Cas9 system, to edit undesirable and/or toxic genes, and
preferably
bacterial pathogenic gene such as bacterial endogenous gene, a single
nucleotide
polymorphism(SNP), an epichromosomal gene, an antibiotic resistance gene, a
gene
encoding one or more virulence factors, a gene encoding one or more toxins,
highly
conserved genes amongst a species, genera or phyla, and/or genes that encode
enzymes
involved in biochemical pathways with products that can modulate host
physiology.
[109] In a preferred embodiment, the CRISPR system targets a toxin and
antitoxin
loci involved in key biological functions including plasmid maintenance,
defense against
phages, persistence and virulence such as those described in Akarsu et. al.,
(2019) PLoS
Comput Biol 15(4): e1006946., Xie et. al., Nucleic Acids Res. 2018;46(M.):D749-
D753.,
Harms et. al., Mol Cell. 2018;70(5):768-784. and do Vale et. al., 2016.
[no] Examples of bacterial pathogens include Escherichia coli,
Shigella
dysenteriae, Yersinia pestis, Francisella tularensis, Bacillus anthracis,
Staphylococcus
aureus, Streptococcus pyogenes, Vibrio cholerae, Pseudomonas aeruginosa,
Klebsiella
pneumoniae, Acinetobacter baumannii, and Salmonella enterica Typhi. In even
further
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preferred embodiments, the bacterium is selected from S. aureus, S.
epidermidis and/or
E. fecalis.
[111] Examples of toxins include pertussis toxin and adenylatecyclase
toxin(ACT)
secreted by Bordetella pertussis, anthrax toxin from Bacillus anthracis and
Staphylococcus aureus leukotoxins, AIP56 from Photobacterium damselaepiscicida
(Phdp), mycolactone, a polyketide molecule produced by Mycobacteriumulcerans,
other
bacterial secreted products not formally termed as toxins, such as S. aureus
super
antigens-like proteins (SSLs) and phenol-soluble modulins (PSMs), Clostridial
C3 toxins,
Shiga toxin, cholera toxin, hemolysins, leucocidin, fimbrial and afimbrial
adhesins,
proteases, lipases, endonucleases, endotoxins and exotoxins cytotoxic factors,
microcins
and colicins. In some embodiments, the toxin is encoded by superantigen
enterotmdn
gene such as the S. aureus Sek gene. In some embodiments, the toxin is a
exotoxin such
as toxic shock syndrome toxin-i (TSST-1); enterotoxins such as SEA, SEB, SECn,
SED,
SEE, SEG, SEH, and SET, and exfoliative toxins, such as ETA and ETB.
[112] Examples of genes that confer virulence traits to Escherichia coli
(e.g.,
0157:H7) include, without limitation, stx1 and 5tx2 (encode Shiga-like toxins)
and espA
(responsible for induction of enterocyte effacement (LEE) A/E lesions). Other
examples
of genes that confer virulence traits to Escherichia coli include fimA
(fimbriae major
subunit), csgD (curli regulator) and csgA. An example of a gene that confers
virulence
traits to Yersinia pestis is yscF (plasmid-borne (pCD1) T3SS external needle
subunit). An
example of a gene that confers virulence traits to Francisella tularensis is
fs1A. An example
of a gene that confers virulence traits to Bacillus anthracis is pag (Anthrax
toxin, cell-
binding protective antigen). Examples of genes that confer virulence traits to
Vibrio
cholerae include, without limitation, ctxA and ctxB (cholera toxin), tcpA
(toxin co-
regulated pilus), and toxT (master virulence regulator).Examples of genes that
confer
virulence traits to Pseudomonas aeruginosa include, without limitation, genes
that
encode for the production of siderophore pyoverdine (e.g., sigma factor pvdS,
biosynthetic genes pvdL, pvdl, pvdJ, pvdH, pvdA, pvdF, pvdQ, pydN, pvdM, pvd0,
pvdP,
transporter genes pvdL, pydR, pvdT and opmQ), genes that encode for the
production of
siderophore pyochelin (e.g., pchD, pchC, pchB, pchA, pchE, pchF and pchG, and
genes
that encode for toxins (e.g., exoU, exoS and exoT). Examples of genes that
confer
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virulence traits to Klebsiella pneumoniae include, without limitation, fimA
(adherence,
type I fimbriae major subunit), and cps (capsular polysaccharide). Examples of
genes that
confer virulence traits to Acinetobacter baumannii include, without
limitation, ptk
(capsule polymerization) and epsA (assembly). Examples of genes that confer
virulence
traits to Salmonella enterica Typhi include, without limitation, hilA
(invasion, SPI-1
regulator), ssrB (SPI-2 regulator), and those associated with bile tolerance,
including
efflux pump genes acrA, acrB and to1C.
[113] Examples of antibiotic resistance genes or various methods for
identifying the
same are known in the art. Genomic methods of identifying resistance phenotype
of strain
are described in the art such as whole-genome sequencing for antimicrobial
susceptibility
testing (WGS-AST) described in Su et. al., Journal of Clinical Microbiology
Feb
2019, 57 (3) e01405-18 and antibiotic resistance determinants (ARDs)
identified from
intestinal microbiota in Ruppe et al., Nat Microbial. 2019;4W:112-123. In some
embodiments, the resistance gene confers resistance to a narrow-spectrum beta-
lactam
antibiotic of the penicillin class of antibiotics. In other embodiments, the
resistance gene
confers resistance to methicillin (e.g., methicillin or oxacillin), or
flucloxacillin, or
dicloxacillin, or some or all of these antibiotics. In selected embodiments,
the CRISPR system is suitable for selectively targeting methicillin-resistant
S.
aureus (MRSA) and/or vancomycin resistant S. aureus (VRSA). In certain
embodiments,
the resistance gene may confer resistance to linezolid, daptomycin,
quinupristin/dalfopristin. In some embodiments, the resistance genes is
selected from
fosfomycin resistance gene fosB, tetracycline resistance gene tetM, kanamycin
nucleotidyltransferase aadD, bifunctional aminoglycoside modifying enzyme
genes aacA-
aphD, chlorampheni col acetyltransferase cat, mupirocin-resistance gene ileS2,
vancomycin resistance genes vanX, vanR, vanH, vraE, vraD, methicillin
resistance factor
femA, fmtA, mecl, streptomycin adenylyltransferase spci, spc2, anti, ant2,
pectinomycin
adenyltransferase spd, ant9, aadA2, and any other resistance gene.
[114] In a preferred embodiment, the CRISPR system targets one or more of
antibiotic resistance genes selected from an extended-spectrum beta-lactamase
resistance factor (ESBL factor), CTX-M-15, beta lactamase, New Delhi metallo-
13-
lactamase (NDM)-1,2,5,6 and tetracycline A (tetA).
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[115] Examples of genes that confer resistance to aminoglycoside include,
without
limitation, aph, aac and aad variants and other genes that encode
aminoglycoside-
modifying enzymes. Examples of genes with SNPs that confer aminoglycoside
resistance
include, without limitation, rpsL, rrnA and rrnB. Examples of genes that
confer beta-
lactam resistance include, without limitation, genes that encode beta-
lactamase (bla)
(e.g., TEM, SHV, crx-m, OXA, AmpC, IMP, VIM, KPC, NDM-1, family beta-
lactamases)
and mecA. Examples of genes with SNPs that confer daptomycin resistance
include,
without limitation, mprF, yycFG, rpoB and rpoC. Examples of genes that confer
macrolide-lincosamide-streptogramin B resistance include, without limitation,
ermA,
ermB and ermC. Examples of genes that confer quinolone resistance include,
without
limitation, qnrA, qnrS, qnrB, qnrC and qnrD. Examples of genes with SNPs that
confer
quinolone resistance include, without limitation, gyrA and parC. Examples of
genes with
SNPs that confer trimethoprim/sulfonamide resistance include, without
limitation, the
dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genes.
Examples
of genes that confer vancomycin resistance include, without limitation, vanA
(e.g., vanRS
and vanHAX), vanB and vanC operons.
[116] In some embodiments, the CRISPR system may be used to target SNPs,
which
cause overexpression of genes that encode multi-drug efflux pumps, such as
acrAB,
mexAB, mexXY, mexCD, mefA, msrA and tetL.
[117] Examples of highly conserved genes that can typify microbial species,
genera
or phyla for the purposes of remodeling complex microbial communities
(referred to
herein as "remodeling" genes) include, without limitation, ribosomal
components rrnA,
rpsL, rpsJ, rp10, rpsM, rp1C, rpsH, rp1P, and rpsK, transcription initiation
factor infB, and
tRNA synthetase pheS.
[118] Examples of genes encoding enzymes involved in biochemical pathways
with
products that can modulate host physiology (referred to herein as "modulatory"
genes
include, without limitation, (1) genes encoding enzymes involved in
deoxycholate
production linked to hepatocellular carcinoma, (2) genes encoding enzymes
involved in
polysaccharide A production by Bacteroides fragilis, leading to development of
regulatory
T cells (TREG), IL-io response and increased TH1 cell numbers, (3) genes
encoding
enzymes involved in butyrate production leading to secretion of inducible
antimicrobial
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peptides, (4) genes encoding enzymes involved in short-chain fatty acid
production
leading to increased energy harvest, obesity, inflammatory modulation and
gastrointestinal wound healing, (5) genes encoding enzymes involved in
transformation
of choline into methylamines, which can disrupt glucose homeostasis, leading
to non-
alcoholic fatty liver disease and cardiovascular disease, (6) genes encoding
enzymes
involved in the generation of neuromodulatory compounds such as y-aminobutyric
acid,
noradrenaline, 5-HT, dopamine and acetylcholine, and (7) genes encoding
enzymes
involved in the formation of lactic acid and propionic acid linked to anxiety.
[119] Besides CRISPR/Cas9 systems, 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, but are not limited to other CRISPR based
systems,
engineered TALEN (Transcription Activator-Like Effector Nuclease) variants,
engineered zinc finger nuclease (ZFN) variants and other systems such as those
described
in Chen et. al., (Front. Microbiol., 03 May 2019) including homologous
recombination-
based technologies, bacteriophage recombineering of electroporated DNA (BRED),
rebooting phages using assembled phage genomic DNA.
[120] As understood herein, terms such as "effective amount" and
"therapeutically
effective amount" of a pharmaceutical composition of the instant invention,
refer to an
amount of a composition suitable to elicit a therapeutically beneficial
response in the
subject, e.g., by eradicating a bacterial pathogen in the subject and/or
altering the
virulence or antibiotic susceptibility of surviving phage-resistant bacterial
pathogens
and/or by providing an added benefit when the composition is simultaneously
administered with either effective and/or ineffective antibiotics. Such
response may
include e.g., preventing, ameliorating, treating, inhibiting, and/or reducing
one of more
pathological conditions associated with a bacterial infection. One of skill in
the art will
appreciate that it is desirable that the initial dose of a composition
described herein be
sufficient to control the bacteria population before it reaches a lethal
threshold. Animal
models suggest that 109 to loll pfu/ml phage particles per dose would likely
be the
maximum dosage tenable based on protein load presented acutely to the liver in
an adult
(which would be scaled down in a pediatric population). It is suspected that
this is enough
of an acute bolus to reduce the bacterial burden sufficiently to potentiate an
immune
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response. Notably, phage "viremia" may be measured in the blood after
administration.
Animal models suggest that viremia is quite transient given the host immune
response
and sequestration in the reticuloendothelial system (liver and spleen).
[121] Suitable effective amounts of the compositions of the instant
invention can
be readily determined by one of skill in the art and can depend upon the age,
weight,
species (if non-human) and medical condition of the subject to be treated. In
addition,
one of skill in the art will appreciate that the type of infection (e.g.,
systemic or localized),
and the accessibility of the infection to treatment may also impact the dosage
amount that
is deemed effective. One of skill in the art will appreciate that initial
information may be
gleaned in laboratory experiments and an effective amount of a composition
described
herein for humans subsequently determined through dosing trials and routine
experimentation.
[122] It is even further contemplated that the compositions of the instant
invention may be administered to a subject by a variety of routes according to
conventional methods, including but not limited to systemic, parenteral (e.g.,
by
intracisternal injection and infusion techniques), intradermal,
transmembranal,
transdermal (including topical), intramuscular, intraperitoneal, intravenous,
intra-
arterial, intralesional, subcutaneous, oral, and intranasal (e.g., inhalation)
routes of
administration. Administration can also be by continuous infusion or bolus
injection.
[123] In addition, the compositions of the instant invention can be
administered
in a variety of dosage forms. These include, e.g., liquid preparations and
suspensions,
including preparations for parenteral, subcutaneous, intradermal,
intramuscular,
intraperitoneal or intravenous administration (e.g., injectable
administration), such as
sterile isotonic aqueous solutions, suspensions, emulsions or viscous
compositions that
may be buffered to a selected pH. In a particular embodiment, it is
contemplated herein
that the compositions of the instant invention are administered to a subject
as an
injectable, including but not limited to injectable compositions for delivery
by
intramuscular, intravenous, subcutaneous, or transdermal injection. Such
compositions
may be formulated using a variety of pharmaceutical excipients, carriers or
diluents
familiar to one of skill in the art.
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[124] In another particular embodiment, the compositions of the instant
invention, and/or pharmaceutical formulations administered in conjunction
therewith,
e.g., antibiotics, may be administered orally. Oral formulations for
administration
according to the methods of the present invention may include a variety of
dosage forms,
e.g., solutions, powders, suspensions, tablets, pills, capsules, caplets,
sustained release
formulations, or preparations which are time-released or which have a liquid
filling, e.g.,
gelatin covered liquid, whereby the gelatin is dissolved in the stomach for
delivery to the
gut. Such formulations may include a variety of pharmaceutically acceptable
excipients
described herein, including but not limited to mannitol, lactose, starch,
magnesium
stearate, sodium saccharine, cellulose, and magnesium carbonate.
[125] In a particular embodiment, it is contemplated herein that a
composition
for oral administration maybe a liquid formulation or as part of a dissolvable
paper placed
on the tongue. Such formulations may comprise a pharmaceutically acceptable
thickening
agent which can create a composition with enhanced viscosity which facilitates
mucosal
delivery of the active agent, e.g., by providing extended contact with the
lining of the
stomach. Such viscous compositions may be made by one of skill in the art
employing
conventional methods and employing pharmaceutical excipients and reagents,
e.g.,
methylcellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl
cellulose, and
carbomer.
[126] Other dosage forms suitable for nasal or respiratory (mucosal)
administration, e.g., in the form of a squeeze spray dispenser, pump dispenser
or aerosol
dispenser, are contemplated herein. Dosage forms suitable for rectal or
vaginal delivery
are also contemplated herein. Where appropriate, compositions for use with the
methods
of the instant invention may also be lyophilized and may be delivered to a
subject with or
without rehydration using conventional methods.
[127] Thus, and as would be understood, the methods of the instant
invention
comprise administering the compositions of the invention to a subject
according to
various regimens, i.e., in an amount and in a manner and for a time sufficient
to provide
a clinically meaningful benefit to the subject. Suitable administration
regimens for use
with the instant invention may be determined by one of skill in the art
according to
conventional methods. For example, it is contemplated herein that an effective
amount
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may be administered to a subject as a single dose, a series of multiple doses
administered
over a period of days, or a single dose followed by one or more additional
"boosting" doses
thereafter. The term "dose" or "dosage" as used herein refers to physically
discrete units
suitable for administration to a subject, each dosage containing a
predetermined quantity
of the active pharmaceutical ingredient calculated to produce a desired
response.
[128] The administrative regimen, e.g., the quantity to be administered,
the
number of treatments, and effective amount per unit dose, etc. will depend on
the
judgment of the practitioner and are peculiar to each subject. Factors to be
considered in
this regard include physical and clinical state of the subject, route of
administration,
intended goal of treatment, as well as the potency, stability, and toxicity of
the particular
composition. As understood by one of skill in the art, a "boosting dose" may
comprise the
same dosage amount as the initial dosage, or a different dosage amount.
Indeed, when a
series of doses are administered in order to produce a desired response in the
subject, one
of skill in the art will appreciate that in that case, an "effective amount"
may encompass
more than one administered dosage amount.
[129] In a preferred embodiment, the compositions described herein can be
administered both topically and systemically, e.g. via IV, intra-articular,
IM, and/or an
injection directly to the site of infection. For example, in the case of a
PJI, the composition
described herein can be directly injected into the infected joint.
Accordingly, the
composition may be formulated as an injectable fluid, semi-solid or depot-type
formulation as will be readily apparent to one of ordinary skill in the art.
However, PJI
could also be treated by IV injection, and/or even as a combination of both
direct injection
to the site of infection along with administration by IV, intra-articular,
and/or IM.
[130] In cases where the infected joint is also being treated by
debridement and/or
replacement of the prosthetic device in a one- or two-stage arthroplasty
exchange, the
composition may be directly administered to the joint during the surgery. For
example,
the composition may be administered on a single occasion (i.e. a "single
shot") or on
multiple occasions as may be required (this "single shot" encompassing both a
direct
injection to the site of infection or by IV). It is however considered that
the composition
may potentially be administered as a single shot and, moreover, may avoid any
requirement for replacing the prosthetic device of the infected joint.
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[131] In some embodiments, the composition may further comprise a further
active agent or preparation such as, for example one or more antibiotics (e.g.
rifampin
and/or a fluoroquinolone such as ciprofloxacin), one or more bactericides,
and/or one or
more other therapeutic molecules such as a small molecule or biologic that has
bactericidal activity. These further agents could be administered as part of
the
composition, or separately yet concurrently with the administration of the
phage
compositions.
[132] In a further aspect, the invention provides a method of treating a
patient
with a prosthetic joint infection (PJI) or at risk of developing a PJI,
comprising
administering a pharmaceutical composition according to the first aspect.
[133] In some embodiments, the patient has a PJI caused by bacteria
selected
from S. aureus, S. epidermidis, E. fecalis, S. caprae, and/or S. lugdunensis.
[134] In other embodiments, the patient has a PJI that has been determined
as
being caused by bacteria selected from S. aureus, S. epidermidis, E. fecalis,
S. caprae,
and/or S. lugdunensis. Said determination may be conducted by, for example,
culturing
synovial fluid obtained from the affected joint by aspiration and determining
the identity
of bacteria in the culture by, for example, 16S rRNA gene sequence analysis.
[135] In other embodiments, the patient is at risk of developing a PJI
caused by
bacteria selected from S. aureus, S. epidermidis, E. fecalis, S. caprae,
and/or S.
lugdunensis.
[136] The method of the invention may be useful for treating or preventing
PJI,
regardless of type. That is, by appropriately selecting the two or more phage
for inclusion
in the composition, the method may be beneficial in treating early, delayed
and late onset
types of PJI, as well as PJI that are the result of a polymicrobial infection.
[137] The method may be performed so as to provide an effective amount of
the
at least two different phage; that is, an amount that is sufficient to cause
lysis (i.e. kill) of
the bacteria present at an infection being treated, such as a PJI being
treated (or in a PJI
that may develop) such that a beneficial or desired clinical result is
achieved and/or to
prevent the occurrence of a bacterial infection. An effective amount can be
administered
in one or more administrations. Typically, an effective amount is sufficient
for treating a
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disease or condition or otherwise to palliate, ameliorate, stabilize, reverse,
slow, delay or
prevent the progression or development of the PJI.
[138] The infection to be treated includes, but is not only limited to, a
bacterial
infection already existing in a patient, but also the prevention of a future
infection that
may or may not occur in a patient with an implantable device. For example,
bacterial
infections of particular strain(s) of bacterium known to frequently occur in a
specific
geographical location (e.g., a geomatched composition) can be treated with the
disclosed
compositions in a prophylactic manner in an attempt to prevent a future
bacterial
infection.
[139] In further details, the method may involve directly administering the
composition to an infected joint or a prosthetic joint at risk of the
development of PJI. In
the latter case, the method may be performed at the time of surgery (i.e. the
composition
may be administered at the time that the joint replacement is being
performed). In cases
where the method is being used to treat an infected joint, the method may
avoid any
requirement for replacing the prosthetic device. That is, the method may be
performed in
lieu of surgery. In other cases, where the infected joint is also being
treated by
debridement and/or replacement of the prosthetic device in a one- or two-stage
arthroplasty exchange, the method may be conveniently performed during the
surgery
(i.e. the composition may be administered to the joint during the surgery).
The
composition may be administered on a single occasion (i.e. a "single shot") or
on multiple
occasions as may be required.
[140] In some embodiments, the method of the invention may be performed as
a
combination therapy. For example, the composition comprising the at least two
different
phage may be administered to the patient as a combination therapy involving
the
administration of a further active agent or preparation such as, for example,
one or more
antibiotics (e.g. rifampin and/or a fluoroquinolone such as ciprofloxacin),
one or more
bactericides, and/or one or more other therapeutic molecules such as a small
molecule or
biologic that has bactericidal activity.
[141] Where performed as a combination therapy with a further active agent
or
preparation, the composition may comprise the at least two different phage
along with
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the further active agent or preparation (i.e. as a single composition), or
otherwise separate
compositions may be used. If administered in separate compositions, the
therapeutic
phage composition and the further active agent or preparation may be
administered
simultaneously or sequentially in any order (e.g. within seconds or minutes
(e.g. 5 to 6o
mins) or even hours (e.g. 2 to 48 hours)).
[142] Although the invention herein has been described with reference to
embodiments, it is to be understood that these embodiments, and examples
provided
herein, are merely illustrative of the principles and applications of the
present invention.
It is therefore to be understood that numerous modifications can be made to
the
illustrative embodiments and examples, and that other arrangements can be
devised
without departing from the spirit and scope of the present invention as
defined by the
appended claims. All patent applications, patents, literature and references
cited herein
are hereby incorporated by reference in their entirety_
EXAMPLES
[143] The invention will now be further illustrated with reference to the
following
example(s). It will be appreciated that what follows is by way of example only
and that
modifications to detail may be made while still falling within the scope of
the invention.
Example 1:
Selection of phage for inclusion in PJI composition
[144] A composition is designed for the treatment of PJI that may be caused
by
one or more of S. aureus (MRSA and/or MSSA), S. epidermidis, E. fecalis, S.
caprae,
and/or S. lugdunensis. Candidate phage for inclusion in the composition are
identified
from those publicly available from the Felix d'Herelle Reference Center for
Bacterial
Viruses of the Universite Laval (Quebec City, Quebec, Canada;
www.phage.ulaval.ca) and
proprietary phage as listed in Table 1 (above).
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Formulation of injectable composition
[145] In one particular example, an injectable fluid composition is
prepared by
combining about loll pfu of each of the following phage, APT-PJI-oi, APT-PJI-
13 and
APT-PJI-15, in a pharmaceutically acceptable diluent (e.g. isotonic saline).
The
composition may be provided in a pre-loaded syringe.
Example 2:
Selection of phage for inclusion in PJI composition
[146] The composition of Example 1 may be readily modified by adding to the
composition, or replacing one or more phage of the composition with, one or
more phage
targeted to lyse other causative bacteria of an implantable device infection
(such as, for
example, PJI) (e.g. S. lugdunensis, E. feciurn, S. agalactiae, P. aeruginosa,
K.
pneumoniae, E. coli and E. clocae). Examples of some candidate phage for
inclusion in
such compositions are identified from those publicly available from the Felix
d'Herelle
Reference Center for Bacterial Viruses of the Universite Laval (Quebec City,
Quebec,
Canada; www.phage.ulaval.ca) and are listed below in Table 2.
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9
APT-14-PCT Final
9 Table 2
Bacterial
Phage Name
GenBank
Bacteria Phage ID code host
Comments
(HER#)* Accession NO.
HER#*
Siphoviridae, A-like
144 (144) 1144
NC oo1416.1
viruses
30 T7 (30) 1024
Podoviridae, species T7 NC_001604.1
E. coil
552 Ebrios (552) 1024
Podoviridae
241 (1392 (241) 1240
Myoviridae NC o23693
F8 Lindberg (10) 1010 Myoviridae DQ163917
P. aeruginosa
369 PP7 (369) 1369
Leviviridae NC 001628
K. pneumoniae 173 1(13 (173) 1173
Myoviridae,
phages
Myoviridae, T4-like
E. dome 67 1 (67) 1067
viruses
ni
Cl,ni
ni
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Example 3:
Prophetic case study
[147] Two months after receiving a ceramic-on-polyethylene replacement hip,
a
55 year old male patient reported to his orthopedic surgeon with considerable
pain in the
joint. He is referred for a CT scan of the affected joint and this confirms
joint distention
and indicates infection of the periprosthetic tissues. Blood tests also
provide further
evidence of PJI. Subsequently, a sample of synovial fluid is drawn from the
hip joint by
joint aspiration (athrocentesis) and the bacteria present in the fluid is
cultured in liquid
media. Extracted bacterial DNA is then subjected to sequence analysis of the
V1-V3 region
of the 16S rRNA gene to identify the bacterial species present using a 16S
rRNA sequence
database. The patient's PJI is found to be caused by S. aureus and E. fecalis.
[148] The patient is administered, by direct injection into the affected
joint, with
a single dose of a therapeutic phage composition including about 109 pfu of
each of the
APT-PJI-oi (targeted to S. aureus) and APT-PJI-15 (targeted to E. fecalis)
phage in a
pharmaceutical saline. Subsequently, the patient is treated with intravenous
(IV) phage
(APT-PJI-oi and/or APT-PJ-15) for 10 days. Following the treatment, the
patient is
closely monitored for clinical response. After 4 weeks, the patient reports
that he had little
or no remaining pain. The joint might be aspirate to determine whether any
infection
remains. A further CT scan may be performed if necessary to determine whether
the joint
is no longer infected.
[149] The invention is not limited to the embodiment herein before
described
which may be varied in construction and detail without departing from the
spirit of the
invention. The entire teachings of any patents, patent applications or other
publications
referred to herein are incorporated by reference herein as if fully set forth
herein.
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