WO2017087679A2

WO2017087679A2 - Bacterial proteases targeting the nf-kb transcription factor family

Info

Publication number: WO2017087679A2
Application number: PCT/US2016/062541
Authority: WO
Inventors: Jorge E. Galan; Hui Sun
Original assignee: Yale University
Current assignee: Yale University
Priority date: 2015-11-17
Filing date: 2016-11-17
Publication date: 2017-05-26
Anticipated expiration: 2018-05-17
Also published as: WO2017087679A3

Abstract

The present invention provides compositions and methods for treating or preventing a disease or disorder associated with increased NF-κΒ signaling. In certain aspects, the invention may be used treating cancer or inflammatory diseases. In one aspect, the invention is an agent which cleaves the NF-κΒ transcription factors RelA, RelB, cRel or a combination thereof. In another aspect, PipA, GtgA or GogA proteins or nucleic acids which encode PipA, GtgA or GogA inhibit NF-κΒ signaling.

Description

TITLE OF THE INVENTION

Bacterial proteases targeting the NF-κΒ transcription factor family

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S Provisional Patent

Application No. 62/256,468, filed November 17, 2015, which is hereby incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

This invention was made with government support under AI055472 awarded by the National Institute of Allergy and Infectious Disease. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Bacterial pathogens that have sustained long-standing associations with their hosts have evolved complex adaptations to modulate host functions to ensure their survival and replication (Issac and Isberg, 2014, Future Microbiol 9:343- 59; Alix et al., 2011, J Cell Biol 195:943-52; Alto and Orth, 2012, Cold Spring Harb Perspect Biol 4:a006114). One of these adaptations is the type III secretions system (T3SS), a complex multi-protein machine that delivers bacterially-encoded proteins into host cells (Galan et al, 2014, Annu Rev Microbiol 68:415-38). These bacterial proteins, known as effectors, have the capacity to modulate or interfere with a variety of cellular processes (Galan, 2007, Cell 130:92). Salmonella enterica serovar Typhimurium (S. Typhimurium), a cause of severe gastroenteritis in humans, encodes two T3SSs within its pathogenicity islands 1 (SPI-1) and 2 (SPI-2), which in a coordinated fashion deliver more than 40 effector proteins into target host cells (Figueira and Holden, 2012, Microbiology 158: 1147-61; Ibarra and Steele-Mortimer, 2009, Cell Microbiol 11: 1579-86; Galan, 2001, Annu Rev Cell Dev Biol 17:53-86). These effectors mediate bacterial entry, survival, and replication within host cells. In addition, through its type III secretion systems, Salmonella stimulates transcriptional responses leading to inflammation (Hobbie et al., 1997, J Immunol 159:5550-9; Chen et al, 1996, Science 274:2115-8; Bruno et al, 2009, PLoS Pathog 5:el000538). Although inflammation is usually viewed as a host defense response that limits pathogen replication, for S. Typhimurium the stimulation of inflammation is necessary to acquire essential nutrients and respiration substrates that become available only in inflamed intestinal tissues (Stecher et al, 2007, PLoS Biol 5:2177- 89; Winter et al., 2010, Nature 467:426-9).

Although the biochemical activities of several effector proteins delivered by these T3SSs are well-characterized (Figueira and Holden, 2012, Microbiology 158: 1147-61 ; Hardet et al, 1998, Cell 93:815-26; Fu and Galan, 1999, Nature 401 :293-7; Zhou et al, 1999, Science 283:2092-5; Du and Galan, 2009, PLoS Pathog 5:el000595; Norris et al., 1998, PNAS 95: 14057-9; Ohlson et al, 2008, Cell host Microbe 4:434-46; Spano and Galan, 2012, Science 338:960-3; Odendall et al, 2012, Cell Host Microbe 12:657-68; Mazurkiewicz et al., 2008, Mol Microbiol 67: 1371-83; Rytkonen et al, 2007, PNAS 104:3502-7), the function of most effector proteins remains unknown. PipA, GtgA, and GogA are three highly related

Salmonella effector proteins encoded within the Salmonella pathogenicity island 5 (PipA) (Wood et al, 1998, Mol Microbiol 29: 883-91) or within lysogenic phages (GtgA and GogA) (Figueroa-Bossi et al, 2001, Mol Microbiol 39:260-71 ; Smith and Ahmer, 2003, J Bacteriol 185: 1357-66). These effectors are highly conserved in Salmonella enterica in that all known isolates encode at least one member of this family. In addition, homologs can also be detected in some strains of pathogenic Escherichia coli and the endosymbiont Arsenophonus nasoniae. Despite their widespread distribution within Salmonellae, nothing is known about their function.

The NF-KB family of transcription factors plays an essential role in inflammation and in many steps of cancer initiation and progression (Hoesel and Schmid, 2013, Mol Cancer 12:86). The most important members of this family are the transcription factors RelA (p65) and RelB, which form homo or hetero dimers with other family members. In non-stimulated cells these dimers are bound to inhibitory molecules of the ΙκΒ family of proteins inhibitors of NF-κΒ. Activation of the NF-kB pathway occurs by the release of the transcription factors from their inhibitory molecules. The transcription factors are then translocated to the nucleus where they stimulate the expression of a variety of gene required for inflammation or cancer progression.

There is a need in the art for compositions and methods for treating diseases and disorders associated with the NF-κΒ pathway. The present invention satisfies this unmet need. SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of inhibiting NF-KB signaling in a subject in need thereof. In one embodiment, the method comprises administering to the subject a therapeutically effective amount of a composition comprising at least one agent that inhibits a NF-κΒ transcription factor, wherein the NF-KB transcription factor is selected from the group consisting of RelA, RelB and cRel.

In one embodiment, the agent comprises at least one isolated peptide. In one embodiment, the at least one isolated peptide comprises an amino acid sequenced selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In one embodiment, the at least one peptide is selected from the group consisting of PipA, GtgA and GogA. In one embodiment, the at least one agent cleaves the NF-κΒ transcription factor.

In another aspect, the invention provides a method of treating or preventing a disease or disorder associated with NF-κΒ in a subject in need thereof. In one embodiment, the method comprises administering to the subject an effective amount of a composition comprising at least one agent that inhibits a NF-KB transcription factor, wherein the NF-κΒ transcription factor is selected from the group consisting of RelA, RelB and cRel.

In one embodiment, the at least one agent cleaves the NF-KB transcription factor.

In one embodiment, the agent comprises at least one isolated peptide. In one embodiment, the at least one isolated peptide comprises an amino acid sequenced selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In one embodiment, the at least one peptide is selected from the group consisting of PipA, GtgA and GogA.

In one embodiment, the at least one agent comprises at least one isolated nucleic acid. In one embodiment, the at least one isolated nucleic acid encodes at least one protein that cleaves the NF-κΒ transcription factor.

In one embodiment, the method further comprises administering to the subject at least one additional therapeutic agent. In one embodiment, the therapeutic agent is selected from the group consisting of anti-cancer agent, and antiinflammatory agent. In one embodiment, disease or disorder is selected from the group consisting of cancer, diabetes, heart disease, asthma, inflammatory bowel disease, lupus, Alzheimer's disease, Huntington's disease, chronic obstructive pulmonary disease (COPD), atopic dermatitis, atopy, allergy, allergic rhinitis, Crohn's disease, and scleroderma.

In another aspect, the invention provides a composition for inhibiting the NF-KB signaling pathway. In one embodiment, the composition comprises at least one agent that inhibits a NF-κΒ transcription factor, wherein the NF-κΒ transcription factor is selected from the group consisting of RelA, RelB, and cRel.

In one embodiment, the at least one agent cleaves the NF-KB

transcription factor.

In one embodiment, the at least one agent comprises at least one peptide. In one embodiment, the at least one peptide is at least one selected from the group consisting of PipA, GtgA and GogA. In one embodiment, the at least one peptide comprises at least one amino acid sequenced selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

In one embodiment, the at least one agent comprises at least one isolated nucleic acid. In one embodiment, the at least one isolated nucleic acid encodes at least one protein that cleaves the NF-κΒ transcription factor. In one embodiment, the at least one isolated nucleic acid encodes at least one full-length protein selected from the group consisting of PipA, GtgA and GogA.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

Figure 1, comprising Figure 1 A through Figure ID, depicts the results of example experiments demonstrating that the absence of the PipA-family of effector proteins increases the mouse virulence of S. Typhimurium without increasing bacterial loads. Figure 1A depicts experimental results of C57/BL6 nramp^+/+ mice orally infected with wild-type S. Typhimurium or the ApipA AgogA AgtgA mutant and bacterial loads in the indicated tissues enumerated 7 days after infection. Each circle represents the bacterial load for an individual animal and horizontal bars indicate geometric means. Figure IB depicts experimental results of C57/BL6 nramp^+/+ mice intraperitoneally infected with wild-type S. Typhimurium or the ΔρίρΑ AgogA AgtgA mutant and bacterial loads in the indicated tissues enumerated 7 days after infection. Each circle represents the bacterial load for an individual animal and horizontal bars indicate geometric means. Figure 1C depicts the survival of animals orally infected with wild-type S. Typhimurium (n=7) or the ApipA AgogA AgtgA mutant (n=8) strains 6 days after infection. The p values of the difference in the survival of animals infected with wild type or mutant strains determined by the log- rank test are shown. Figure ID depicts the survival of animals intraperitoneally infected with wild-type S. Typhimurium (n=7) or the ApipA AgogA AgtgA mutant (n=8) strains 6 days after infection. The p values of the difference in the survival of animals infected with wild type or mutant strains determined by the log-rank test are shown.

Figure 2, comprising Figure 2A and Figure 2B, depicts the results of example experiments demonstrating that the absence of the PipA-family of effector proteins increases the ability of S. Typhimurium to stimulate pro-inflammatory cytokine production in the mouse intestine. Figure 2A depicts experimental results of C57/BL6 nramp+/+ mice orally infected with wild type (n=5) or ApipA AgogA AgtgA (n=5) S. Typhimurium strains and 24 hours after infection the relative levels of the indicated cytokines in the intestine were measured by quantitative PCR. Figure 2B depicts experimental results of C57/BL6 nramp+/+ mice orally infected with wild type (n=5) or ApipA AgogA AgtgA (n=5) S. Typhimurium strains and 48 hours after infection the relative levels of the indicated cytokines in the intestine were measured by quantitative PCR. Data were normalized to the levels of GAPDH and are expressed relative to uninfected control animals (n=5). The data shown were compiled from two independent experiments of three measurements each, p values of the indicated differences determined by the Student t test are shown.

Figure 3, comprising Figure 3A through Figure 3F, depicts the results of example experiments demonstrating that the PipA family of effector proteins negatively regulates Salmonella-induced NF-κΒ signaling. Figure 3A depicts experimental results of HEK293T cells transfected with a plasmid encoding an Elkl signaling reporter construct. Eighteen hours after transfection, cells were infected with the indicated S. Typhimurium strains at a MOI=5 and the reporter activity was measured at 8 hours after infection. Data are shown relative to the activity of the reporter in uninfected control cells and represent the mean ± standard deviation of three independent measurements. Figure 3B depicts experimental results of HEK293T cells transfected with a plasmid encoding a STAT3 signaling reporter construct. Eighteen hours after transfection, cells were infected with the indicated S.

Typhimurium strains at a MOI=5 and the reporter activity was measured at 8 hours after infection. Data are shown relative to the activity of the reporter in uninfected control cells and represent the mean ± standard deviation of three independent measurements. Figure 3C depicts experimental results of HEK293T cells transfected with a plasmid encoding a NF-κΒ signaling reporter construct. Eighteen hours after transfection, cells were infected with the indicated S. Typhimurium strains at a MOI=5 and the reporter activity was measured at 8 hours after infection. Data are shown relative to the activity of the reporter in uninfected control cells and represent the mean ± standard deviation of three independent measurements. Figure 3D depicts experimental results of HEK293T cells transfected with a plasmid encoding a NF-κΒ signaling reporter construct. Eighteen hours after transfection, cells were infected with the indicated S. Typhimurium strains at a MOI=5 and the reporter activity was measured at the indicated times after infection. Data are shown relative to the activity of the reporter in uninfected control cells and represent the mean ± standard deviation of three independent measurements. Figure 3E depicts experimental results of HEK293T cells transfected with a plasmid encoding a NF-κΒ signaling reporter construct. Eighteen hours after transfection, cells were infected with the indicated S. Typhimurium strains at a MOI=5 and the reporter activity was measured at 8 hours after infection. Data are shown relative to the activity of the reporter in uninfected control cells and represent the mean ± standard deviation of three independent measurements. Figure 3F depicts experimental results of HEK293T cells transfected with a plasmid encoding a NF-κΒ reporter construct along with 25 or 50 ng of a plasmid encoding PipA, GtgA, or GogA. Eighteen hours after transfection, cells were infected with S. Typhimurium AgogA AgtgA ApipA at a MOI = 5 and the reporter activity was measured 8 hours after infection. Data are shown relative to the activity of the reporter in uninfected control cells and represent the mean ± standard deviation of three independent measurements.

Figure 4, comprising Figure 4A through Figure 4C, depicts the results of example experiments demonstrating that the PipA family of effector proteins can directly inhibit NF-κΒ signaling. Figure 4A depicts a simplified diagram of the TRIF signaling pathway. Figure 4B depicts experimental results of HEK293T cells transfected with a plasmid encoding a NF-κΒ reporter construct along with plasmids encoding the indicated proteins (horizontal axis) in conjunction with a plasmid encoding PipA or the empty vector (mock). The reporter activity was subsequently measured 18 hours after transfection. Data are shown relative to the activity of the reporter in control cells (transfected with the empty vector) and represent the mean ± standard deviation of three independent measurements. Figure 4C depicts

experimental results of HEK293T cells transfected with a plasmid encoding a NF-κΒ reporter construct along with 25 or 50 ng of a plasmid encoding PipA, GtgA or GogA. Eighteen hours after transfection, cells were treated with TNFa (10 ng/ml) or infected with the ApipA AgogA AgtgA S. Typhimurium at a MOI = 5 and the reporter activity was measured 8 hours after treatment. Data are shown relative to the activity of the reporter in uninfected control cells and represent the mean ± standard deviation of three independent measurements.

Figure 5, comprising Figure 5A through Figure 5F, depicts the results of example experiments demonstrating that PipA, GtgA and GogA inhibit the NF-KB pathway by directly targeting RelA. Figure 5A depicts experimental results of HEK293T cells transiently transfected with plasmids encoding M45-tagged RelA or HA-tagged TRAF2, along with the indicated amounts of a plasmid encoding FLAG- tagged PipA or the empty vector (mock). Eighteen hours after transfection, cell lysates were analyzed by Western blot with anti-M45, anti-HA, anti-FLAG, and anti- tubulin antibodies (as loading control). Figure 5B depicts experimental results of HEK293T cells transiently transfected with plasmids encoding M45-tagged RelA, along with plasmids encoding FLAG-tagged GogA, or GtgA or the empty vector (control). Eighteen hours after transfection, cell lysates were analyzed by Western blot with anti-M45 and anti-FLAG antibodies. Figure 5C depicts experimental results of HEK293T cells transiently transfected with plasmids encoding M45-tagged STAT3, or c-Jun along with plasmids encoding FLAG-tagged PipA, GogA, or GtgA or the empty vector (mock). Eighteen hours after transfection, cell lysates were analyzed by Western blot with anti-M45 and anti-FLAG antibodies. Figure 5D depicts experimental results of HEK293T cells transiently transfected with a plasmid encoding M45-tagged RelA. Eighteen hours after transfection, cells were infected with wild type or the ApipA AgogA AgtgA S. Typhimurium strains with a MOI=10. Cell lysates were analyzed by Western blot with anti-M45 or anti tubulin (as loading control) antibodies at the indicated times after infection. Figure 5E depicts experimental results of HeLa cells infected with wild type or the ΔρίρΑ AgogA AgtgA S. Typhimurium strains with a MOI = 10. Six hours after infection, cell lysates were analyzed by Western blot with anti-RelA, and anti-tubulin antibodies (as loading control). Figure 5F HeLa cells were infected with wild type or the ApipA AgogA AgtgA S. Typhimurium strains with a MOI = 10. At indicated times after infection, cell lysates were analyzed by Western blot with anti-phosphorylated STAT3 and anti- tubulin antibodies (as loading control).

Figure 6, depicts the results of example experiments demonstrating that PipA, GtgA and GogA are specific proteases for transcription factors of the RelA family. Immunofluorescence staining of HeLa cells transfected with a plasmid encoding FLAG-tagged PipA, GogA, or GtgA or infected with S. Typhimurium encoding FLAG-tagged PipA, GogA, or GtgA. Eighteen hours after transfection and 4 hours after bacterial infection, cells were stained with an anti FLAG antibody (to visualize the effector proteins) and 4',6-diamidino-2-phenylindole (DAPI) to visualize nuclear DNA. Scale bars: 5 and 10 μιτι for PipA and GogA/GtgA, respectively.

Figure 7, comprising Figure 7A through Figure 7E, depicts the results of example experiments demonstrating that PipA, GtgA and GogA are specific proteases for transcription factors of the RelA family. Figure 7 A depicts experimental results of HEK293T cells transiently transfected with plasmids encoding M45-tagged RelA, along with plasmids encoding either FLAG-tagged PipA or its catalytic mutant PipA^E181A. Eighteen hours after transfection, cell lysates were analyzed by Western blot with anti-M45, anti-FLAG, and anti-tubulin (as loading control) antibodies. Figure 7B depicts experimental results of HEK293T cells transiently transfected with a plasmid encoding a NF-κΒ reporter construct, along with 25, 50 and 100 ng of plasmid DNA encoding either FLAG-tagged PipA or its catalytic mutant PipA^E181A. Eighteen hours after transfection, cells were infected with the AgogA/AgtgA/ApipA S. Typhimurium mutant strain at MOI=5 and the reporter activity measured 8 hours after infection. Data are the reporter activity relative to control cells (no infection) and represent the mean ± standard deviation of three independent experiments. Figure 7C depicts purified RelA_1-21o was incubated with purified PipA or PipA^E181A in a reaction buffer in the presence [50 (+) or 100 (++) mM] or absence (-) of EDTA. The reaction was stopped by addition of SDS loading buffer, proteins separated by SDS-PAGE and visualized by Coomassie blue staining. RelA_1-21o and its cleaved product are indicated. Figure 7D depicts purified RelA_1-21o was incubated with purified GogA, GtgA, or PipA in a reaction buffer. The reaction was stopped by addition of SDS loading buffer, proteins separated by SDS-PAGE and visualized by Coomassie blue staining. RelA_1-21o and its cleaved product are indicated. Figure 7E depicts experimental results of HEK293T cells transiently transfected with plasmids encoding M45-tagged RelB, pl05, or plOO, along with plasmids encoding FLAG-tagged PipA, GogA, or GtgA or the empty vector (mock). Eighteen hours after transfection, cell lysates were analyzed by Western blot with anti-M45 and anti-FLAG antibodies.

Figure 8 depicts the amino acid sequence alignment of the S.

Typhimurium PipA family of effector proteins. GtgA, GogA, and PipA are from S. Typhimurium strains SL1344. The Escherichia coli (WP 044710484.1) and

Arsenophonus nasoniae (WP_026822997.1) sequences were obtained from National Center for Biotechnology Information data base.

Figure 9, comprising Figure 9A and Figure 9B, depicts the results of example experiments demonstrating that the absence of the PipA-family of effector proteins does not increase the bacterial loads of S. Typhimurium in NRAMPl- deficient mice. Figure 9A depicts experimental results of C57BL/6 (nrampl^"7") mice orally infected with wild-type S. Typhimurium or the ApipA/AgogA/AgtgA mutant and bacterial loads in the indicated tissues enumerated 6 days after infection. Figure 9B depicts experimental results of C57BL/6 (nrampl^"7") mice intraperitoneally infected with wild-type S. Typhimurium or the ApipA/AgogA/AgtgA mutant and bacterial loads in the indicated tissues enumerated 6 days after infection.

Figure 10, comprising Figure 10A and Figure 1 OB, depicts the results of example experiments demonstrating the survival of nrampl^+/+ or nrampl^"7" mice after oral infection with the S. Typhimurium ApipA AgogA AgtgA mutant strain. Figure 10A depicts experimental results of C57BL/6 nrampl^+/+ mice orally infected with 5 x 10⁸ c. f. u. of wild type S. Typhimurium (n=10) or the ApipA/AgogA/AgtgA triple mutant (n=l l) and survival was recorded over time. Figure 10B depicts

/ 8 experimental results of C57BL/6 nrampl^{" "} mice orally infected with 5 x 10 c. f. u. of wild type S. Typhimurium (n=10) or the ApipA/AgogA/AgtgA triple mutant (n=l 1) and survival was recorded over time. The p values of the difference in the survival of animals infected with wild type or mutant strains are shown.

Figure 11 depicts the results of example experiments demonstrating that the absence of GtgA, GogA and PipA results in increased intestinal inflammation. C57BL/6 nrampl mice were either mock infected (control) or infected orally with 10^s wild type or AgtgA/AgogA/ApipA S. Typhimurium strains. Four days after infection, ceca were removed, fixed, and embedded in paraffin, and tissue sections were stained with hematoxylin and eosin. Each photograph was obtained from a different animal. Similar results were obtained in four independent animals for each group.

Figure 12, comprising Figure 12A and Figure 12B, depicts the results of example experiments demonstrating that Absence of the PipA-family of type III secretion effector proteins does not affect the ability of S. Typhimurium to invade and replicate within cultured epithelial cells. Figure 12A depicts experimental results of Henle-407 cells infected with S. Typhimurium wild type, or the AgogA, AgtgA, ApipA, or AgogA/AgtgA/ApipA triple mutant strains at MOI=5. Bacterial invasion was measured by the gentamicin protection assay as indicated in Material and Methods. Values represent the % of the inoculum that survive the gentamicin treatment due to bacterial internalization and have been standardized relative to the levels of invasion of wild-type S. Typhimurium, which was considered to be 100%. The results represent the mean ± standard deviation of three independent experiments. Figure 12B depicts experimental results of Henle-407 cells infected with S.

Typhimurium wild type, or the AgogA, AgtgA, ApipA, or AgogA/AgtgA/ApipA triple mutant strains at MOI=5. The number of c.f.u was enumerated 30 minutes and 8 hours after infection. Values are the fold change after 8 hours of infection (relative to the values at 30 minutes after infection) and represent the mean ± standard deviation of three independent experiments.

Figure 13 depicts the conservation of a protease motif in the PipA family of effector proteins. Shown is the amino acid sequence alignment of the S. Typhimurium PipA family of effector proteins depicting the location of a conserved metaloprotease Zn-binding motif.

Figure 14 depicts the absence of the PipA-family of effector proteins. Shown is the C57BL/6 (nramp^+/+) mice orally infected with wild-type S.

Typhimurium or the ApipA/AgogA/AgtgA mutant and bacterial loads in the indicated tissues enumerated 15 days after infection. Each circle represents the bacterial load for an individual animal and horizontal bars indicate geometric means. The differences between the means of the wild type and mutant c.f.u in the different tissues were not statistically significant (p > 0.5). Figure 15 depicts serum cytokine levels in mice infected with wild type S. Typhimurium or the ΔρίρΑ AgogA AgtgA isogenic mutant strain. C57/BL6 nramp^+/+ mice were orally infected with wild type (n = 4) or ApipA AgogA AgtgA (n = 4) S. Typhimurium strains and 4 days after infection the levels of the indicated cytokines in the serum measured by ELISA. Values represent the mean ± standard deviations of the measurements.

DETAILED DESCRIPTION

The present invention provides compositions and methods for treating and preventing diseases or disorders which are associated with NF-κΒ activity. The present invention is based, in part, upon the unexpected finding that a family of related Salmonella Typhimurium effector proteins PipA, GogA and GtgA specifically and redundantly target components of the NF-κΒ signaling pathway to inhibit transcriptional responses leading to inflammation. These effector proteins are highly specific proteases that cleave both the RelA (p65) and RelB transcription factors, but do not target plOO (NF- κΒ2) or pl05 (NF- κΒΙ). Thus, the present invention provides compositions and methods for treating or preventing diseases or disorders which are associated with NF-κΒ activity by inducing the proteolytic cleavage of RelA, RelB, or cRel with effector proteins. For example, in certain aspects the present invention comprises the use of PipA, GogA or GtgA peptides, agents which proteolytically cleave RelA, RelB, and cRel, to treat and prevent diseases and disorders associated with NF- κΒ activity.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, each of the following terms has the meaning associated with it in this section.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

"About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

The term "abnormal" when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the "normal" (expected) respective characteristic. Characteristics which are normal or expected for one cell or tissue type, might be abnormal for a different cell or tissue type.

The terms "cells" and "population of cells" are used interchangeably and refer to a plurality of cells, i.e., more than one cell. The population may be a pure population comprising one cell type. Alternatively, the population may comprise more than one cell type. In the present invention, there is no limit on the number of cell types that a cell population may comprise.

The term "cancer" as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, head and neck cancers, lymphoma, leukemia, lung cancer and the like.

An "inflammatory disease" is used herein to refer to a state in which there is a response to tissue damage, cell injury, an antigen, and/or an infectious disease. In some cases, causation will not be able to be established. The symptoms of inflammation may include, but are not limited to cell infiltration and tissue swelling. Disease states contemplated under the definition of inflammatory disease include asthma, chronic obstructive pulmonary disease, interstitial lung disease, chronic obstructive lung disease, chronic bronchitis, eosinophilic bronchitis, eosinophilic pneumonia, pneumonia, inflammatory bowel disease, atopy dermatitis, atopy, allergy, allergic rhinitis, idiopathic pulmonary fibrosis, scleroderma, and emphysema.

A "disease" is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate. In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

A disease or disorder is "alleviated" if the severity of a sign or symptom of the disease or disorder, the frequency with which such a sign or symptom is experienced by a patient, or both, is reduced.

"Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA

corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

An "effective amount" or "therapeutically effective amount" of a compound is that amount of compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered.

"Expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

As used herein, the term "fragment," as applied to a nucleic acid, refers to a subsequence of a larger nucleic acid. A "fragment" of a nucleic acid can be at least about 15 nucleotides in length; for example, at least about 50 nucleotides to about 100 nucleotides; at least about 100 to about 500 nucleotides, at least about 500 to about 1000 nucleotides; at least about 1000 nucleotides to about 1500 nucleotides; about 1500 nucleotides to about 2500 nucleotides; or about 2500 nucleotides (and any integer value in between). As used herein, the term "fragment," as applied to a protein or peptide, refers to a subsequence of a larger protein or peptide. A "fragment" of a protein or peptide can be, for example, at least about 5 amino acids in length; for example, at least about 10 amino acids in length; for example, at least about 20 amino acids in length; for example, at least about 50 amino acids in length; at least about 100 amino acids in length; at least about 200 amino acids in length; at least about 300 amino acids in length; or at least about 400 amino acids in length (and any integer value in between).

"Homologous" refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared X 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.

"Isolated" means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

The terms "patient," "subject," "individual," and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

"Parenteral" administration of a composition includes, e.g., subcutaneous (s.c), intravenous (i.v.), intramuscular (i.m), or intrasternal injection, or infusion techniques.

The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric "nucleotides." The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

As used herein, the term "promoter/regulatory sequence" means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.

A "constitutive" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

An "inducible" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.

A "tissue-specific" promoter is a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

"Proliferation" is used herein to refer to the reproduction or multiplication of similar forms, especially of cells. That is, proliferation encompasses production of a greater number of cells, and can be measured by, among other things, simply counting the numbers of cells, measuring incorporation of H-thymidine into the cell, and the like.

As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, a subject is preferably a mammal such as a non- primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) and a primate (e.g., monkey and human), most preferably a human. A "therapeutic" treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.

As used herein, "treating a disease or disorder" means reducing the frequency with which a symptom of the disease or disorder is experienced by a patient.

The phrase "therapeutically effective amount," as used herein, refers to an amount that is sufficient or effective to prevent or treat (delay or prevent the onset of, prevent the progression of, inhibit, decrease or reverse) a disease or disorder, including alleviating symptoms of such diseases.

To "treat" a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

As used herein, the phrase "a tumor site" refers to any site or region within a subject which a tumor has formed, may be expected to form, or was previously located. In certain embodiments, the tumor site is in need of anti-tumor activity.

A "vector" is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or a virus. The term should also be understood to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Description

The present invention is based, in part, on the discovery that

Salmonella Typhimurium effector proteins PipA, GogA and GtgA specifically cleave components of the NF-κΒ signaling pathway. Specifically, it is demonstrated herein that PipA, GogA, and GtgA cleave RelA,RelB, and cRel transcription factors, thereby inhibiting NF-κΒ activity or NF-κΒ signaling.

In one embodiment, the invention provides compositions for inhibiting NF-KB signaling pathway, where the composition comprises an agent which cleaves NF-KB transcription factors. In one embodiment, the composition comprises at least one of PipA, GogA, GtgA, or a combination thereof. In one embodiment, the composition comprises at least one isolated nucleic acid encoding at least one of PipA, GogA, GtgA, or a combination thereof. In certain embodiments composition comprises at least one amino acid sequence selected from SEQ ID NO: l, SEQ ID NO:2, or SEQ ID NO:3. In certain embodiments composition comprises at least one isolated nucleic acid encoding an amino acid sequence selected from SEQ ID NO: l, SEQ ID NO:2, or SEQ ID NO:3.

In one embodiment, the invention provides a method of inhibiting NF- KB activity or NF-κΒ signaling in a cell. For example, in one embodiment, the method comprises administering to the cell an effective amount of a composition comprising an agent that cleaves a NF-κΒ transcription factor. In one embodiment, the composition comprises at least one of PipA, GogA, GtgA, or a combination thereof. In one embodiment, the composition comprises one or more nucleic acid molecules encoding PipA, GogA, GtgA, or a combination thereof.

In one embodiment, the invention provides a method of inhibiting NF- KB activity or NF-κΒ signaling in a subject in need thereof. For example, in one embodiment, the method comprises administering to the subject a therapeutically effective amount of a composition comprising at least one agent that cleaves a NF-KB transcription factor. In one embodiment, the composition comprises at least one of PipA, GogA, GtgA, or a combination thereof. In one embodiment, the composition comprises one or more nucleic acid molecules encoding PipA, GogA, GtgA, or a combination thereof. In one embodiment, the invention provides a method for treating or preventing a disease or disorder associated with NF-κΒ activity or NF-κΒ signaling in a subject in need thereof. In one embodiment, administering to the subject an effective amount of a composition comprising at least one agent that cleaves a NF-KB transcription factor. In one embodiment, the composition comprises at least one of PipA, GogA, GtgA, or a combination thereof. In one embodiment, the composition comprises one or more nucleic acid molecules encoding PipA, GogA, GtgA, or a combination thereof.

In one embodiment, the invention provides a method for treating a disease or disorder associated with NF-κΒ activity, including, but not limited to cancer, diabetes, heart disease, asthma, inflammatory bowel disease, lupus,

Alzheimer's, Huntington's disease, chronic obstructive pulmonary disease (COPD), atopic dermatitis, atopy, allergy, allergic rhinitis, scleroderma, Crohn's disease, and the like. In certain aspects, the compositions and methods of the present invention can reduce NF-κΒ pathway activation, thereby treating a disease or disorder associated with NF-KB.

Compositions

In one aspect, the present invention comprises compositions for treating and preventing cancer and inflammatory diseases. In one embodiment, the composition comprises at least one agent that cleaves NF-κΒ transcription factors, such as RelA, RelB or cRel, thereby inhibiting the NF-κΒ signaling pathway.

Exemplary agents, include, but are not limited to, isolated nucleic acids, vectors, isolated peptides, peptide mimetics, small molecules, and the like. In certain embodiments the at least one agent comprises PipA, GogA, GtgA, or a combination thereof. In certain embodiments, the agent comprises at least one nucleic acid molecule encoding at least one of PipA, GogA, GtgA, or a combination thereof.

An agent that decreases NF-κΒ signaling is any agent that decreases the normal endogenous activity associated with NF-κΒ signaling. In certain embodiments, the agent modulates the level or activity of the NF-κΒ pathway by modulating the transcription, translation, splicing, degradation, enzymatic activity, binding activity, or combinations thereof, of NF-κΒ. In certain embodiments, the agent cleaves the RelA, RelB and cRel NF-κΒ subunits, thereby decreasing NF-KB activity. In one embodiment, the composition of the present invention comprises an at least one isolated peptide comprising PipA, GogA or GtgA, or biologically functional fragment thereof. The composition may comprise, for example, any isoform of PipA, GogA or GtgA, including PipA, GogA or GtgA from any organism. In one embodiment, the composition comprises full-length PipA, GogA or GtgA. In one embodiment, the composition comprises recombinant PipA, GogA or GtgA.

An exemplary amino acid sequence of PipA is:

MLPVTYRLIPQSGVSTYRLNTADTPVFPDIPEHAPNPSRLRLAHDSLAINSEFR LEPECVVEYLISGAGGIDPDTEIDDDTYDECYDELSSVLQNAYTQSETFRRLM NYAYEKELHDVEQRWLLGAGEAFETTVAQEHFKLSEGRKVICLNLDDSDDS YTEHYESNEGRQLFDTKRSFIHEVVHALTHLQDKEENHPRGPVVEYTNIILKE MGHPSPPRMVYIFNK (SEQ ID NO: 1).

An exemplary amino acid sequence of GtgA is:

MPTGIKPIFINNMMSTYGL SHPHD SKVFPDLPEHQDNP S QLRLQHD GL ATDDK ARLEPMCL AEYLI S GPGGMDPDIEIDDDTYDECREVL SRILED AYTQ S GTFRRL MNYAYDQELHDVEQRWLLGAGENFGTTVTDEDLESSEGRKVIALNLDDTDD DSIPEYYESNDGPQQFDTTRSFIHEVVHALTHLQDKEDSNPRGPVVEYTNIILK EMGHTSPPRIAYEFSN (SEQ ID NO: 2).

An exemplary amino acid sequence of GogA is:

MPAGIKPIFINNMMSIYGLSHPHDSKVFPDLPEHQDNPSQLRLQHDGLATDDK ARLEPMCL AEYLI S GPGGMDPDIEIDDDTYDECREVL SRILED AYTQ S GTFRRL MNYAYDQELHDVEQRWLLGAGENFGTTVTDEDLESSEGRKVIALNLDDTDD DSIPECYESNDGPQPFDTTRSFIHEVVHALTHLQDKEDNNPRGPVVEYTNIILK EMGHTSPPRIAYESSN (SEQ ID NO: 3).

In one embodiment, the at least one isolated peptide comprises Salmonellae Typhimurium PipA, GogA or GtgA, or biologically functional fragment thereof. Exemplary S. Typhimurium PipA, GogA or GtgA amino acid sequences include, but are not limited to, amino acid sequences of GenBank Accession No. NC_003197.1, GenBank Accession No. NC_010393.1, and GenBank Accession No. NC_010392.1 However, the present invention is not limited to these particular sequences. Rather the present invention encompasses any PipA, GogA or GtgA isoform from any source. The peptides of the present invention may be made using chemical methods. For example, peptides can be synthesized by solid phase techniques (Roberge J Y et al (1995) Science 269: 202-204), cleaved from the resin, and purified by preparative high performance liquid chromatography. Automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.

The invention should also be understood to include any form of a peptide having substantial homology to PipA, GogA or GtgA. Preferably, a peptide which is "substantially homologous" is about 50% homologous, more preferably about 70% homologous, even more preferably about 80% homologous, more preferably about 90% homologous, even more preferably, about 95% homologous, and even more preferably about 99% homologous to amino acid sequence of PipA, GogA or GtgA disclosed herein.

The peptide may alternatively be made by recombinant means or by cleavage from a longer polypeptide. The composition of a peptide may be confirmed by amino acid analysis or sequencing.

The variants of the peptides according to the present invention may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which there are one or more modified amino acid residues, e.g., residues that are modified by the attachment of substituent groups, (iii) one in which the peptide is an alternative splice variant of the peptide of the present invention, (iv) fragments of the peptides and/or (v) one in which the peptide is fused with another peptide, such as a leader or secretory sequence or a sequence which is employed for purification (for example, His-tag) or for detection (for example, Sv5 epitope tag). The fragments include peptides generated via proteolytic cleavage (including multi-site proteolysis) of an original sequence. Variants may be post- translationally, or chemically modified. Such variants are deemed to be within the scope of those skilled in the art from the teaching herein.

As known in the art the "similarity" between two peptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to a sequence of a second polypeptide. Variants are defined to include peptide sequences different from the original sequence, preferably different from the original sequence in less than 40% of residues per segment of interest, more preferably different from the original sequence in less than 25% of residues per segment of interest, more preferably different by less than 10% of residues per segment of interest, most preferably different from the original protein sequence in just a few residues per segment of interest and at the same time sufficiently homologous to the original sequence to preserve the functionality of the original sequence. The present invention includes amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% similar or identical to the original amino acid sequence. The degree of identity between two peptides is determined using computer algorithms and methods that are widely known for the persons skilled in the art. The identity between two amino acid sequences is preferably determined by using the BLASTP algorithm [BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al, J. Mol. Biol. 215: 403-410 (1990)].

The peptides of the invention can be post-translationally modified. For example, post-translational modifications that fall within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc. Some modifications or processing events require introduction of additional biological machinery. For example, processing events, such as signal peptide cleavage and core glycosylation, are examined by adding canine microsomal membranes or Xenopus egg extracts (U.S. Pat. No. 6,103,489) to a standard translation reaction.

The peptides of the invention may include unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation. A variety of approaches are available for introducing unnatural amino acids during protein translation.

A peptide or protein of the invention may be conjugated with other molecules, such as proteins, to prepare fusion proteins. This may be accomplished, for example, by the synthesis of N-terminal or C-terminal fusion proteins provided that the resulting fusion protein retains the functionality of PipA, GogA or GtgA.

A peptide or protein of the invention may be phosphorylated using conventional methods such as the method described in Reedijk et al. (The EMBO Journal 11(4): 1365, 1992). Cyclic derivatives of the peptides of the invention are also part of the present invention. Cyclization may allow the peptide to assume a more favorable conformation for association with other molecules. Cyclization may be achieved using techniques known in the art. For example, disulfide bonds may be formed between two appropriately spaced components having free sulfhydryl groups, or an amide bond may be formed between an amino group of one component and a carboxyl group of another component. Cyclization may also be achieved using an azobenzene-containing amino acid as described by Ulysse, L., et al, J. Am. Chem. Soc. 1995, 117, 8466-8467. The components that form the bonds may be side chains of amino acids, non-amino acid components or a combination of the two. In an embodiment of the invention, cyclic peptides may comprise a beta-turn in the right position. Beta-turns may be introduced into the peptides of the invention by adding the amino acids Pro-Gly at the right position.

It may be desirable to produce a cyclic peptide which is more flexible than the cyclic peptides containing peptide bond linkages as described above. A more flexible peptide may be prepared by introducing cysteines at the right and left position of the peptide and forming a disulphide bridge between the two cysteines. The two cysteines are arranged so as not to deform the beta-sheet and turn. The peptide is more flexible as a result of the length of the disulfide linkage and the smaller number of hydrogen bonds in the beta-sheet portion. The relative flexibility of a cyclic peptide can be determined by molecular dynamics simulations.

The invention also relates to peptides comprising PipA, GogA or GtgA fused to, or integrated into, a target protein, and/or a targeting domain capable of directing the chimeric protein to a desired cellular component or cell type or tissue. The chimeric proteins may also contain additional amino acid sequences or domains. The chimeric proteins are recombinant in the sense that the various components are from different sources, and as such are not found together in nature (i.e., are heterologous).

In one embodiment, the targeting domain can be a membrane spanning domain, a membrane binding domain, or a sequence directing the protein to associate with for example vesicles or with the nucleus. In one embodiment, the targeting domain can target a peptide to a particular cell type or tissue. For example, the targeting domain can be a cell surface ligand or an antibody against cell surface antigens of a target tissue (e.g., liver, intestines, kidney). A targeting domain may target the peptide of the invention to a cellular component.

A peptide of the invention may be synthesized by conventional techniques. For example, the peptides or chimeric proteins may be synthesized by chemical synthesis using solid phase peptide synthesis. These methods employ either solid or solution phase synthesis methods (see for example, J. M. Stewart, and J. D. Young, Solid Phase Peptide Synthesis, 2^nd Ed., Pierce Chemical Co., Rockford 111. (1984) and G. Barany and R. B. Merrifield, The Peptides: Analysis Synthesis, Biology editors E. Gross and J. Meienhofer Vol. 2 Academic Press, New York, 1980, pp. 3-254 for solid phase synthesis techniques; and M Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin 1984, and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, suprs, Vol 1, for classical solution synthesis). By way of example, a peptide of the invention may be synthesized using 9-fluorenyl methoxycarbonyl (Fmoc) solid phase chemistry with direct incorporation of phosphothreonine as the N-fluorenylmethoxy-carbonyl-O-benzyl-L-phosphothreonine derivative.

N-terminal or C-terminal fusion proteins comprising a peptide or chimeric protein of the invention conjugated with other molecules may be prepared by fusing, through recombinant techniques, the N-terminal or C-terminal of the peptide or chimeric protein, and the sequence of a selected protein or selectable marker with a desired biological function. The resultant fusion proteins contain the PipA, GogA or GtgA peptide fused to the selected protein or marker protein as described herein. Examples of proteins which may be used to prepare fusion proteins include immunoglobulins, glutathione-S -transferase (GST), hemagglutinin (HA), and truncated myc.

Peptides of the invention may be developed using a biological expression system. The use of these systems allows the production of large libraries of random peptide sequences and the screening of these libraries for peptide sequences that bind to particular proteins. Libraries may be produced by cloning synthetic DNA that encodes random peptide sequences into appropriate expression vectors (see Christian et al 1992, J. Mol. Biol. 227:711 ; Devlin et al, 1990 Science 249:404;

Cwirla et al 1990, Proc. Natl. Acad, Sci. USA, 87:6378). Libraries may also be constructed by concurrent synthesis of overlapping peptides (see U.S. Pat. No.

4,708,871). The peptides and chimeric proteins of the invention may be converted into pharmaceutical salts by reacting with inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, etc., or organic acids such as formic acid, acetic acid, propionic acid, gly colic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benezenesulfonic acid, and toluenesulfonic acids.

In one embodiment, the present invention provides a composition comprising at least one isolated nucleic acid encoding PipA, GogA or GtgA, or a biologically functional fragment thereof.

In certain embodiments, the composition increases the expression of a biologically functional fragment of PipA, GogA or GtgA. For example, in one embodiment, the composition comprises an isolated nucleic acid sequence encoding a biologically functional fragment of PipA, GogA or GtgA. As would be understood in the art, a biologically functional fragment is a portion or portions of a full length sequence that retain the biological function of the full length sequence. Thus, a biologically functional fragment of PipA, GogA or GtgA comprises a peptide that retains the function of full length PipA, GogA or GtgA.

In one embodiment, the at least one isolated nucleic acid sequence encodes at least one of PipA, GogA or GtgA. In one embodiment, the at least one isolated nucleic acid sequence encodes at least one peptide comprising an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. Further, the invention encompasses an isolated nucleic acid encoding a peptide having substantial homology to PipA, GogA or GtgA disclosed herein. In certain embodiments, the isolated nucleic acid sequence encodes a peptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with an amino acid sequence selected from SEQ NOs: 1-3.

The isolated nucleic acid sequence encoding PipA, GogA or GtgA can be obtained using any of the many recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than cloned.

The isolated nucleic acid may comprise any type of nucleic acid, including, but not limited to DNA and RNA. For example, in one embodiment, the composition comprises an isolated DNA molecule, including for example, an isolated cDNA molecule, encoding PipA, GogA or GtgA, or functional fragment thereof. In one embodiment, the composition comprises an isolated RNA molecule encoding PipA, GogA or GtgA, or a functional fragment thereof.

The nucleic acid molecules of the present invention can be modified to improve stability in serum or in growth medium for cell cultures. Modifications can be added to enhance stability, functionality, and/or specificity and to minimize immunostimulatory properties of the nucleic acid molecule of the invention. For example, in order to enhance the stability, the 3 '-residues may be stabilized against degradation, e.g., they may be selected such that they consist of purine nucleotides, particularly adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogues, e.g., substitution of uridine by 2'- deoxythymidine is tolerated and does not affect function of the molecule.

In one embodiment of the present invention the nucleic acid molecule may contain at least one modified nucleotide analogue. For example, the ends may be stabilized by incorporating modified nucleotide analogues.

Non-limiting examples of nucleotide analogues include sugar- and/or backbone-modified ribonucleotides (i.e., include modifications to the phosphate-sugar backbone). For example, the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. In some backbone- modified ribonucleotides the phosphoester group connecting to adjacent

ribonucleotides is replaced by a modified group, e.g., of phosphothioate group. In some sugar-modified ribonucleotides, the 2' OH-group is replaced by a group selected from H, OR, R, halo, SH, SR, NH₂, NHR, NR₂ or ON, wherein R is Ci-C₆ alkyl, alkenyl or alkynyl and halo is F, CI, Br or I.

Other examples of modifications are nucleobase-modified ribonucleotides, i.e., ribonucleotides, containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. Bases may be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and/or cytidine modified at the 5-position, e.g., 5-(2- amino)propyl uridine, 5-bromo uridine; adenosine and/or guanosines modified at the 8 position, e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. It should be noted that the above modifications may be combined. In some instances, the nucleic acid molecule comprises at least one of the following chemical modifications: 2'-H, 2'-0-methyl, or 2'-OH modification of one or more nucleotides. In certain embodiments, a nucleic acid molecule of the invention can have enhanced resistance to nucleases. For increased nuclease resistance, a nucleic acid molecule, can include, for example, 2'-modified ribose units and/or phosphorothioate linkages. For example, the 2' hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents. For increased nuclease resistance the nucleic acid molecules of the invention can include 2'-0-methyl, 2'-fluorine, 2'-0-methoxy ethyl, 2'-0-aminopropyl, 2'-amino, and/or phosphorothioate linkages. Inclusion of locked nucleic acids (LNA), ethylene nucleic acids (ENA), e.g., 2'-4'-ethylene-bridged nucleic acids, and certain nucleobase modifications such as 2-amino-A, 2-thio (e.g., 2-thio-U), G-clamp modifications, can also increase binding affinity to a target.

In one embodiment, the nucleic acid molecule includes a 2' -modified nucleotide, e.g., a 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-0-methyl, 2'-0-methoxyethyl (2'- O-MOE), 2'-0-aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-DMAOE), 2'-0-dimethylaminopropyl (2'-0-DMAP), 2'-0-dimethylaminoethyloxyethyl (2'-0- DMAEOE), or 2'-0-N-methylacetamido (2'-0-NMA). In one embodiment, the nucleic acid molecule includes at least one 2'-0-methyl-modified nucleotide, and in some embodiments, all of the nucleotides of the nucleic acid molecule include a 2'-0- methyl modification.

In certain embodiments, the nucleic acid molecule of the invention preferably has one or more of the following properties:

Nucleic acid agents discussed herein include otherwise unmodified RNA and DNA as well as RNA and DNA that have been modified, e.g., to improve efficacy, and polymers of nucleoside surrogates. Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, preferably as occur naturally in the human body. The art has referred to rare or unusual, but naturally occurring, RNAs as modified RNAs, see, e.g., Limbach et al. (Nucleic Acids Res., 1994, 22:2183-2196). Such rare or unusual RNAs, often termed modified RNAs, are typically the result of a post-transcriptional modification and are within the term unmodified RNA as used herein. Modified RNA, as used herein, refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occur in nature, preferably different from that which occurs in the human body. While they are referred to as "modified RNAs" they will of course, because of the modification, include molecules that are not, strictly speaking, RNAs. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows the bases to be presented in the correct spatial relationship such that hybridization is substantially similar to what is seen with a ribophosphate backbone, e.g., non-charged mimics of the ribophosphate backbone.

Modifications of the nucleic acid of the invention may be present at one or more of, a phosphate group, a sugar group, backbone, N-terminus, C-terminus, or nucleobase.

The present invention also includes a vector in which the isolated nucleic acid of the present invention is inserted. The art is replete with suitable vectors that are useful in the present invention.

In brief summary, the expression of natural or synthetic nucleic acids encoding PipA, GogA or GtgA is typically achieved by operably linking a nucleic acid encoding the PipA, GogA or GtgA or portions thereof to a promoter, and incorporating the construct into an expression vector. The vectors to be used are suitable for replication and, optionally, integration in eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

The vectors of the present invention may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U. S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties. In another

embodiment, the invention provides a gene therapy vector.

The isolated nucleic acid of the invention can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

Further, the vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U. S. Pat. No. 6,326, 193).

A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In one embodiment, lentivirus vectors are used.

For example, vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity. In one embodiment, the composition includes a vector derived from an adeno-associated virus (AAV). Adeno-associated viral (AAV) vectors have become powerful gene delivery tools for the treatment of various disorders. AAV vectors possess a number of features that render them ideally suited for gene therapy, including a lack of pathogenicity, minimal immunogenicity, and the ability to transduce postmitotic cells in a stable and efficient manner. Expression of a particular gene contained within an AAV vector can be specifically targeted to one or more types of cells by choosing the appropriate combination of AAV serotype, promoter, and delivery method

In certain embodiments, the vector also includes conventional control elements which are operably linked to the transgene in a manner which permits its transcription, translation and/or expression in a cell transfected with the plasmid vector or infected with the virus produced by the invention. As used herein, "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A great number of expression control sequences, including promoters which are native, constitutive, inducible and/or tissue-specific, are known in the art and may be utilized.

Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Growth Factor -l a (EF-l a). However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

Enhancer sequences found on a vector also regulates expression of the gene contained therein. Typically, enhancers are bound with protein factors to enhance the transcription of a gene. Enhancers may be located upstream or downstream of the gene it regulates. Enhancers may also be tissue-specific to enhance transcription in a specific cell or tissue type. In one embodiment, the vector of the present invention comprises one or more enhancers to boost transcription of the gene present within the vector.

In order to assess the expression of PipA, GogA or GtgA, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co- transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.

Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al, 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter- driven transcription. Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes,

nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a "collapsed" structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20°C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. "Liposome" is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al, 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a mi cellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine- nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids into a host cell, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, "molecular biological" assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.

In one embodiment, the present invention provides a delivery vehicle comprising at least one of PipA, GogA or GtgA, or a nucleic acid molecule encoding PipA, GogA or GtgA. Exemplary delivery vehicles include, but are not limited to, microspheres, microparticles, nanoparticles, polymerosomes, liposomes, and micelles. For example, in certain embodiments, the delivery vehicle is loaded with PipA, GogA or GtgA, or a nucleic acid molecule encoding PipA, GogA or GtgA. In certain embodiments, the delivery vehicle provides for controlled release, delayed release, or continual release of its loaded cargo. In certain embodiments, the delivery vehicle comprises a targeting moiety that targets the delivery vehicle to a treatment site.

The present invention provides a scaffold or substrate composition comprising PipA, GogA or GtgA, a nucleic acid molecule encoding PipA, GogA or GtgA, or a combination thereof.

For example, in one embodiment, PipA, GogA or GtgA, a nucleic acid molecule encoding PipA, GogA or GtgA, or a combination thereof is incorporated within a scaffold. In another embodiment, PipA, GogA or GtgA, a nucleic acid molecule encoding PipA, GogA or GtgA, or a combination thereof, is applied to the surface of a scaffold. The scaffold of the invention may be of any type known in the art. Non-limiting examples of such a scaffold includes a, hydrogel, electrospun scaffold, foam, mesh, sheet, patch, and sponge.

The present invention also provides pharmaceutical compositions comprising one or more of the compositions described herein. Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for administration to the wound or treatment site. The pharmaceutical compositions may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, and/or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.

Administration of the compositions of this invention may be carried out, for example, by parenteral, by intravenous, intratumoral, subcutaneous, intramuscular, or intraperitoneal injection, or by infusion or by any other acceptable systemic method.

As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" that may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Genaro, ed. (1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

The composition of the invention may comprise a preservative from about 0.005% to 2.0% by total weight of the composition. The preservative is used to prevent spoilage in the case of exposure to contaminants in the environment.

Examples of preservatives useful in accordance with the invention included but are not limited to those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea and combinations thereof. In one embodiment, the preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.

In an embodiment, the composition includes an anti-oxidant and a chelating agent that inhibits the degradation of one or more components of the composition. In various embodiments, antioxidants for some compounds are BHT, BHA, alpha-tocopherol and ascorbic acid in a range of about 0.01% to 0.3% and more preferably BHT in the range of 0.03% to 0.1 % by weight by total weight of the composition. In some embodiments, the chelating agent is present in an amount of from 0.01 % to 0.5% by weight by total weight of the composition. Chelating agents include edetate salts (e.g. disodium edetate) and citric acid in the weight range of about 0.01 % to 0.20% and more preferably in the range of 0.02% to 0.10% by weight by total weight of the composition. The chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are used in some embodiments as the antioxidant and chelating agent respectively for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art.

Liquid suspensions may be prepared using conventional methods to achieve suspension of PipA, GtgA, GogA or other composition of the invention in an aqueous or oily vehicle. Aqueous vehicles include, for example, water, and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate,

polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to,

naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., poly oxy ethylene stearate,

heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and

polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin, and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl-para- hydroxybenzoates, ascorbic acid, and sorbic acid.

Treatment Methods

The present invention provides a method for the treatment or prevention of a disease or disorder associated with NF-κΒ activity or NF-κΒ signaling in a subject in need thereof. For example, in certain embodiments, the method treats or prevents a disease or disorder associated with increased NF-κΒ activity. Exemplary diseases or disorders treated or prevented by way of the present invention includes, but is not limited to cancer, diabetes, heart disease, asthma, inflammatory bowel disease, lupus, Alzheimer's disease, Huntington's disease, COPD, atopic dermatitis, atopy, allergy, allergic rhinitis, Crohn's disease, and scleroderma.

Cancers that may be treated include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors. The cancers may comprise non-solid tumors (such as hematological tumors, for example, leukemias and lymphomas) or may comprise solid tumors. Types of cancers to be treated with a composition of the invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas. Adult tumors/cancers and pediatric tumors/cancers are also included.

Hematologic cancers are cancers of the blood or bone marrow.

Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma,

pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and central nervous system (CNS) tumors (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases).

In certain embodiments, the method comprises administering an effective amount of a composition described herein to a subject diagnosed with cancer, or suspected of having cancer. In certain aspects, the composition is contacted to a cell or tissue where cancer is present or at risk for developing. In one

embodiment, the composition is administered systemically to the subject.

In certain aspects, for in vivo treatment methods, delivery the nucleic acid is injected directly into the subject. For example, in one embodiment, the nucleic acid is delivered at the site where the composition is required.

In vivo nucleic acid transfer techniques include, but is not limited to, transfection with viral vectors such as adenovirus, Herpes simplex I virus, adeno- associated virus), lipid-based systems (useful lipids for lipid-mediated transfer of the gene are DOTMA, DOPE and DC-Choi, for example), naked DNA, and transposon- based expression systems. Exemplary gene therapy protocols see Anderson et al., Science 256: 808-813 (1992). See also WO 93/25673 and the references cited therein. In certain embodiments, the method comprises administering of RNA, for example mRNA, directly into the subject (see for example, Zangi et al, 2013 Nature

Biotechnology, 31 : 898-907).

For ex vivo treatment, an isolated cell is modified in an ex vivo or in vitro environment. In one embodiment, the cell is autologous to a subject being treated with the composition of the invention. Alternatively, the cell can be allogeneic, syngeneic, or xenogeneic with respect to the subject. The modified cells may then be administered to the subject directly, or within a scaffold as described elsewhere herein. For example, the modified cell may be seeded on or within a scaffold to be administered to the subject.

One skilled in the art recognizes that different methods of delivery may be utilized to administer an isolated nucleic acid into a cell. Examples include: (1) methods utilizing physical means, such as electroporation (electricity), a gene gun (physical force) or applying large volumes of a liquid (pressure); and (2) methods wherein the nucleic acid or vector is complexed to another entity, such as a liposome, aggregated protein or transporter molecule.

Furthermore, the actual dose and schedule can vary depending on whether the compositions are administered in combination with other pharmaceutical compositions, or depending on interindividual differences in pharmacokinetics, drug disposition, and metabolism. Similarly, amounts can vary in in vitro applications depending on the particular cell line utilized (e.g., based on the number of vector receptors present on the cell surface, or the ability of the particular vector employed for gene transfer to replicate in that cell line). Furthermore, the amount of vector to be added per cell will likely vary with the length and stability of the therapeutic gene inserted in the vector, as well as also the nature of the sequence, and is particularly a parameter which needs to be determined empirically, and can be altered due to factors not inherent to the methods of the present invention (for instance, the cost associated with synthesis). One skilled in the art can easily make any necessary adjustments in accordance with the exigencies of the particular situation.

Genetically modified cells may also contain a suicide gene i.e., a gene which encodes a product that can be used to destroy the cell. In many gene therapy situations, it is desirable to be able to express a gene for therapeutic purposes in a host, cell but also to have the capacity to destroy the host cell at will. The therapeutic agent can be linked to a suicide gene, whose expression is not activated in the absence of an activator compound. When death of the cell in which both the agent and the suicide gene have been introduced is desired, the activator compound is administered to the cell thereby activating expression of the suicide gene and killing the cell.

Examples of suicide gene/prodrug combinations which may be used are herpes simplex virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase thymidilate kinase (Tdk: :Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside.

The composition of the invention may be administered to a patient or subject in need in a wide variety of ways. Modes of administration include intraoperatively intravenous, intravascular, intramuscular, subcutaneous, intracerebral, intraperitoneal, soft tissue injection, surgical placement, arthroscopic placement, and percutaneous insertion, e.g., direct injection, cannulation or catheterization. Any administration may be a single application of a composition of invention or multiple applications. Administrations may be to single site or to more than one site in the individual to be treated. Multiple administrations may occur essentially at the same time or separated in time.

In certain embodiments, the composition of the invention is administered during surgical resection or debulking of a tumor or diseased tissue. For example, in subjects undergoing surgical treatment of diseased tissue or tumor, the composition may be administered to the site in order to further treat the tumor or promote bone growth.

Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.

Pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the subject, and the type and severity of the subject's disease, although appropriate dosages may be determined by clinical trials.

When "therapeutic amount" is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, disease type, extent of disease, and condition of the patient (subject).

The administration of the subject compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the compositions of the present invention are preferably administered by i.v. injection.

In certain embodiments of the present invention the composition is administered to a subject in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities, including but not limited surgery, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. In a further embodiment, the cell compositions of the present invention are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or

CAMPATH. In another embodiment, the cell compositions of the present invention are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan. For example, in one embodiment, subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In an additional embodiment, the composition is administered before or following surgery.

Methods of treatment of the diseases and disorders encompassed by the invention can comprise the transplantation of single cells, cell lines, compositions, or cell populations of the invention into a subject in need thereof. In certain

embodiments, the subject is a human.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore, specifically point out some embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure. Example 1 : A family of Salmonella type III secretion effector proteins selectively targets the NF-κΒ signaling pathway to preserve host homeostasis

Microbial infections usually lead to host innate immune responses and inflammation. These responses most often limit pathogen replication although they can also result in host-tissue damage. The enteropathogenic bacteria Salmonella Typhimurium utilizes a type III secretion system to induce intestinal inflammation by delivering specific effector proteins that stimulate signal transduction pathways resulting in the production of pro-inflammatory cytokines. It is demonstrated herein that a family of related Salmonella Typhimurium effector proteins PipA, GogA and GtgA specifically and redundantly target components of the NF-κΒ signaling pathway to inhibit transcriptional responses leading to inflammation. It is shown that these effector proteins are highly specific proteases that cleave both the RelA (p65) and RelB transcription factors but do not target pi 00 (NF-KB2) or pi 05 (NF-κΒ 1). A Salmonella Typhimurium strain lacking these effector proteins is defective in its ability to inhibit NF-κΒ activity and, paradoxically, exhibits increased virulence in an animal model of infection. The results demonstrated herein thus indicate that bacterial pathogens can also evolve determinants to preserve host homeostasis even if at the expense of decreasing its virulence.

The materials and methods employed in these experiments are now described.

Plasmids

Human TRIF, TRAF2, RIP1, IKKa, pi 05 or RelA were amplified from a human cDNA library and cloned into the vectors pCMV-3XHA, pRK5-M45 or pET15b to generate HA-tagged versions of TRIF, TRAF2, RIP1, IKKa, and RelA, M45 -tagged versions of pi 05 and RelA, and His-tagged RelA (all tags are located at the amino terminus of the respective proteins). gogA, gtgA and pipA were amplified from the chromosome of S. Typhimurium strain SL1344 and cloned into the vectors pRK5-Flag or pET15b to generate Flag-tagged or His-tagged versions of the proteins. For S. Typhimurium mutant complementation studies, gogA, gtgA or pipA were cloned into the pBAD24 vector placing their expression under the control of an arabinose inducible promoter (Guzman et al, 1995, J Bacteriol 177:4121-30) (Table 1). The plasmids pCMV4-human pi 00 and pCDNA3-mouse RelB were purchased from Addgene and were subcloned into the vector pRK5- M45 to generate M45- taggged versions of these proteins. The catalytic mutant Pip^AE181A was generated using PCR-mediated mutagenesis.

Table 1. Bacterial strains and lasmids used in this study

S. Typhimurium strains and growth condition

All of S. Typhimurium strains were derived from S. Typhimurium strain SL1344 (Hoiseth and Stocker. 1981 , Nature 291 :238-9). Bacterial mutants were constructed by allelic exchange as previously described (Kaniga et al, 1994, Mol Microbiol 13:555-68). S. Typhimurium strains were cultured in LB containing 0.3M NaCl to induce the expression of the SPI-1 T3SS (Galan and Curtiss III, 1990, Infect Immun 58: 1879-85).

Cell culture and bacterial infection

The human embryonic kidney epithelial HEK 293T and human epithelial Henle-407 cell lines were cultured in antibiotic free Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% bovine calf (Henle-407) or bovine fetal (HEK293T) sera. For bacterial infections, 2 x 10⁵ HEK293T cells or 7 x 10⁴ Henle-407 cells were seeded into each well of a 24-well plate. Eighteen hours later, cells were infected for 1 hour with the different S. Typhimurium strains at the multiplicities of infection (MOI) indicated in the figure legends. Cells were then treated with gentamicin (100 μg/ml) for 1 hour to kill extra cellular bacteria. In experiments involving longer infection times, the infected cells were cultured in medium with low concentration gentamicin (10 μg/ml) for the indicated times.

Immunofluorescence staining

HeLa cells grown on glass coverslips were transiently transfected using Lipofectamine 2000 (Invitrogen) with 0.5 μg of plasmid encoding FLAG- epitope-tagged PipA or infected with S. Typhimurium strain expressing FLAG-tagged PipA as described above. Eighteen hours after transfection and 4 hours after bacterial infection, cells were washed once with PBS and fixed in 4% PF A/PBS for 20 minutes at room temperature (RT). FLAG-tagged PipA was stained with mouse monoclonal anti-FLAG M2 (Sigma; 1: 10,000 dilution) and secondary anti-mouse antibody conjugated to Alexa 488 (Invitrogen, 1 : 1,000). Images were acquired with an inverted microscope (Eclipse TE2000-U; Nikon) equipped with a CCD camera (MicroMAX RTE/CCD-1300Y; Princeton Instruments).

Bacteria internalization and intracellular replication

Cultured epithelial Henle-407 cells grown in a 24-well plate were infected for 1 hour and incubated in the presence of gentamicin as described above. Thirty minutes or 8 hours after gentamicin treatment, cells were washed twice with HBSS and then lysed in 300 μΐ 0.1% Sodium Deoxycholate (DOC) in HBSS to release their bacterial content. Multiple dilutions were then plated onto LB plates containing streptomycin to determine colony forming units (c.f.u.). Lucif erase reporter activity

Dual luciferase assay was performed following the manufacturer's instructions (Promega). To monitor the activation of the Erk pathway, HEK293T cells plated in 24-well plates were co-transfected with 0.4 μg of Gal4-Elk, 0.4 μg of Gal4- luc, 20 ng of pRLActin as internal control. To measure NFKB activation, HEK293T cells were co-transfected with 20 ng of the pG13-luc reporter plasmid encoding a NFKB responsive element and 20 ng of pRLActin as internal control. Eighteen hours after transfection, cells were infected with bacteria or were lysed to measure luciferase activity as previously described (Xu et al, 2014, Nature 513:237-41).

In vitro protease activity

Purified His-tagged-RelAi_-2io was mixed with purified His-tagged- GogA, His-tagged-GtgA, His-tagged-PipA or His-tagged-PipA^E181A in 40 μΐ of reaction buffer (50mM Tris-HCl pH 7.5, 2mM CaCl₂, 50mM NaCl) in the absence of EDTA or in the presence of EDTA. Reactions were carried out for 1 hour at room temperature and stopped by the addition of SDS loading buffer. Digestion products were analyzed by SDS-PAGE followed by Coomassie staining.

Quantitative PCR

Total RNA from mouse tissues (cecum) were isolated using TRIzol (Invitrogen) reagent according to the manufacture's protocol and were reversed- transcribed with the iScript reverse transcriptase (BIORAD). Quantitative PCR was performed using iQ SYBR Green Supermix (BIORAD) in an iCycler real time PCR machine (Bio-Rad) with the following primers:

GAPDH fw ATTGTCAGCAATGCATCCTG (SEQ ID NO: 4) GAPDH re ATGGACTGTGGTCATGAGCC (SEQ ID NO: 5) TNFa fw CCACCACGCTCTTCTGTCTAC (SEQ ID NO: 6) TNFa re AGGGTCTGGGCCATAGAACT (SEQ ID NO: 7)

KC re TCTCCGTTACTTGGGGACAC (SEQ ID NO: 8) KC fw ACCCAAACCGAAGTCATAGC (SEQ ID NO: 9)

ILip fw: GCAACTGTTCCTGAACTCAACT (SEQ ID NO: 10) ILi re : ATCTTTTGGGGTCCGTCAACT (SEQ ID NO: 11) ΜΙΡΙα fw ACCATGACACTCTGCAACCA (SEQ ID NO: 12) ΜΙΡΙα re GTGGAATCTTCCGGCTGTAG (SEQ ID NO: 13)

Mouse infections

A C57BL/6 mouse line carrying a wild type allele of Nrampl

(Slcl lal) (from the 129/Svj mouse) was generated by backcrossing to C57BL/6 for 12 generations. The Nrampl (Slcl lal) alleles were verified by PCR amplification and nucleotide sequencing as previously described (Lara-Tejero et al., 2006, J Exp Med 203: 1407-12). Groups of age- and sex- matched C57BL/6 Nrampl ^_/" and C57BL/6 Nrampl ^+/+ mice were infected at 8-12 weeks of age. For oral infections food was removed 4 hours prior to inoculation, and mice were administered (by stomach gavage) 100 μΐ of 10% bicarbonate solution (to buffered the stomach pH) followed by the indicated bacterial dose in 100 μΐ PBS. For intraperitoneal injection, the indicated bacterial dose was administered in 100 μΐ PBS. To determine bacterial loads, tissues were mechanically homogenized in 3 ml PBS containing 0.05% sodium deoxycholate, and dilutions were plated on LB plates containing streptomycin to determine colony- forming units as previously described (Lara-Tejero et al, 2006, J Exp Med 203: 1407- 12).

Histology

Histopathological analysis of intestinal tissues was carried out as previously described (Lara-Tejero et al, 2006, J Exp Med 203: 1407-12). Briefly, a portion of the distal cecal tip of experimental animals was fixed in 3.7% formalin for 72 hours, then transferred to 70% ethanol for 48 hours prior to paraffin embedding, sectioning, and hematoxylin-eosin staining.

The results of the experiments are now described.

Absence of PipA. GtgA and GogA results in increased S. Typhimurium virulence in a mouse model of infection

In an effort to gain insight into the potential function of the PipA, GtgA and GogA effectors, a S. Typhimurium strain was constructed simultaneously lacking these three effector proteins and its phenotype was examined in mouse models of infection. Mice (C57BL/6) expressing either wild type (resistant) or mutant (susceptible) alleles of NRAMP1 (SLC11A1), a divalent metal ion transporter that is known to significantly enhance resistance to S. Typhimurium infection (Blackwell et al., 2001, Cell Microbiol 3:773-84), were inoculated orally or intraperitoneally. There were no significant differences in the levels of colony forming units (c. f. u.) of the wild type and ΔρίρΑ AgtgA AgogA S. Typhimurium mutant strains in the different tissues of both mouse strains after oral or intraperitoneal infection (Figure 1A- Figure IB; Figure 9A- Figure B and Figure 14). Surprisingly, despite the presence of equivalent bacterial burden a significant proportion of mice orally inoculated with the S. Typhimurium ΔρίρΑ AgtgA AgogA mutant strains succumbed to infection earlier than animals inoculated with wild type (Figure 1C and Figure 10A). This difference, however, was not apparent when animals were inoculated via the intraperitoneal route (Figure 1C- Figure ID). Even more unexpected was the observation that this phenotype was apparent in mice that express a wild type allele of NRAMP1

(SLC11A1) (Figure 1C and Figure 10A) but not in mice expressing a mutant allele of this transporter and therefore more susceptible to wild type S. Typhimurium (Figure 10B).

Since equivalent number of bacterial loads in tissues infected with wild type or the mutant strain was observed, it was hypothesized that the inability of the S. Typhimurium ΔρίρΑ AgtgA AgogA mutant to inhibit the NF-κΒ signaling pathway might lead to increased inflammation and increased production of pro-inflammatory cytokines in the intestinal epithelium, which may result in increased lethality.

Consistent with this hypothesis higher levels of pro-inflammatory cytokines (Figure 2) and more severe inflammation (Figure 11) in the intestine of mice infected with the S. Typhimurium ΔρίρΑ AgtgA AgogA mutant strain than in those infected with wild- type bacteria. The levels of a selected group of cytokines in the serum of infected animals were also measured, and although animals infected with the AgogA AgtgA ApipA mutant strain exhibited higher levels of the measured cytokines than those infected with wild type, the differences did not approach statistical significance (Figure 15). These results indicate that the absence of GogA, GtgA and PipA results in a more severe inflammatory response to S. Typhimurium, with increased cytokine production early in infection leading to more severe lethality. Infection of cultured cells with the S. Typimurium AgogA AgtgA ApipA mutant strain results in increased NF-κΒ signaling

In an effort to better understand the phenotype of the S. Typhimurium AgogA AgtgA ApipA mutant observed in experimental animals, this mutant strain was examined with several assays for phenotypes that previous studies have shown to be dependent on the function of the SPI-1- and/or SPI-2-encoded T3SSs (Figueira and Holden, 2012, Microbiology 158: 1147-61 ; Galan, 2001, Annu Rev Cell Dev Biol 17:53-86; Ibarra and Steele-Mortimer, 2009, Cell Microbiol 11 : 1579-86). S.

Typhimurium ΔρίρΑ AgtgA AgogA mutant strain exhibited wild type levels of invasion and replication within cultured cells, phenotypes that are strictly dependent on the SPI-1 and SPI-2 T3SSs (Figure 12). Also through the activity of its T3SSs, S. Typhimurium stimulates a profound reprogramming of gene expression of the infected cells by stimulating mitogen activated protein (MAP) kinase, NF-κΒ and signal transducer and activator of transcription 3 (STAT-3) signaling pathways (Hobbie et al, 1997, J Immunol 159:5550-9; Chen et al, 1996, Science 274:2115-8; Bruno et al, 2009, PLoS Pathog 5:el000538; Hannemann et al., 2013, PLoS Pathog 9:el003668). The S. Typhimurium ApipA AgtgA AgogA mutant strain activated MAP kinase and STAT-3 signaling in a manner indistinguishable from wild type (Figure 3 A- Figure 3B). In contrast, the S. Typhimurium ApipA AgtgA AgogA mutant strain activated the NF-κΒ signaling pathway significantly more robustly than wild type (Figure 3C). The enhanced activation of NF-κΒ was seen starting at 4 hours after infection and was more apparent later (8 hours) in infection (Figure 3D). In contrast, S. Typhimurium strains carrying single deletion mutations in pip A, gtgA, or gogA showed little (AgogA) to no (AgtgA or ApipA) enhancement in their ability to activate NF-KB (Figure 3C- Figure 3D). The phenotype of the ApipA AgtgA AgogA triple mutant could be complemented in trans by expressing plasmid-borne pipA, gtgA or gogA (Figure 3E). Furthermore, transient expression of PipA in cultured mammalian cells completely abolished NF-κΒ activation by the S. Typhimurim ApipA AgtgA AgogA mutant strain (Figure 3F). These results indicate that these three effectors work in a redundant fashion to inhibit Salmonella-induced NF-κΒ signaling.

PipA can directly inhibit NF-κΒ signaling Whether the PipA family members by themselves were able to inhibit NF-KB signaling in a context different from bacterial infection was then investigated. Therefore the ability of PipA to prevent NF-κΒ activation by the transient expression of TRIF, an adaptor for Toll-like receptors and a potent activator of this pathway (Yamamoto et al, 2002, J Immunol 169:6668-72) was studied (Figure 4A). Transient expression of PipA completely abolished TRIF-induced (Figure 4B) or TNFa-induced (Figure 4C) NF-κΒ activation. These results indicate that these effector proteins are not only necessary but also sufficient to inhibit NF-κΒ signaling and that inhibition occurs even when this pathway is activated by an agonist other than S. Typhimurium. The location in the NF-κΒ signaling pathway where these effectors exert their function was investigated by examining the effect of PipA expression on the activation of NF-κΒ by downstream components of the TRIF signaling pathway (Cohen, 2014, J Cell Sci 127:2383-90) (Figure 4A). Expression of PipA inhibited an NF-KB-dependent reporter when activated by the expression of TRAF2, RIP1, IKK or even the transcription factor RelA itself (Figure 4B). These results indicate that PipA, and by extension the other members of this protein family, must exert their function at the level of or downstream from RelA.

PipA. GtgA and GogA inhibit the NF-κΒ pathway by directly targeting RelA

The effect of PipA on RelA expression was studied by transiently co- expressing differentially epitope-tagged PipA and RelA. Expression of PipA led to a drastic reduction in the levels of RelA expression (Figure 5A). In contrast, expression of PipA had no effect on the expression of TRAF2 (Figure 5A). Similar results were obtained after co-expression of GtgA or GogA (Figure 5B). In contrast, expression of PipA, GtgA or GogA did not alter the levels of other transcription factors activated by Salmonella infection such as c-Jun or STAT3 (Figure 5C). The levels of RelA after S. Typhimurium infection were then examined. Consistent with the transient expression experiments, infection of cultured cells with wild-type S. Typhimurium resulted in a marked decreased in the levels of RelA (Figure 5D). In contrast, infection with the S. Typhimurium ΔρίρΑ AgtgA AgogA mutant strain did not alter the levels of RelA in infected cells (Figure 5D). These results indicate that the PipA family of effector proteins redundantly targets RelA to inhibit its expression or to stimulate its degradation. PipA localizes to the nucleus of infected cells

To gain insight into the mechanism by which the PipA family of effectors targets RelA, the localization of PipA was examined bother after transient transfection or bacterial infection. PipA localized to the nucleus both after transient expression or bacterial infection (Figure 6). These results indicate that these effectors must target RelA and RelB subsequent to activation of the NF-κΒ signaling pathway, which results in the nuclear translocation of the transcription factors. This observation is consistent with the kinetics of the PipA/GtgA/GogA-dependent inhibition of NF-KB during bacterial infection (Figure 3D) since the phenotype was only apparent later in infection and subsequent to the Salmonella-induced NF-κΒ activation.

PipA. GtgA and GogA are specific proteases for transcription factors of the RelA family

Primary amino acid sequence similarity searches did not yield proteins with significant similarity to the PipA family of effectors other than true homologs in other bacteria. However, structural similarity searches identified features present in metalloproteinases such as a region that fits the consensus for the zinc-binding site that is a signature for these proteinases (Figure 13). Therefore mutations were introduced in the predicted zinc-binding site of PipA and the effect of this mutation on the expression of RelA was examined. Introduction of this mutation effectively prevented the ability of PipA to inhibit Salmonella-induced NF-κΒ signaling (Figure 7A) or reduce the levels of RelA in transient co-transfection experiments (Figure 7B).

To ascertain if the observed proteolytic degradation of RelA was the direct result of the proteolytic activity of the PipA family of effector proteins, PipA, GogA, GtgA and their catalytic mutants were purified and examined for their proteolytic activity towards purified RelA in vitro. Purified effectors were able to cleave RelA in the presence but not in the absence of divalent cations (Figure 7C and Figure 2D). To identify the precise site of cleavage, the amino-terminal sequence of the RelA cleavage product was determined after PipA digestion. PipA cleaves between residues Gly40 and Arg41 of RelA (Table 2). The cleavage products, which are presumably degraded in vivo, are nonetheless expected to be non-functional since they would lack critical domains necessary for transcriptional activity. These results indicate that PipA, and by extension the other family members, are proteases that directly target RelA for cleavage and subsequent degradation.

In addition to RelA (p65), there are other members of the NF-κΒ family of transcription factors that share the Rel-homology domain and form homo or heterodimers with one another thus adding complexity to the transcriptional outputs (Thanos and Maniatis, 1995, Cell 80:529-32). The ability of the PipA family of effectors to target other members of the NF-κΒ family was tested. PipA, GtgA and GogA were able to effectively cleave RelB but did not cleave pi 00 (NF-KB2) or pi 05 (NF-κΒΙ) (Figure 7E). These results indicate that the PipA family of effector proteases selectively and specifically targets a subset of the NF-κΒ transcription factors.

Table 2. RelA amino terminal sequence after digestion with PipA, GogA or GtgA

PipA. GtgA. and GogA and homeostasis of S. Typhimurium

Host-pathogen interactions shaped by long-standing associations have evolved to maximize pathogen replication while preserving host homeostasis. S. Typhimurium, for example, can induce its own internalization into non-phagocytic cells through the activity of the effector proteins SopE and SopE2, which are exchange factors and thus activators of Rho-family GTPases (Hardet et al, 1998, Cell 93:815-26; Stender et al, 2000, Mol Microbiol 36: 1206-11). These responses are subsequently reversed by the delivery of SptP, an effector with an opposing GAP activity (Fu and Galan, 1999, Nature 401 :293-7). Similarly, other effectors oppose Salmonella-induced signaling pathways leading to nuclear responses (Du and Galan, 2009, PLoS Pathog 5:el000595; Pilar et al, 2012, PLoS Pathog 8:el002773).

Reported herein is the discovery of a family of effector proteins that proteolytically and specifically targets a subset of NF-κΒ transcription factors thus inhibiting a key signaling pathway in the inflammatory responses to microbial pathogens. S.

Typhimurium is known to potently activate the NF-κΒ signaling pathway and induce intestinal inflammation (Hobbie et al., 1997, J Immunol 159:5550-9; Chen et al, 1996, Science 274:2115-8; Bruno et al, 2009, PLoS Pathog 5:el000538), which is required for its ability to acquire essential nutrients in the gut (Stecher et al., 2007, PLoS Biol 5:2177-89; Winter et al, 2010, Nature 467:426-9). Paradoxically, absence of the PipA, GtgA, and GogA resulted in increased virulence, indicating that S. Typhimurium has evolved these effector proteins to preserve host homeostasis at the expense of increasing its potential virulence. This is a remarkable example of pathogen adaptation to maximize its long-term survival.

It is well established that in the mouse, the divalent metal ion transporter NRAMP1 (SLC11A1) confers resistant to S. Typhimurium infections (Blackwell et al., 2001, Cell Microbiol 3:773-84). Thus, NRAMP1 -deficient animals are -1,000 fold more susceptible to S. Typhimurium regardless the inoculation route. Therefore it is noteworthy that the hypervirulent phenotype of the S. Typhimurium AgogA AgtgA ApipA mutant strain is paradoxically manifested only in wild type animals, which are more resistant to infection. However, the results presented herein indicate that the increased NF-κΒ activation stimulated by the infection with the mutant strain does not result in higher bacterial loads but rather in a significant increase in the levels of pro-inflammatory cytokine in the intestine. The early death observed in a significant proportion of the animals infected with the S. Typhimurium AgogA AgtgA ApipA mutant strain may be the result of a "cytokine storm" triggered by the increased cytokine production in the intestine of the wild type animals infected by the mutant. Consistent with this hypothesis only the increased virulence phenotype after oral administration of the bacterial mutant strains but not after intraperitoneal infection was observed. Previous studies have indeed shown that the intestinal inflammatory response to S. Typhimurium early during infection is higher in nrampl^+/+ than in nrampl^{" "} mice (Valdez et al., 2009), which is consistent with this hypothesis. Why the absence of NRAMPl (SLC11A1) results in an increased inflammatory response to Salmonella is unclear but it is intriguing that previous studies have shown that this transporter modulates signal transduction pathways leading to inflammation (Gomez et al., 2007, J Biol Chem 282:36190-8; Hedges et al, 2013, J Immunol 190:4263-73).

It is widely believed that the remarkable advances in the understanding of the mechanisms of pathogenesis of the most important pathogens will result in the development of novel antimicrobial strategies aimed at specifically targeting those mechanisms. In this context, type III secretion system machines and their effector proteins are viewed as potential targets for the development of such type of drugs (Patel et al, 2005, Trends Pharmacol Sci 26:564-70). However, the findings reported here showing that absence of a family of effector proteins results in increased virulence indicates that caution should be exercised before targeting biochemical activities of effectors whose functions have not been thoroughly characterized. In summary, these findings revealed a remarkable adaptation of a bacterial pathogen to secure its own long-term survival even if at the expense of decreasing its virulence.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed is:

1. A method of inhibiting NF-κΒ signaling in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition comprising at least one agent that inhibits a NF-KB transcription factor, wherein the NF-κΒ transcription factor is selected from the group consisting of RelA, RelB and cRel.

2. The method of claim 1 , wherein the at least one agent comprises at least one isolated peptide.

3. The method of claim 2, wherein the at least one isolated peptide comprises an amino acid sequenced selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ ID NO: 3.

4. The method of claim 1, wherein the at least one agent cleaves the NF-KB transcription factor.

5. The method of claim 2, wherein the at least one peptide is selected from the group consisting of PipA, GtgA and GogA.

6. A method of treating or preventing a disease or disorder associated with NF-κΒ in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising at least one agent that inhibits a NF-κΒ transcription factor, wherein the NF-κΒ transcription factor is selected from the group consisting of RelA, RelB and cRel.

7. The method of claim 6, wherein at least one agent comprises an isolated peptide.

8 The method of claim 7, wherein the at least one isolated peptide comprises an amino acid sequenced selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

9. The method of claim 6, wherein the at least one agent cleaves the NF-KB transcription factor.

10. The method of claim 7, wherein the at least one isolated peptide is selected from the group consisting of PipA, GtgA and GogA.

11. The method of claim 6, wherein the at least one agent comprises at least one isolated nucleic acid.

12. The method of claim 11, wherein the at least one isolated nucleic acid encodes at least one protein that cleaves the NF-κΒ transcription factor.

13. The method of claim 12, wherein the at least one protein selected from the group consisting of PipA, GtgA and GogA.

14. The method of claim 8, wherein the method further comprises administering to the subject at least one additional therapeutic agent.

15. The method of claim 14, wherein the therapeutic agent is selected from the group consisting of anti-cancer agent, and anti-inflammatory agent.

16. The method of claim 14, wherein the composition and the additional therapeutic agent are co-administered.

17. The method of claim 6, wherein the disease or disorder is selected from the group consisting of cancer, diabetes, heart disease, asthma, inflammatory bowel disease, lupus, Alzheimer's disease, Huntington's disease, chronic obstructive pulmonary disease (COPD), atopic dermatitis, atopy, allergy, allergic rhinitis, Crohn's disease, and scleroderma.

18. A composition for inhibiting the NF-κΒ signaling pathway, wherein the composition comprises at least one agent that inhibits a NF-KB transcription factor, wherein the NF-κΒ transcription factor is selected from the group consisting of RelA, RelB, and cRel.

19. The composition of claim 18, wherein the at least one agent comprises at least one peptide.

20. The composition of claim 18, wherein the at least one agent cleaves the NF-κΒ transcription factor.

21. The composition of claim 19, wherein the at least one peptide is at least one selected from the group consisting of PipA, GtgA and GogA.

22. The composition of claim 18, wherein the agent comprises at least one isolated nucleic acid.

23. The composition of claim 22, wherein the at least one isolated nucleic acid encodes at least one protein that cleaves the NF-κΒ transcription factor.

24. The composition of claim 22, wherein the at least one isolated nucleic acid encodes at least one full-length protein selected from the group consisting of PipA, GtgA and GogA.

25. The composition of claim 20, wherein the at least one peptide comprises at least one amino acid sequenced selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.