Engineered bacteria and uses thereof

Abstract

The present disclosure relates to engineered bacteria, compositions and methods for immune modulation, specifically Bacteroides ovatus strains locked in a defined orientation at a genomic locus undergoing phase variation. The resulting "ON" and "OFF" mutants showed distinct immunomodulatory effects in vitro and in vivo, including differential cytokine secretion, regulatory T cell induction, and antitumor activity. These engineered strains offer a novel platform for modulating immune responses in cancer, autoimmune, and inflammatory diseases.

Description

[0001] ENGINEERED BACTERIA AND USES THEREOF

[0002] TECHNOLOGICAL FIELD

[0003] The present disclosure relates to the field of immune modulation. More specifically, the present disclosure relates to engineered bacteria, specifically engineered bacteria “locked” in a desired orientation in a genomic locus displaying phase variation, and uses thereof.

[0004] BACKGROUND ART

[0005] References considered to be relevant as background to the presently disclosed subject matter are listed below:

[0006] 1. Carasso, S. et al. Inflammation and bacteriophages affect DNA inversion states and functionality of the gut microbiota. Cell Host Microbe 32, 322-334 e329 (2024).

[0007] 2. Ben-Assa, N. et al. Analysis of a phase-variable restriction modification system of the human gut symbiont Bacteroides fragilis. Nucleic Acids Res 48, 11040-11053 (2020).

[0008] 3. Jiang, X. et al. Invertible promoters mediate bacterial phase variation, antibiotic resistance, and host adaptation in the gut. Science 363, 181-187 (2019).

[0009] 4. Garcfa-Bayona, L. & Comstock, L. E. Streamlined Genetic Manipulation of Diverse Bacteroides and Parabacteroides Isolates from the Human Gut Microbiota. mBio 10 (2019).

[0010] Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.

[0011] BACKGROUND

[0012] Emerging evidence is accumulating on the effects of the gut microbiome on the host's immune system. The bacteria components with immune-modulatory effects span from capsular polysaccharides on the bacterial surface, fermenting nutrients and secreting metabolites. To adapt to ever-changing environments, bacteria exhibit various functional plasticity through mechanisms that allow changes in its phenotype based on cues from the environment and neighboring microbes. These phenotypic changes can, in turn, affect the microbial ecosystem, as well as the host. One such mechanism is phase variation caused by DNA inversions, where the inversions often occur in functional regions of genes or in promotor areas which causes changes in expression and function of the genes. Such alterations were shown by the inventors and others to have immunomodulatory effects on the host [REF 1, 2].

[0013] The gut microbiome composition and function were shown to correlate with immune-modulation and also with immunotherapy response in various cancers, not only in colorectal cancer but also in cancers located in distant locations from the gut. The link between microbiome and cancer (in general) as well as cancer immunotherapy was even recognized as a hallmark for cancer therapy. However, the mechanisms that contribute to the microbial effect on the response are not yet completely understood.

[0014] Cancer immunotherapy has revolutionized cancer treatment and predicted survival, as well as progression-free survival were elongated in various cancer types. However, not all patients respond to the treatment. In some patients, the gut microbiome was shown to correlate with improved response to melanoma immunotherapy. However, correlation analysis across various studies reveals different significant bacteria. In some studies, attempts to use “off the shelf probiotics” were found to reduce immunotherapy efficiency.

[0015] In different diseases such as inflammatory bowel diseases (IBD) and autoimmune diseases the host has overactivation of the immune system.

[0016] GENERAL DESCRIPTION

[0017] A first aspect of the present disclosure relates to a genetically engineered bacterium or a population comprising said bacterium. The genetically engineered bacterium is locked in a desired orientation in at least one genomic locus displaying phase variation. This bacterium comprises at least one modification in a target nucleic acid sequence that comprises or encodes at least one element. The element induces directly or indirectly phase variation in the at least one genomic locus.

[0018] Another aspect of the present disclosure relates to a composition comprising a genetically engineered bacterium or a population comprising the bacterium. More specifically, the genetically engineered bacterium is locked in a desired orientation in at least one genomic locus displaying phase variation. This bacterium comprises at least one modification in a target nucleic acid sequence that comprises or encodes at least one element that induces directly or indirectly phase variation in the at least one genomic locus. The composition further comprising at least one of acceptable carrier / s, diluent / s, excipient / s and additive / s.

[0019] Another aspect of the present disclosure relates to a method for modulating an immune response in a subject in need thereof. The method comprises the step of administrating to the subject at least one genetically engineered bacterium, a population comprising the bacterium, or any composition thereof, wherein the bacterium is locked in a desired orientation in at least one genomic locus displaying phase variation. This bacterium comprises at least one modification in a target nucleic acid sequence that comprises or encodes at least one element that induces directly or indirectly phase variation in the at least one genomic locus. The present disclosure further provides at least one genetically engineered bacterium, a population comprising the bacterium, or any composition thereof, as disclosed herein, for use in a method for modulating an immune response in a subject in need thereof.

[0020] Another aspect of the present disclosure relates to a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one pathologic disorder in a subject. The method comprising the steps of administrating to the subject at least one genetically engineered bacterium, or a population comprising said bacterium, wherein the genetically engineered bacterium is locked in a desired orientation in at least one genomic locus displaying phase variation. This bacterium comprises at least one modification in a target nucleic acid sequence that comprises or encodes at least one element that induces directly or indirectly phase variation in the at least one genomic locus. The present disclosure further provides at least one genetically engineered bacterium, a population comprising the bacterium, or any composition thereof, as disclosed herein, for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one pathologic disorder in a subject.

[0021] Another aspect of the present disclosure relates to a method for the preparation of an immunomodulatory composition comprising the following steps: Step (a) includes genetically modifying at least one target nucleic acid sequence in a bacterium. The target sequence comprises or encodes at least one element that induces directly or indirectly phase variation in at least one genomic locus displaying phase variation. The modification results in locking the genomic locus in a desired orientation. Step (b) includes admixing a bacterium locked in a desired orientation of the genomic locus obtained by step (a), with at least one of carrier / s, diluent / s, excipient / s and additive / s. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0023] Figure 1. Percentage (%) of 'ON' orientation for I PxY gene promoter of B. ovatus in responders vs. non-responder patients to immunotherapy treatment

[0024] A metagenomic dataset of human stool samples from 161 adult melanoma patients collected prior to immunotherapy initiation was used for computational analysis (Spencer et. al., 2021, Science DOI: 10.1126 / science.aaz7015).

[0025] Each dot represents one patient’ s bacterial phase variation in the UPxY gene promoter ratio of 'ON' orientation before immunotherapy (p=0.018).

[0026] Figure 2A-2B. Graphical abstract of bacterial engineering process (created with BioRender, com )

[0027] Fig. 2A. demonstrates manipulated bacterial genomic region (by knocking out tyrosine invertase to prevent the inversion in the promoter).

[0028] Fig. 2B. demonstrates the process of engineering the bacteria to lock the promoter region of UPxY gene of B. ovatus. Step 1 includes plasmid linearization (step la) and amplification of down steam (DS) and upstream (US) sections of the target gene to be deleted (in this case tyrosine invertase in B. ovatus) (step lb). In step 2, the amplified DS and US sections are added to the linearized plasmid and a circular plasmid is created using Gibson. In step 3, the plasmid is transformed into E. coli (e.g. by electroporation or heat shock) for amplification and then mating of the E. coli with the target bacteria (e.g. B. ovatus) is performed for transferring the plasmid from the E. coli to the target bacteria. In step 4, E. coli is eliminated as well as target bacteria with incorrect plasmid insertion. In step 5, colonies are selected and in step 6 are tested for losing the target gene and not reverting to WT. Step 7 includes final checking. Details of each step are included in the Experimental Procedure.

[0029] Figure 3. The effect on the growth rate in two different growth media, for the different mutants Wild-type B. ovatus and mutants harboring the UPxY promoter region locked in either the 'OFF' or 'ON' orientation were cultured in basal and BHIS media for 24 hours, and their growth rates were measured. Y axis represents the optical density (OD) measurement, and the X axis represents time in seconds, offl.l, offl.2, offl.3, off2.1, etc., represents different clones of the corresponding mutant or WT). No significant change in growth rate between the various mutants was observed. Figure 4A-4B. Biofilm formation is reduced for engineered locked 'ON' bacteria

[0030] Quantification of biofilm formation by the wild-type B. ovatus and its mutants, in basal (Fig. 4A) and YCFA (Fig. 4B) media, was tested using the crystal violet (CV) staining method as described in the Experimental Procedure. The X axis represents the 'OFF' mutants, 'ON' mutants, WT or blank control, and the Y axis represents CV absorption following biofilm staining normalized to mean of WT. EXP. (experiment) D, E, F represents different repeats of the same experiment.

[0031] Figure 5A-5C. Change in TNF-a, IL-6 cytokine secretion after 24h and IL-10 after 4 days, incubation of splenocytes with PF A fixed engineered bacteria

[0032] Splenocytes were induced with anti-CD3and incubated with either fixed wild-type (WT) B. ovatus, fixed mutants harboring the UPxY promoter in the 'ON' locked orientation or fixed mutants harboring the UPxY promoter in the 'OFF' locked orientation. Cytokines secretion was quantified using EEISA kit after incubation for 24h (TNF-a (Fig. 5A), IL-6 (Fig. 5B)) or 4 days (IL- 10, (Fig. 5C)) and normalized to anti-CD3 / LPS controls. Y axis represents the fold change from the anti- CD3 / LPS control. EXP. (experiments) 1, 2, 3 represents different repeats of the same experiment. Figure 6A-6B (i)-(vi). Immunophenotyping of monocolonized mice with engineered bacteria Germ-free (GF) mice (C57BL / 6J01aHsd) were monocolonized once at 4 weeks with lOOul of o / n grown bacteria (about 10A9 CFU per mouse). The mice were then kept in sterile conditions for 14 days and sacrificed. Immune phenotype was tested using FACS.

[0033] Fig. 6A. Heatmap for Z-score of different immune parameters in the blood.

[0034] Fig. 6B(i)-6B(vi). Box plots of immune cell populations in blood or inguinal lymph nodes (ILN). X axis represents tested groups (GF - no bacteria added, 'ON' - locked on bacteria, 'OFF' - locked off bacteria). Y axis represents the percentage of the specific cell type from the parent cell population. The following cell types were evaluated: Ki67 high out of CD4+ cells in the blood (Fig. 6B(i)), Foxp3+ out of CD4+ cells in the blood (Treg) (Fig. 6B(ii)), innate immune cells with no CD 1 lb and no CD 11c that have low expression of MHC class2 and PDL- 1 in ILN (Fig. 6B(iii)) , innate immune cells with CDl lb+ and CD11C+ with high expression of MHC class 2 and PDL- 1 in blood (Fig. 6B(iv)), CD8+ with low Ki67 expression in blood (Fig. 6B(v)) and innate immune cells with no CD1 lb and no CD11c that have high MHC class2 expression (Fig. 6B(vi)). Figure 7. Colonization of engineered bacteria in different organs in GF mice

[0035] After two weeks of introducing engineered bacteria to Germ-Free (GF) mice, the number of bacteria in feces, cecum and small intestine (SI) content were determined. X axis represents the mice number: mice # 4 to #8 were introduced with locked ‘ON’ mutant while mice #9 to #10 were introduced with locked ‘OFF’ mutant. Y axis represents the colony forming unit per milligram (CFU / mg).

[0036] Figure 8A-8C. Relationship between engineered bacteria’s OD to colony forming unit (CFU)

[0037] The number of bacteria (CFU) at different optical density (OD) during the growth of bacteria of the "ON" orientation (Fig. 8A), "OFF" orientation (Fig. 8B) or WT bacteria (Fig. 8C) was determined. X axis represents the OD at 600nm. Y axis represents the number of bacteria in the logarithm scale log(CFU).

[0038] Figure 9. The colony forming unit of mutants in different parts of the digestive system of GF mice

[0039] The CFU of the content of feces, cecum and SI of GF mice was tested. The results were normalized to the weight of the content. The bacteria colonized the cecum and colon (feces) more than the SI and there was no difference between the 'ON' and 'OFF' mutants.

[0040] Figure 10. An additional version of bacterial engineering

[0041] Here the inventors scrambled the invertible region rather than erasing the Tyrosine invertase gene. In this configuration, the tyrosine invertase enzyme is expressed but cannot invert the relevant sequence (in this case the promoter), hence forming a stable configuration of the engineered bacteria.

[0042] Figure 11. RNA expression of the UPxY gene

[0043] Results of qPCR amplification of the UPxY gene after the engineered promoter. RNA was extracted from bacteria, transformed to cDNA and used to quantify UPxY gene relative to a reference gene. The results demonstrate that the UPxY gene is expressed significantly higher for the engineered “ON” orientation in comparison to the engineered 'OFF' orientation or the wildtype bacteria (WT).

[0044] Round dots represent mutants engineered using the scramble method, “X” are mutants engineered using the deletion method, and triangles are WT.

[0045] Figure 12. Experimental design for cancer model

[0046] Bacteria or only media were administered orally for 3 consecutive days to specific pathogen free (SPF) mice. After a week from the last gavage, YUMM1.7 cells (500,000 cells / mouse) were injected to the flank. After 5 days and 7 days from tumor injection, mice were treated with anti- PD1 (250ug / mouse) as well as additional oral gavage with the bacteria. At day 10 from tumor injection, mice were sacrificed, tumor weights were recorded and draining lymph nodes were analyzed using flow cytometry immune characterization.

[0047] Figure 13. Tumor wight monitoring

[0048] The graph demonstrates net tumor weight in mg (Y axis) of mice treated as described in Figure 12. In white - SPF (mice with no added bacteria), in striped pattern - ON (SPF mice supplemented with engineered mutants bacteria in the 'ON' orientation), in dotted pattern - OFF (SPF mice supplemented with engineered mutants bacteria in the 'OFF' orientation).

[0049] Figure 14. Immunophenotyping of tumor draining lymph node - CD11B+ cells

[0050] Graph shows CD11B+ cells percentage out of the CD45+ CD3- cells. Significant cell percentage elevation is detected for the 'ON' group, in comparison to both the 'OFF' and 'SPF' groups.

[0051] Figure 15. Immunophenotyping of tumor draining lymph node - CD11B+ F4 / 80+

[0052] Graph shows CD11B+ F4 / 80+ (macrophages) cells percentage out of the CD45+ CD3- cells. Significant cell percentage elevation is detected for the 'ON' group, in comparison to both the 'OFF' and 'SPF' groups.

[0053] Figure 16. Immunophenotyping of tumor draining lymph node - CD4+ CD49b+

[0054] Graph shows CD4+ CD49b+ (NK) cells percentage out of CD45+ CD3+ cells. Significant cell percentage reduction is detected for the 'OFF' group, in comparison to both the 'ON' and 'SPF' groups.

[0055] DETAILED DESCRIPTION OF EMBODIMENTS

[0056] The success rate of immunotherapy is difficult to predict, and many of the contributing factors remain unknown. Bacterial composition is a candidate factor for influencing immunotherapy success rate. Previous studies have shown that responders to immunotherapy have different microbiomes compared to non-responders and that fecal transplantation of the responders’ stool into non-responders can markedly improve the patients’ response to treatment. However, these are very “general” solutions without robustness. Additionally, probiotics from the shelf, are very generic, without a tailor to specific diseases or to companion therapeutics. Moreover, the “off-the- shelf’ probiotics were not only not shown to improve immunotherapy but also have been shown to deteriorate the disease state of the patients and their potential response to therapy. Currently, there are no known bacterial compositions to improve cancer immunotherapy. Unsuccessful attempts have been made to improve immunotherapy by introducing bacterial composition to patients in various clinical trials.

[0057] The inventors identified DNA inversions within the gut microbiome that might affect patients’ response to melanoma immunotherapy. The inventors apply microbial genetic engineering to alter the causal and immune-modulatory effects of the bacteria on the host, both as potential therapy and as potential companion therapeutics.

[0058] The genetic engineered bacteria are based on computational findings on bacterial DNA inversions in patients (either with regards to disease state or with regards to response to treatment). Engineered bacteria modulate the host immune system differently and can be relevant to various disease states and treatments.

[0059] The inventors aim to tailor beneficial bacteria to harbor specific immunomodulatory effects based on engineering of their DNA inversion states. Using such engineered bacteria can potentially improve response rates of immunotherapy as well as alleviate flares of autoimmune or immune mediated diseases.

[0060] The inventors' engineered bacteria can be used as tailored “probiotics” to alter the immune response towards therapeutics and companion therapeutics. The engineered bacteria “locked” in the certain DNA inversion can either induce immune suppressive effects, which are relevant to disease conditions such as IBD, or autoimmune / autoinflammatory diseases, or to induce immune activating effects, which are relevant as for cancer treatment, either alone or as companion therapeutics to biological treatments for cancer patients. The inventors have shown that the 'OFF' and 'ON' mutants can induce either of these effects, and this invention can be tailored to the designated disease condition, depending on the engineered orientation in the bacteria.

[0061] More specifically, the present disclosure demonstrates two distinct genetic strategies for locking Bacteroides ovatus in a desired promoter orientation to modulate host immune responses. The first, termed herein the "deletion approach", involves knocking out the tyrosine recombinase gene responsible for invertible promoter switching. The second, referred to herein as the "scrambled approach", achieves orientation locking by modifying the invertase recognition (IR) sequences within the promoter of the target UPxY locus, used here as a proof of concept. Both methods successfully produced bacterial strains stably fixed in either an "ON" or "OFF" configuration without affecting bacterial growth (Fig. 3). In vitro and ex vivo characterization showed that locked "ON" mutants exhibited a significant reduction in biofilm formation (Fig. 4) and enhanced cytokine secretion, specifically IU-6, IU-10, and TNF-a, upon immune stimulation (Fig. 5), whereas "OFF" mutants induced a lower immune response. In vivo experiments in germ-free mice revealed that locked "OFF" mutants induced a more regulatory immune profile, marked by an increase in CD4+Foxp3+regulatory T cells and PDL-1+expressing cells (Fig. 6B), suggesting reduced immune activation. These strains colonized the colon and cecum more efficiently than the small intestine, with no significant differences between "ON" and "OFF" orientations (Figs. 7, 9) and showed no alterations in growth rate (Figs. 3, 8).

[0062] The results obtained with the "scrambled approach" confirmed the alignment between engineered promoter orientation and gene expression: reverse transcriptase real-time qPCR showed that "OFF" mutants had reduced UPxY RNA expression, while "ON" mutants displayed a 4-8-fold increase in expression compared to WT, consistent across both engineering methods (Fig. 11). In a melanoma tumor model, mice colonized with the "ON"-locked strain exhibited significantly reduced tumor weight compared to the "OFF" group (Fig. 13). Immune profiling of tumor-draining lymph nodes revealed higher levels of CDllb+cells (Fig. 14) and macrophages (Fig. 15) in the "ON" group, while the "OFF" group showed reduced frequencies of CD4+NK cells (Fig. 16). These findings collectively highlight that locking bacterial gene expression in a stable orientation using either genetic strategy can influence immune outcomes and tumor progression, supporting the potential application of engineered B. ovatus strains in immune modulation and cancer immunotherapy .

[0063] A first aspect of the present disclosure relates to a genetically engineered bacterium or a population comprising said bacterium. The genetically engineered bacterium is locked in a desired orientation in at least one genomic locus displaying phase variation. This bacterium comprises at least one modification in a target nucleic acid sequence that comprises, comprised within, or encodes at least one element. The element induces directly or indirectly phase variation in at least one genomic locus.

[0064] The term “genetically engineered bacterium” , as used herein, refers to a bacterial organism whose genome has been modified through recombinant DNA technologies, such as gene deletion, insertions, rearrangements, site-directed mutagenesis, or homologous or non-homologous recombination, for example, by using a gene editing system (e.g., the CRISPR / Cas system), to introduce specific changes at one or more loci. These modifications alter or eliminate the natural stochastic switching (ON / OFF states) commonly observed in genomic loci undergoing phase variation. The present disclosure relates the genetically engineered bacterium or a population comprising such bacterium.

[0065] The term “population” , as used herein, refers to a group of bacteria that may be clonally derived or genetically diverse, but which are collectively characterized by comprising at least one genetically engineered bacterium as described. Such populations may occur in vitro, in vivo, or ex vivo and may be homogeneous (all bacteria bearing the same locked orientation) or heterogeneous (the bacteria comprising different engineered variants). In certain embodiments, the cell population is predominantly composed of the disclosed genetically engineered bacterium, while in other embodiments, the genetically engineered bacterium forms a minor component of the population. Accordingly, the population may comprise from about 10% to about 100% of the engineered bacterial strain, based on the total number of bacterial cells present. In certain embodiments, the engineered bacterium constitutes at least 10%, 20%, 30%, 40%, or 50% of the total bacterial population. In more enriched preparations, the engineered strain may represent at least 60%, 70%, 80%, or 90% of the population. In particular pure or monoclonal preparations, the engineered bacterium may comprise at least 95%, 98%, 99%, or even 100% of the total bacterial content.

[0066] The genetically engineered bacterium of the present disclosure is locked in a desired orientation in at least one genomic locus displaying phase variation.

[0067] The term "phase variation " (also referred to as antigenic variation) as used herein, refers to a reversible switch between an “all-or-none” ('ON' / 'OFF') expressing states of a gene, operon, or locus. This process results in differential expression of one or more proteins among individual cells of a clonal population. Phase variation mechanisms generate variations in the sequence of surface proteins resulting in the expression of different forms and structures of the antigenic proteins on the cell surface. Genetic modifications mechanism of phase variation includes for example DNA inversion, DNA recombination, transposition mechanism, slipped strand mispairings (SSM) and phase variation via differential methylation, as detailed herein below.

[0068] The genetically engineered bacterium of the present disclosure is thus rendered locked in a desired orientation in at least one genomic locus displaying phase variation.

[0069] The term "locked in a desired orientation" refers to genetically engineered state in which the natural ability of a phase-variable locus to switch between 'ON' and 'OFF' states is permanently, irreversibly, and / or stably fixed in one preferred specific configuration (e.g., 'ON' or 'OFF'). This "locking" may be achieved through targeted genetic modifications that disrupt, inhibit, attenuate, decrease or remove the underlying switching mechanism, such as by deleting an invertase gene or mutating any sequence involved in the inversion process, for example, any sequence involved with the targeting, recognition, binding of the invertase and / or any sequence or region involved with or subjected to the inversion, and / or a sequence that is subjected for inversion by the invertase and / or recombinase, for example, the inverted repeats of the relevant genomic region of interest, thereby stabilizing the resulting gene expression (as a result of either the "ON" or "OFF" orientation) to above 50%, specifically, between about 50% to 100%, more specifically, above 98% of the bacterial population (qPCR measurement).

[0070] Still further, the term "a desired orientation” , in connection with the preset disclosure refers to the specific directional arrangement of the target sequence, for example, the target promoter region within the target genomic locus, such that it either enables ("ON") or prevents ("OFF") transcription of an adjacent coding sequence (e.g., the UPxY coding sequence). Given the inherent 5' to 3' polarity of DNA, transcription requires that the promoter be oriented in a direction that aligns its core elements, such as the TATA box, transcription start site (TSS), and upstream regulatory sequences, appropriately relative to the coding region, allowing RNA polymerase and associated transcription factors to initiate and proceed through the open reading frame in the correct direction. In the context of the present disclosure, the desired orientation may be either an "ON" orientation, in which the promoter is aligned to direct transcription toward the coding sequence in the proper reading frame, or an "OFF" orientation, in which the promoter is arranged in the reverse direction or otherwise misaligned, thereby preventing or significantly reducing transcription. For the "ON" orientation, promoter elements must be positioned upstream (5') of the coding sequence, with their sequence motifs facing toward the open reading frame, ensuring accessibility and correct assembly of the transcriptional machinery. In contrast, in the "OFF" orientation, the promoter faces away from the coding sequence or has an inverted configuration, such that transcription initiation is blocked or yields nonfunctional transcripts. The genetically engineered bacteria disclosed herein have a locked orientation in a specific genomic locus that naturally display phase variation. The term "genomic locus displaying phase variation", also referred to as "phase variation locus" or "phase variation region" refers herein to segment of the bacterial genome that is inherently capable of undergoing genetic or epigenetic changes that result in phase variation. Structurally, such regions may include structural features like inverted repeats, short sequence repeats, insertion elements, recombination sites, amplifications, deletions, or methylation- sensitive sequences. These features contribute to the reversible or semi-reversible expression of genes under selective or stochastic control. Non-limiting examples of such loci include the upxY locus in Bacteroides ovatus and Bacteroides fragilis, which regulates capsular polysaccharide expression through reversible promoter inversion and is involved in complex phase- variable expression of surface polysaccharides; the ypxY locus in Bacillus subtilis, which undergoes phase-variable control via site-specific recombination; the fim switch region in Escherichia coli, which regulates type 1 fimbriae expression through promoter inversion; the lid locus in Haemophilus influenzae, which modulates expression of lipooligosaccharide biosynthetic genes through slipped- strand mispairing; the mod gene loci in Neisseria species, which control phase- variable DNA methyltransferases leading to phasevarion-based gene regulation; and the bvgAS locus in Bordetella pertussis, which undergoes large-scale inversions and epigenetic modifications that toggle between virulent and avirulent phases.

[0071] As described, the engineered bacterium of the present disclosure comprises at least one modification in at least one target nucleic acid sequence, or in one or more target nucleic acid sequence. The term “modification”, in this context, refers to any deliberate change made to a target nucleic acid sequence within the bacterial genome and any alteration to the nucleotide sequence or structural organization of a defined genomic region. Such modifications may include, but are not limited to, deletions, insertions, point mutations, inversions, substitutions, or rearrangements of one or more nucleotides within the locus. They can be introduced by targeted genome-editing techniques such as CRISPR / Cas systems, TALENs, or zinc finger nucleases, or by conventional recombinant DNA approaches involving homologous recombination or transposon-mediated integration. The modification may involve the introduction of an exogenous sequence, such as a regulatory element, or coding sequence, at a defined position within the locus, or the replacement of an endogenous sequence with a replacement sequence. Alternatively, the modification may entail the disruption or silencing of an endogenous gene, for instance through frameshift mutations or promoter deletions, in order to modulate gene expression or abolish protein function. Epigenetic modifications, such as targeted methylation or demethylation of specific CpG sites, may also be encompassed, as they can influence transcriptional activity without altering the underlying DNA sequence. In some embodiments, the modification results in the generation of a knock-in or knock-out allele, a conditional or inducible expression construct, or a variant allele carrying specific point mutations. Overall, the term “modification" is intended to encompass any molecular change, structural, sequence-based, or regulatory, that alters the native configuration or function of the locus in a controlled and definable manner. The intent of the modification may be to disable phase switching, alter gene expression, or fix a promoter or regulatory element in a predefined orientation. The phrase “target nucleic acid sequence / s” used herein refers to the specific genomic segment, or nucleic acid sequence, selected for modification because of its role or effect in enabling, modulating or regulating phase variation. The target sequence may either encode a product. For example, a protein, such as a DNA invertase or recombinase, or encode a regulatory nucleic acid sequence (e.g., miRNA, IncRNA). Alternatively, the target sequence may act as, may be comprised within, or may comprise a non-coding sequence, for example, a regulatory element such binding site for any effector, for example, as an invertase binding site, recombination site, binding site for transcription factors, DNA sequence motif involved in sitespecific rearrangement, a promoter, enhancer, splice donor and / or acceptor sites and the like. The one or more target sequence may be located within, adjacent to, or distal from the locus in which phase variation occurs, provided that it functionally regulates, affects or enables such phase variation of the indicated locus.

[0072] Thus, the target nucleic acid sequence comprises, comprised within, or encodes at least one element, which induces directly or indirectly phase variation in the at least one genomic locus.

[0073] The term “element”, in this context, refers to a nucleic acid regulatory component, or a product (e.g., a protein product), that is either encoded by, or comprises, or included or comprised, at least partially, within the target nucleic acid sequence, and which serves to initiate, enable, or promote phase variation at a designated genomic locus. Examples of such elements include nucleic acid sequences, or any encoded products (e.g., protein or nucleic acid products), without limitation, to protein products such as invertases (e.g., serine or tyrosine site-specific recombinases or MPIs), invertase recognition, binding or targeting sites, or sequences participating directly or indirectly in the inversion process (e.g., inverted repeats sequences), or recombinase binding, recognition or targeting sites (typically flanking an invertible promoter), transposases or transposase sequences, homopolymeric tracts susceptible to slipped- strand mispairing, or DNA motifs that serve as substrates for epigenetic modification such as methylation. The element may exert its effect directly, by catalyzing a recombination or inversion event, or by participating in, or being targeted by the inversion process (e.g., inverted repeats), or indirectly, by modulating the expression or function of proteins or nucleic acid sequences involved in such inversion events.

[0074] The phrase “induces directly or indirectly” phase variation refers to the capacity of the aforementioned element to either cause or contribute to the initiation or extent of phase variation at a genomic locus. "Direct induction" includes the catalysis of inversion, recombination, or other rearrangement events by an enzyme such as an invertase. Alternatively, nucleic acid sequence that functions as a binding site for such invertase or recombinase, is also considered as directly inducing the phase variation. "Indirect induction" encompasses regulatory effects that alter the transcription, translation, or activity of such enzymes or their cofactors, thereby modulating the frequency or directionality of phase variation without acting as the immediate effector.

[0075] In some embodiments, the phase variation in the genomic locus of the disclosed genetically engineered bacterium, is associated with at least one physiological state and / or condition in a subject.

[0076] The term “associated with ” as used herein, refers to the existence of any relationship, linkage, correlation, dependency, influence, or interaction between two or more entities, events, or conditions. This relationship may be causal or non-causal, direct or indirect, and may be based on experimental evidence, statistical correlation, mechanistic inference, predictive modeling, or other recognized scientific or empirical observations.

[0077] In some embodiments, the phase variation in the genomic locus of the disclosed genetically engineered bacterium is associated with at least one physiological state and / or condition in a subject. In some embodiment, the subject is or can be the host of the bacteria displaying the indicated phase variation. As used in this context, “associated with” encompasses any biological, functional, or regulatory relationship in which the phase state of the genomic locus correlates with, responds to, or influences a physiological condition present in the host. This association may be substantiated by experimental data, mechanistic insight, or predictive modeling, and may be leveraged in certain embodiments for diagnostic, therapeutic, or regulatory purposes.

[0078] It should be understood that the "physiological state and / or condition" of a subject of the present disclosure comprise pathological condition / s and / or health condition / s in a subject. More specifically, a “pathological condition”, refers to a condition, in which there is a disturbance of normal functioning, any abnormal condition of the body or mind that causes discomfort, dysfunction, or distress to the person affected or those in contact with that person. Pathologic condition as used herein is any condition or pathology caused by or associated with a pathogenic agent (including biotic or abiotic agents), a physical or metabolic chronic or acute stress, tissue and / or organ injury, disfunction or hyperfunction, and any of the conditions disclosed herein.

[0079] In contrast, a “health condition” , as used herein, refers to any non-pathological physiological state, transient or chronic deviation from baseline, or biologically relevant context that does not meet the criteria of a disease state but may nonetheless influence or reflect the function of the microbiome, immune system, or other physiological systems. Non-limiting examples include pregnancy, hormonal cycles, dietary states (e.g., fasting, ketogenic diet), behavioral or emotional states (e.g., stress, sleep deprivation), physical exertion, aging, microbiota composition, vaccination status, or environmental exposures. A health condition may also refer to a baseline or normal physiological status in a subject, wherein modulation of the microbiome is still desirable for preventative, diagnostic, or enhancement purposes. It should be understood that the phase variation in the genomic locus may be associated with any physiological state, including both health and disease.

[0080] In some further embodiments, the physiological state and / or condition associated with the phase variation comprises at least one of: an immunological state and / or condition, a metabolic state, diet, behavioral / mental state and / or condition in the subject.

[0081] Thus, in some specific embodiments, the physiological state and / or condition associated with the phase variation comprises an immunological state and / or condition in the subject.

[0082] As used herein, an “immunological state and / or condition” refers to the status, reactivity, or dysregulation of the subject’s immune system, encompassing both normal immune physiology and pathological immune responses. This may include immune activation, suppression, hypersensitivity, tolerance, or exhaustion, and further encompasses immunopathological conditions such as autoimmune diseases, chronic inflammation, immune deficiency syndromes, infectious diseases, and immune responses to malignancies or immunotherapies.

[0083] In some other specific embodiments, the physiological state and / or condition associated with the phase variation comprises a metabolic state in the subject.

[0084] A “metabolic state”, as used herein, refers to the current or chronic state of the subject’s biochemical processes related to energy production, nutrient utilization, or macromolecule synthesis and degradation. This includes, but is not limited to, states of insulin sensitivity or resistance, glucose or lipid metabolism profiles, and altered metabolic setpoints as seen in obesity, diabetes, cachexia, or metabolic syndrome.

[0085] In some further embodiments, the physiological state and / or condition associated with the phase variation comprises a diet state in the subject.

[0086] The term “diet” refers to the subject’s nutritional intake pattern and composition, whether transient (e.g., fasting, ketogenic, high-fat, or fiber-rich diets) or habitual. Diet can profoundly influence microbiome composition and metabolic signaling, and may therefore affect, or be affected by, phase variation in engineered bacteria. In some embodiments, the physiological state and / or condition associated with the phase variation comprises a behavioral / mental state and / or condition in the subject.

[0087] The phrase “behavioral and / or mental state” encompasses neurological, psychological, cognitive, and emotional attributes or conditions of the subject, including stress, anxiety, depression, mood, attention, and circadian rhythms.

[0088] In some specific embodiments, the immunological state and / or condition associated with phase variation comprises and / or reflects at least one immune-related disorder in the subject and / or an immune-response of the subject to the immune-related disorder. An 'Immune-related disorder” or " Immune-mediated disorder”, as used herein encompasses any condition that is associated with dysregulation, dysfunction, or altered activity of the immune system of a subject, more specifically through inappropriate activation, suppression, or failure of immune surveillance, or that can be treated, prevented, or ameliorated by modulating components of the immune system, including the innate or adaptive immune response. Non-limiting examples of immune-related disorders include infectious diseases (e.g., by a pathogen, specifically, viral, bacterial, or fungal infections), inflammatory diseases (e.g., inflammatory bowel disease or chronic systemic inflammation), autoimmune disorders (e.g., rheumatoid arthritis, lupus, type 1 diabetes), immunodeficiency conditions (either primary, such as severe combined immunodeficiency (SCID), or secondary, such as viral or chemotherapy-induced immunosuppression), metabolic disorders with immune involvement (e.g., obesity-associated inflammation, type 2 diabetes), and proliferative disorders, particularly cancer, in which immune evasion or immune exhaustion plays a critical role.

[0089] The specific term “immune-response of the subject to the immune-related disorder”, as used herein, refers to the physiological, cellular, and molecular reaction of the subject’s immune system to the presence, progression, or treatment of an immune-related disorder. This response may involve the activation, suppression, recruitment, or exhaustion of immune cells, the production of cytokines or antibodies, or changes in immune checkpoints and regulatory pathways. In some embodiments, the disclosed genetically engineered bacterium is locked in a desired orientation in a genomic locus associated with at least one immune-related disorder.

[0090] In some specific embodiments, an immune-related disorder comprises at least one of: a proliferative disorder, an inflammatory disorder, an infectious disease, an autoimmune disorder, an immune-deficiency condition, a neurodegenerative and / or cognitive and / or mental disorder, a metabolic disorder, and a condition involving at least one wound in at least one tissue and / or organ of the subject. As indicated by the disclosed examples, the genetically engineered bacteria of the present disclosure may be applicable specifically for melanoma patients. In some specific embodiments, the proliferative disorder is at least one malignant neoplastic disorder. In more specific embodiments, the malignant neoplastic disorder comprises melanoma. More specifically, melanoma is a malignant tumor arising from melanocytes, the pigment-producing cells found predominantly in the skin but also present in the eyes (uveal melanoma), mucous membranes, and, rarely, internal organs. It is considered the most aggressive form of skin cancer due to its high potential for metastasis if not detected and treated at an early stage. Clinically, melanoma is classified into several types based on histopathological and anatomical features, including superficial spreading melanoma, which is the most common and typically presents as a flat or slightly raised discolored patch; nodular melanoma, a more aggressive subtype characterized by rapid vertical growth; lentigo maligna melanoma, which tends to develop in sun-damaged skin of older individuals; and acral lentiginous melanoma, more commonly seen on the palms, soles, and under the nails, especially in individuals with darker skin. Melanoma is also staged to reflect its progression and to guide treatment strategies. Stage 0, or melanoma in situ, is confined to the epidermis without invasion. Stage I and II represent localized disease with increasing tumor thickness and possible ulceration but no lymph node involvement. Stage III indicates regional spread to nearby lymph nodes or skin, while Stage IV denotes distant metastases to organs such as the lungs, liver, brain, or bones. It should be understood that the term melanoma as used herein encompass any of the melanoma types, stages or associated malignancies discussed herein.

[0091] As described, in some embodiments, the pathologic disorder applicable in the present disclosure may be any proliferative disorder. As used herein to describe the present disclosure, “proliferative disorder'", " malignant neoplastic disorder”, “cancer”, “tumor” and “malignancy” all relate equivalently to a hyperplasia of a tissue or organ. If the tissue is a part of the lymphatic or immune systems, malignant cells may include non-solid tumors of circulating cells. Malignancies of other tissues or organs may produce solid tumors. Malignancy, as contemplated in the present disclosure may be any one of melanomas, carcinomas, lymphomas, leukemia, myeloma, and sarcomas.

[0092] Leukemia refers to progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease-acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in the number of abnormal cells in the blood-leukemic or aleukemic (subleukemic).

[0093] Sarcoma is a cancer that arises from transformed connective tissue cells. These cells originate from embryonic mesoderm, or middle layer, which forms the bone, cartilage, and fat tissues. This is in contrast to carcinomas, which originate in the epithelium. The epithelium lines the surface of structures throughout the body, and is the origin of cancers in the breast, colon, and pancreas.

[0094] Myeloma as mentioned herein is a cancer of plasma cells, a type of white blood cell normally responsible for the production of antibodies. Collections of abnormal cells accumulate in bones, where they cause bone lesions, and in the bone marrow where they interfere with the production of normal blood cells. Most cases of myeloma also feature the production of a paraprotein, an abnormal antibody that can cause kidney problems and interferes with the production of normal antibodies leading to immunodeficiency. Hypercalcemia (high calcium levels) is often encountered.

[0095] Lymphoma is a cancer in the lymphatic cells of the immune system. Typically, lymphomas are present as a solid tumor of lymphoid cells. These malignant cells often originate in lymph nodes, presenting as an enlargement of the node (a tumor). It can also affect other organs in which case it is referred to as extranodal lymphoma. Non limiting examples of lymphoma include Hodgkin's disease, non-Hodgkin's lymphomas and Burkitt's lymphoma.

[0096] In some embodiments, the present disclosure may be applicable for any solid tumor. In more specific embodiments, the methods disclosed herein may be applicable for any malignancy that may affect any organ or tissue in any body cavity, for example, the peritoneal cavity (e.g., liposarcoma), the pleural cavity (e.g., mesothelioma, invading lung), any tumor in distinct organs, for example, the urinary bladder, ovary carcinomas, and tumors of the brain meninges.

[0097] It should be understood that the present disclosure may be applicable for any type and / or stage and / or grade of any of the malignant disorders discussed herein or any metastasis thereof. Still further, it must be appreciated that the present disclosure may be applicable for invasive as well as non-invasive cancers. When referring to "non-invasive" cancer it should be noted as a cancer that do not grow into or invade normal tissues within or beyond the primary location. When referring to "invasive cancers" it should be noted as cancer that invades and grows in normal, healthy adjacent tissues.

[0098] As used herein the term "metastatic cancer" or "metastatic status" refers to a cancer that has spread from the place where it first started (primary cancer) to another place in the body. A tumor formed by metastatic cancer cells originated from primary tumors or other metastatic tumors, that spread using the blood and / or lymph systems, is referred to herein as a metastatic tumor or a metastasis. More specifically, further malignancies that may find utility in the present disclosure can comprise but are not limited to hematological malignancies (including lymphoma, leukemia, myeloproliferative disorders, Acute lymphoblastic leukemia; Acute myeloid leukemia), hypoplastic and aplastic anemia (both virally induced and idiopathic), myelodysplastic syndromes, all types of paraneoplastic syndromes (both immune mediated and idiopathic) and solid tumors (including GI tract, colon, lung, liver, breast, prostate, pancreas and Kaposi's sarcoma. The present disclosure may be applicable as well for the treatment or inhibition of solid tumors such as tumors in lip and oral cavity, pharynx, larynx, paranasal sinuses, major salivary glands, thyroid gland, esophagus, stomach, small intestine, colon, colorectum, anal canal, liver, gallbladder, extrahepatic bile ducts, ampulla of vater, exocrine pancreas, lung, pleural mesothelioma, bone, soft tissue sarcoma, carcinoma and malignant melanoma of the skin, breast, vulva, vagina, cervix uteri, corpus uteri, ovary, fallopian tube, gestational trophoblastic tumors, penis, prostate, testis, kidney, renal pelvis, ureter, urinary bladder, urethra, carcinoma of the eyelid, carcinoma of the conjunctiva, malignant melanoma of the conjunctiva, malignant melanoma of the uvea, retinoblastoma, carcinoma of the lacrimal gland, sarcoma of the orbit, brain, spinal cord, vascular system, hemangiosarcoma, Adrenocortical carcinoma; AIDS- related cancers; AIDS -related lymphoma; Anal cancer; Appendix cancer; Astrocytoma, childhood cerebellar or cerebral; Basal cell carcinoma; Bile duct cancer, extrahepatic; Bladder cancer; Bone cancer, Osteosarcoma / Malignant fibrous histiocytoma; Brainstem glioma; Brain tumor; Brain tumor, cerebellar astrocytoma; Brain tumor, cerebral astrocytoma / malignant glioma; Brain tumor, ependymoma; Brain tumor, medulloblastoma; Brain tumor, supratentorial primitive neuroectodermal tumors; Brain tumor, visual pathway and hypothalamic glioma; Breast cancer; Bronchial adenomas / carcinoids; Burkitt lymphoma; Carcinoid tumor, childhood; Carcinoid tumor, gastrointestinal; Carcinoma of unknown primary; Central nervous system lymphoma, primary; Cerebellar astrocytoma, childhood; Cerebral astrocytoma / Malignant glioma, childhood; Cervical cancer; Childhood cancers; Chronic lymphocytic leukemia; Chronic myelogenous leukemia; Chronic myeloproliferative disorders; Colon Cancer; Cutaneous T-cell lymphoma; Desmoplastic small round cell tumor; Endometrial cancer; Ependymoma; Esophageal cancer; Ewing's sarcoma in the Ewing family of tumors; Extracranial germ cell tumor, Childhood; Extragonadal Germ cell tumor; Extrahepatic bile duct cancer; Eye Cancer, Intraocular melanoma; Eye Cancer, Retinoblastoma; Gallbladder cancer; Gastric (Stomach) cancer; Gastrointestinal Carcinoid Tumor; Gastrointestinal stromal tumor (GIST); Germ cell tumor: extracranial, extragonadal, or ovarian; Gestational trophoblastic tumor; Glioma of the brain stem; Glioma, Childhood Cerebral Astrocytoma; Glioma, Childhood Visual Pathway and Hypothalamic; Gastric carcinoid; Hairy cell leukemia; Head and neck cancer; Heart cancer; Hepatocellular (liver) cancer; Hodgkin lymphoma; Hypopharyngeal cancer; Hypothalamic and visual pathway glioma, childhood; Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas); Kaposi sarcoma; Kidney cancer (renal cell cancer); Laryngeal Cancer; Leukemias; Leukemia, acute lymphoblastic (also called acute lymphocytic leukemia); Leukemia, acute myeloid (also called acute myelogenous leukemia); Leukemia, chronic lymphocytic (also called chronic lymphocytic leukemia); Leukemia, chronic myelogenous (also called chronic myeloid leukemia); Leukemia, hairy cell; Lip and Oral Cavity Cancer; Liver Cancer (Primary); Lung Cancer, Non-Small Cell; Lung Cancer, Small Cell; Lymphomas; Lymphoma, AIDS-related; Lymphoma, Burkitt; Lymphoma, cutaneous T-Cell; Lymphoma, Hodgkin; Lymphomas, Non- Hodgkin (an old classification of all lymphomas except Hodgkin's); Lymphoma, Primary Central Nervous System; Marcus Whittle, Deadly Disease; Macroglobulinemia, Waldenstrom; Malignant Fibrous Histiocytoma of Bone / Osteosarcoma; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular (Eye); Merkel Cell Carcinoma; Mesothelioma, Adult Malignant; Mesothelioma, Childhood; Metastatic Squamous Neck Cancer with Occult Primary; Mouth Cancer; Multiple Endocrine Neoplasia Syndrome, Childhood; Multiple Myeloma / Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplastic Syndromes; Myelodysplastic / Myeloproliferative Diseases; Myelogenous Leukemia, Chronic; Myeloid Leukemia, Adult Acute; Myeloid Leukemia, Childhood Acute; Myeloma, Multiple (Cancer of the Bone-Marrow); Myeloproliferative Disorders, Chronic; Nasal cavity and paranasal sinus cancer; Nasopharyngeal carcinoma; Neuroblastoma; Non-Hodgkin lymphoma; Non-small cell lung cancer; Oral Cancer; Oropharyngeal cancer; Osteosarcoma / malignant fibrous histiocytoma of bone; Ovarian cancer; Ovarian epithelial cancer (Surface epithelial- stromal tumor); Ovarian germ cell tumor; Ovarian low malignant potential tumor; Pancreatic cancer; Pancreatic cancer, islet cell; Paranasal sinus and nasal cavity cancer; Parathyroid cancer; Penile cancer; Pharyngeal cancer; Pheochromocytoma; Pineal astrocytoma; Pineal germinoma; Pineoblastoma and supratentorial primitive neuroectodermal tumors, childhood; Pituitary adenoma; Plasma cell neoplasia / Multiple myeloma; Pleuropulmonary blastoma; Primary central nervous system lymphoma; Prostate cancer; Rectal cancer; Renal cell carcinoma (kidney cancer); Renal pelvis and ureter, transitional cell cancer; Retinoblastoma; Rhabdomyosarcoma, childhood; Salivary gland cancer; Sarcoma, Ewing family of tumors; Sarcoma, Kaposi; Sarcoma, soft tissue; Sarcoma, uterine; Sezary syndrome; Skin cancer (nonmelanoma); Skin cancer (melanoma); Skin carcinoma, Merkel cell; Small cell lung cancer; Small intestine cancer; Soft tissue sarcoma; Squamous cell carcinoma - see Skin cancer (nonmelanoma); Squamous neck cancer with occult primary, metastatic; Stomach cancer; Supratentorial primitive neuroectodermal tumor, childhood; T-Cell lymphoma, cutaneous (Mycosis Fungoides and Sezary syndrome); Testicular cancer; Throat cancer; Thymoma, childhood; Thymoma and Thymic carcinoma; Thyroid cancer; Thyroid cancer, childhood; Transitional cell cancer of the renal pelvis and ureter; Trophoblastic tumor, gestational; Unknown primary site, carcinoma of, adult; Unknown primary site, cancer of, childhood; Ureter and renal pelvis, transitional cell cancer; Urethral cancer; Uterine cancer, endometrial; Uterine sarcoma; Vaginal cancer; Visual pathway and hypothalamic glioma, childhood; Vulvar cancer; Waldenstrom macroglobulinemia and Wilms tumor (kidney cancer).

[0099] In some embodiments, the immune-related disorder applicable in the present disclosure may be an inflammatory disease. The terms "inflammatory disease” or "inflammatory-associated condition" refers to any disease or pathologically condition which can benefit from the reduction or induction of at least one inflammatory parameter, for example, induction of an inflammatory cytokine such as IFN-gamma and IU-2 and reduction in IU-6 levels. The condition may be caused (primarily) from inflammation, or inflammation may be one of the manifestations of the diseases caused by another physiological cause. In some embodiments, an inflammatory disease that may be applicable for the present disclosure may be inflammatory bowel disease (IBD).

[0100] In some embodiments, the immune-related disorder applicable in the present disclosure comprises an autoimmune disorder.

[0101] An "autoimmune disorder" is state in which the immune system gets directed against self-cells or tissues. Autoimmune disorders include for example, but not limited to inflammatory bowel disease (IBD), ulcerative colitis (UC), Crohn's disease (CD), Systemic Uupus Erythematosus (SEE), Rheumatoid Arthritis (RA), fatty liver disease, Lymphocytic colitis, Ischaemic colitis, Diversion colitis, Behcet's syndrome, Indeterminate colitis, Graft versus Host Disease (GvHD), Eaton- Lambert syndrome, Goodpasture's syndrome, Greave's disease, Guillain-Barr syndrome, autoimmune hemolytic anemia (AIHA), hepatitis, insulin-dependent diabetes mellitus (IDDM) and NIDDM, multiple sclerosis (MS), myasthenia gravis, plexus disorders e.g. acute brachial neuritis, polyglandular deficiency syndrome, primary biliary cirrhosis, scleroderma, thrombocytopenia, thyroiditis e.g. Hashimoto's disease, Sjogren's syndrome, allergic purpura, psoriasis, mixed connective tissue disease, polymyositis, dermatomyositis, vasculitis, polyarteritis nodosa, arthritis, alopecia areata, polymyalgia rheumatica, Wegener's granulomatosis, Reiter's syndrome, ankylosing spondylitis, pemphigus, bullous pemphigoid, dermatitis herpetiformis, psoriatic arthritis, reactive arthritis, and ankylosing spondylitis, inflammatory arthritis, including juvenile idiopathic arthritis, gout and pseudo gout, as well as arthritis associated with colitis or psoriasis, Pernicious anemia, some types of myopathy and Lyme disease (Late).

[0102] More specifically, in some embodiments, the immune-related disorder applicable in the disclosure may be an inflammatory bowel disease (IBD). "Inflammatory bowel disease (IBD)" is characterized by repetitive episodes of inflammation of the gastrointestinal tract caused by an abnormal immune response to gut microflora. Inflammatory bowel disease encompasses two types of idiopathic intestinal disease that are differentiated by their location and depth of involvement in the bowel wall. Ulcerative colitis (UC) involves diffuse inflammation of the colonic mucosa. Most often UC affects the rectum (proctitis), but it may extend into the sigmoid (proctosigmoiditis), beyond the sigmoid (distal ulcerative colitis), or include the entire colon up to the cecum (pancolitis). Crohn disease (CD) results in transmural ulceration of any portion of the gastrointestinal tract (GI) most often affecting the terminal ileum and colon. Both diseases are classified by extent (mild, moderate, or severe) and location. CD also is classified by phenotype- inflammatory, stricturing, or penetrating. Besides the GI tract, both Crohn disease and ulcerative colitis have many extraintestinal manifestations.

[0103] In some embodiments, the immune-related disorder applicable in the present disclosure may be immunodeficiency. "Immunodeficiency" (or immune deficiency) is a state in which the immune system's ability to fight infectious disease and cancer is compromised or entirely absent. Most cases of immunodeficiency are acquired ("secondary") due to extrinsic factors that affect the patient's immune system. Examples of these extrinsic factors include viral infection, specifically HIV, extremes of age, and environmental factors, such as nutrition. In the clinical setting, the immunosuppression by some drugs, such as steroids, can be either an adverse effect or the intended purpose of the treatment. Examples of such use are in organ transplant surgery as an anti-rejection measure and in patients suffering from an overactive immune system, as in autoimmune diseases. Immunodeficiency also decreases cancer immune surveillance, in which the immune system scans the cells and kills neoplastic ones. Still further, Primary immunodeficiencies (PH)). also termed innate immunodeficiencies, are disorders in which part of the organism immune system is missing or does not function normally. To be considered a primary immunodeficiency, the cause of the immune deficiency must not be caused by other disease, drug treatment, or environmental exposure to toxins. Most primary immune deficiencies are genetic disorders; the majority is diagnosed in children under the age of one, although milder forms may not be recognized until adulthood. While there are over 100 recognized PIDs, most are very rare. Secondary immunodeficiencies occur when the immune system is compromised due to environmental factors. Such factors include but are not limited to chemotherapy, radiotherapy, biological therapy, bone marrow transplantation, gene therapy, adoptive cell transfer or any combinations thereof.

[0104] In some embodiments, the immune-related disorder applicable in the present disclosure may be at least one infectious disease. An "infectious disease" as used herein encompasses any infectious disease caused by a pathogenic agent, specifically, a pathogen. More specifically, such infectious disease may be any pathological disorder caused by a pathogen. As used herein, the term “pathogen” refers to an infectious agent that causes a disease in a subject host. Pathogenic agents include prokaryotic microorganisms (e.g., bacteria, archea), lower eukaryotic microorganisms, complex eukaryotic organisms, viruses, fungi, mycoplasma, prions, parasites, for example, a parasitic protozoan, yeasts, or a nematode, as well as toxins and venoms.

[0105] In some embodiments, the pathologic disorder applicable in the present disclosure may be at least one neurodegenerative disorder. "Neurodegeneration" is the umbrella term for the progressive loss of structure or function of neurons, including synaptic dysfunction and death of neurons. Many neurodegenerative diseases including Parkinson’s and Alzheimer’s are associated with neurodegenerative processes. Other examples of neurodegeneration that may be also applicable herein may include Friedreich's ataxia, Lewy body disease, spinal muscular atrophy, multiple sclerosis, frontotemporal dementia, corticobasal degeneration, progressive supranuclear palsy, multiple system atrophy, hereditary spastic paraparesis, amyloidosis, Amyotrophic lateral sclerosis (ALS), and Charcot Marie Tooth. It should not be overlooked that normal aging processes include progressive neurodegeneration, specifically, age-related cognitive decline (ACD) and mild cognitive impairment (MCI) are also applicable in the present disclosure. More specifically, the term "neurodegenerative diseases" is the general term for the progressive loss of structure or function of neurons, leading to their death. The major risk factor for neurodegenerative diseases is aging. Mitochondrial DNA mutations as well as oxidative stress both contribute to aging. Many of these diseases are late onset, meaning there are some factors that change as a person ages, for each disease. One constant factor is that in each disease, neurons gradually lose function as the disease progresses with age. Still further, in some embodiments, the pathologic disorder applicable in the present disclosure may be any mental condition. A "mental disorder" is characterized by a clinically significant disturbance in an individual’ s cognition, emotional regulation, or behavior. It is usually associated with distress or impairment in important areas of functioning. Mental disorders may also be referred to as mental health conditions. The latter is a broader term covering mental disorders, psychosocial disabilities and (other) mental states associated with significant distress, impairment in functioning, or risk of self-harm. There are many different types of mental disorders including for example anxiety disorders, depression, bipolar disorder, post- traumatic stress disorder (PTSD), schizophrenia, eating disorders, such as anorexia nervosa and bulimia nervosa, disruptive behavior and dissocial disorders and neurodevelopmental disorders such as autism spectrum disorder (ASD), and attention deficit hyperactivity disorder (ADHD) amongst others.

[0106] In some embodiments, the pathologic disorder applicable in the present disclosure may be at least one metabolic disorder. "Metabolic disorders" may include atherosclerosis and peripheral vascular diseases, as well as cardiovascular diseases such as coronary artery diseases (CAD). Of particular interest in connection with metabolic disorders are conditions associated with obesity, hypertension, elevated cholesterol (combined hyperlipidemia), such conditions often termed metabolic syndrome (it is also known as Syndrome X, Reavan's syndrome, or CHAOS). It should be noted that the disclosed conditions may be congenital or acquired conditions.

[0107] In some embodiments, the immune-related disorder used by the present disclosure refers to a condition involving at least one wound in at least one tissue and / or organ of a subject. A "wound" is any disruption of or damage to living tissue, such as skin, mucous membranes, or organs. Wounds can either be the sudden result of direct trauma (mechanical, thermal, chemical), or can develop slowly over time due to underlying disease processes such as diabetes mellitus, venous / arterial insufficiency, or immunologic disease. In certain specific embodiments, the disclosed method may be applicable for inflammatory conditions such as inflammatory bowel disease (IBD).

[0108] Still further, in some embodiments, the disclosed genetically engineered bacterium is locked in a desired orientation in a genomic locus associated with an immune response of a subject.

[0109] In some embodiments, the immune response of the subject comprises responsiveness of the subject to at least one therapeutic compound and / or therapeutic regimen.

[0110] The term "responsiveness" or "responder" to a certain treatment, refers to an improvement in at least one relevant clinical parameter as compared to an untreated subject diagnosed with the same pathology (e.g., the same type, stage, degree and / or classification of the pathology), or as compared to the clinical parameters of the same subject prior to treatment with said medicament. The term "non-responsiveness" or "non responder” or "drug resistance” to treatment with a specific medicament, refers to a patient not experiencing an improvement in at least one of the clinical parameter and is diagnosed with the same condition as an untreated subject diagnosed with the same pathology (e.g., the same type, stage, degree and / or classification of the pathology), or experiencing the clinical parameters of the same subject prior to treatment with the specific medicament. In yet some further embodiments the subject may be further sub classified with respect to the expected degree, depth or extent and / or duration of responsiveness, for example as a poor responder, a responder displaying mild response, a responder displaying a good response or even a responder displaying excellent response, and the like. Loss of responsiveness refers to a situation wherein a responder experiences a decline in at least one of the clinical parameters that showed an improvement in a past examination. Responsiveness may include, but is not limited to: a reduction in disease symptoms; a measurable biological or molecular change (e.g., modulation of cytokine levels, immune cell activation, tumor regression, or biomarker expression); improved clinical outcome; prevention of disease progression; enhanced survival; or reversal of drug resistance. Responsiveness may be evaluated through clinical assessments, laboratory markers, imaging, functional tests, or other diagnostic indicators.

[0111] In some embodiments, the immune response comprises responsiveness of the subject to at least one therapeutic compound.

[0112] The term “therapeutic compound'' . as used herein, refers to any agent, whether a small molecule, biologic, antibody, peptide, nucleic acid, live organism, engineered entity, or chemical formulation, that is administered to a subject for the purpose of treating, ameliorating, preventing or modulating a disease, disorder, or abnormal physiological condition. Therapeutic compounds may act systemically or locally and may exert their effect by interacting with host cells, microbial populations, immune components, signaling pathways, or disease-causing agents. The term encompasses conventional pharmaceuticals, targeted biologies, immunotherapies, metabolic regulators, and live bio therapeutic products. A therapeutic compound may be used alone or in combination with other compounds or as part of a broader therapeutic regimen.

[0113] In some further embodiments, the immune response comprises responsiveness of the subject to a therapeutic regimen. "Therapeutic regimen” or "treatment regimen" as used herein refers to the course of treatment type including the drugs to be used, their dosage, the frequency and duration of treatments, and other considerations defined based on medical decisions. Therefore, in some embodiments, a treatment regimen for a subject displaying a disease relapse and / or loss of responsiveness, nonresponsiveness, poor-responsiveness and / or drug-resistance may be ceased (i.e. stopped) and optionally replaced by an alternative treatment regimen.

[0114] In some embodiments, the therapeutic compound comprises immune-checkpoint inhibitors.

[0115] "Immune checkpoint inhibitors" are immunotherapy drugs regulating (stimulating or inhibiting) the immune system, and / or an immune response. Stimulatory checkpoint molecules are members of the tumor necrosis factor (TNF) receptor superfamily, CD27, CD40, 0X40, GITR and CD137, while inhibitory include CTLA-4, PD1, PDL-1, B7-H3 (CD276), B7-H4 (VTCN1), BTLA, (CD272), IDO, KIR, LAG3, N0X2, TIM-3, VISTA, (SIGLEC9). Cancer therapy involves the use of compounds that inhibit the action of the inhibitory immune-checkpoint molecules, specifically, any of those indicated above. In some embodiments, the therapeutic compound may be an inhibitor of PD-1L. For example, at least one anti-PD-lL antibody.

[0116] In some other embodiments, the disclosed genetically engineered bacterium is locked in a desired orientation in a genomic locus associated with an inflammatory disorder, for example, inflammatory bowel disease (IBD).

[0117] As mentioned above, the present disclosure relates to a genetically engineered bacterium. In some embodiments, the bacterium belongs to at least one bacterial species residing within at least one microbiome community of the subject.

[0118] "Microbiome community" refers to a community of commensal, symbiotic, and / or pathogenic microorganisms (such as bacteria, archea, fungi, viruses, algae, protists and / or bacteriophages) that coexist in a particular environment or host. Human microbiomes include for example gut, skin, genital, and oral microbiome communities. Still further, in some embodiments, the microbiome may be the gut microbiome. Thus, the genetically engineered bacterium of the present disclosure may be of any bacteria residing in the gut microbiome. "Gut microbiota" , "gut microbiome" , or "gut flora" , are the microorganisms, including bacteria, archaea, fungi, and viruses that live in the digestive tracts of animals. It should be understood however that microorganisms of any habitat and / or microbiome may be used in the present disclosure, for example, the skin microbiome, the genital microbiome, the oral microbiome etc. In some embodiments, the gut microbiome may comprise at least one of: bacteria, archaea, fungi, algae, protists, viruses, and bacteriophages. The gut is the main location of the human microbiome. In humans, gut microbiota has the largest number and species of bacteria compared to other areas of the body. The gut microbiota has broad impacts, including effects on colonization, resistance to pathogens, maintaining the intestinal epithelium, metabolizing dietary and pharmaceutical compounds, controlling immune function, and even behavior through the gut-brain axis.

[0119] As mentioned, the bacterium of the present disclosure belongs to at least one bacterial species residing within at least one microbiome community which comprises bacteria, archea, fungi, viruses, algae, protists and / or bacteriophages.

[0120] The term “bacteria” (singular: bacterium), as used herein, refers to microscopic, single-celled prokaryotic organisms that lack a true nucleus and membrane-bound organelles. Bacteria exist in a wide range of shapes (e.g., cocci, bacilli, spirilla) and can be classified based on their Gramstaining properties as Gram-positive, Gram-negative, or Gram-variable. The term includes both free-living and intracellular species, facultative and obligate anaerobes, and bacteria forming part of complex biofilms or symbiotic consortia.

[0121] The term “archaea”, as used herein, refers to a distinct domain of single-celled prokaryotic microorganisms that are genetically and biochemically separate from bacteria. Archaea are notable for their ability to thrive in extreme environments (e.g., high temperature, salinity, or acidity), but are also present in mesophilic conditions, including the human gut. Representative groups of archaea include methanogens, which produce methane under anaerobic conditions, halophiles, which inhabit high-salt environments, and thermophiles, which tolerate high temperatures.

[0122] The term “fungi”, as used herein, refers to a diverse kingdom of eukaryotic organisms that includes unicellular yeasts, multicellular molds, and filamentous fungi, as well as complex structures such as mushrooms. Fungi may exist as saprophytic, parasitic, commensal, or mutualistic organisms and are integral members of both environmental and host-associated microbiomes. In the human microbiome, fungi such as Candida species may reside in the gut, skin, or mucosal tissues. Fungi reproduce via spores and contribute to nutrient recycling, hostmicrobiota dynamics, and immune modulation.

[0123] The term “viruses”, as used herein, refers to non-cellular infectious agents composed of genetic material (DNA or RNA) enclosed within a protein capsid, and sometimes surrounded by a lipid envelope. Viruses require host cells to replicate and may infect bacteria (bacteriophages), archaea, fungi, protists, animals, or plants. In the context of the human microbiome, viruses can influence microbial community dynamics and host immune responses. The virome is a subset of the microbiome that includes both eukaryotic viruses and bacteriophages.

[0124] The term “algae”, as used herein, refers to a broad group of photosynthetic eukaryotic organisms, including both unicellular forms (e.g., Chlorella, Dunaliella) and multicellular forms (e.g., seaweeds). Algae are found in aquatic environments and moist terrestrial habitats. While not typically dominant members of the human microbiome, certain algal species or their derivatives may be present in environmental or aquatic microbiomes relevant to therapeutic or ecological applications. Some algae may establish symbiotic relationships with other microorganisms or hosts.

[0125] The term “protists”, as used herein, refers to a diverse category of unicellular eukaryotic organisms that do not fall under the kingdoms of animals, plants, or fungi. Protists include amoebae, flagellates, ciliates, and apicomplexans, and may exhibit heterotrophic, autotrophic, or mixotrophic lifestyles. In the microbiome context, protists may serve as microbial predators, symbionts, or even pathogens. Their interactions with bacteria and other microbiota members can influence microbial composition and functional outcomes.

[0126] The term “bacteriophages” (or phages) refers to viruses that specifically infect and replicate within bacterial hosts. Phages may exhibit lytic or lysogenic life cycles and play a central role in shaping bacterial community structure, gene transfer (via transduction), and microbial competition. In the human gut and other microbiomes, phages constitute a significant part of the virome.

[0127] As indicated above, in some embodiments, the genetically engineered bacterium disclosed herein belongs to the gut microbiome.

[0128] In some further embodiments, the at least one engineered bacterium of the present disclosure belongs to at least one phylum selected from Bacteroidota, Verrucomicrobiota, proteobacteria, actinobacteria, firmicutes and Tenericutes.

[0129] The phylum "Bacteroidota" (synonym Bacteroidetes) is composed of three large classes of Gramnegative, nonsporeforming, anaerobic or aerobic, and rod-shaped bacteria that are widely distributed in the environment, including in the guts and on the skin of animals. "Verrucomicrobiota” is a phylum of Gram-negative bacteria that contains only a few described species, which have been isolated from fresh water, marine and soil environments and human feces. This phylum is considered to have two sister phyla: Chlamydiota (formerly Chlamydiae) and Lentisphaerota (formerly Lenlisphaerae) within the PVC superphylum, all are encompassed by the present disclosure. In some embodiments, Verrucomicrobiota may comprise Akkermansia muciniphila, or any species or isolate thereof. Still further, "Proteobacteria”, also called Pseudomonadota is a major phylum of Gram-negative bacteria, which includes a wide variety of pathogenic genera, such as Escherichia, Salmonella, Vibrio, Yersinia, Legionella, and many others. " Actinobacteria' ' also called Actinomycetota are a diverse phylum of Gram-positive bacteria with high G+C content found in soil. "Firmicutes " also called Bacillota are a phylum of bacteria, most of which have gram-positive cell wall structure, and they are all defined as the core group of related forms called the low-G+C group, in contrast to the Actinomycetota. "Tenericutes” or Mycoplasmatota is a phylum of gram-negative bacteria consisting of cells bounded by a plasma membrane, and they are devoid of cell walls. This phylum contains the class Mollicutes. Notable genera that may be applicable in the present disclosure, include Mycoplasma, Spiroplasma, Ureaplasma, and Candidates Phytoplasma.

[0130] In some embodiments, the Bacteroidota engineered bacterium is of the genus of at least one of: Bacteroides, Bacteroidia, Bacteroidales, Bacteroidaceae and Phocaeicola.

[0131] "Bacteroides" is a genus of Gram-negative, obligate anaerobic bacteria. Bacteroides species are non endo spore-forming bacilli, and their membranes contain sphingolipids, and also meso- diaminopimelic acid in their peptidoglycan layer. Bacteroides species form the most substantial portion of the mammalian gastrointestinal microbiota. In yet some further embodiments, the bacteria of the preset disclosure may comprise at least one of Bacteroides ovatus, Bacteroides fragilis and Bacteroides thetaiotaomicron, or any isolate or species thereof.

[0132] More specifically, in some embodiments, bacterial isolates or species applicable in the present disclosure may comprise Bacteroides ovatus. Still further, in some embodiments, any Bacteroidota bacteria may be useful in the present disclosure, such Bacteroidota bacteria may comprise in some embodiments, at least one of Bacteroides fragilis, Bacteroides uniformis, Bacteroides ovatus, Bacteroides stercoris, Bacteroides cellulosilyticus, Bacteroides caccae, Bacteroides eggerthii, Bacteroides thetaiotaomicron, Bacteroides intestinalis, Bacteroides clarus, Bacteroides fragilis_A, Bacteroides finegoldii, Bacteroides faecis, Bacteroides massiliensis, Bacteroides togonis, Bacteroides nordii, Bacteroides salyersiae, Bacteroides intestinalis _A, Bacteroides ndongoniae, Bacteroides sp003545565, Bacteroides sp905207245, Bacteroides bouchesdurhonensis, Bacteroides fluxus, Bacteroides gallinarum, Bacteroides stercorirosoris, Bacteroides graminisolvens, Bacteroides pyogenes, Bacteroides oleiciplenus, Bacteroides sp002491635, Bacteroides cutis, Bacteroides sp900547205, Bacteroides acidifaciens, Bacteroides sp905197435, Bacteroides neonati, Bacteroides sp014385165 and / or Parabacteroides distasonis, and any species and isolates thereof.

[0133] Still further, in some embodiments, Bacteroides fragilis, or any species or isolates thereof, may be useful in the present disclosure. Bacteroides fragilis is an anaerobic, Gram-negative, pleomorphic to rod-shaped bacterium. It belongs to the Bacteroides genus, and Bacteroidota phylum. Bacteroides fragilis resides in the human gastrointestinal tract and is essential to healthy gastrointestinal function such as mucosal immunity and host nutrition. B. fragilis utilizes a complex series of surface proteins, lipopolysaccharide chains, and outer membrane vesicles.

[0134] Still further, in some embodiments, Bacteroides ovatus, Bacteroides thetaiotaomicron, Phocaeicola dorei, and Bacteroides cellulosilyticus or any species or isolates thereof, may be useful in the present disclosure. Specifically, "Bacteroides ovatus", is an anaerobe, Gramnegative, rod-shaped bacterium that was residing in human feces. "Bacteroides thetaiotaomicron" is a gram-negative, rod shaped obligate anaerobic bacterium that is a prominent member of the normal gut microbiome in the distal intestines. "Phocaeicola dorei" is a gram negative, rod-shaped bacterium that found and contributes to normal intestinal functionality. Phocaeicola dorei is a non- spore-forming, non-motile, and anaerobic bacterium with a G+C DNA content of 43%. "Bacteroides cellulosilyticus" (identified as strain CRE21) are a species of bacteria within the Bacteroides genus, degrading cellulose (cellulolytic) within the human microbiota. Its genome is characterized with G-C content of 43.05%. Still further, in the disclosed method any isolate or species of Bacteroides ovatus, Bacteroides fragilis, Bacteroides thetaiotaomicron, Phocaeicola dorei, Bacteroides cellulosilyticus, Bacteroides ovatus, Bacteroides stercorirosoris, may be applicable.

[0135] In some specific embodiments, the genetically engineered bacterium is Bacteroides ovatus or any isolate or species thereof.

[0136] The term "isolate" or a "genetic isolate" as used herein refers to a population of organisms with little genetic mixing with other organisms within the same species due to geographic isolation or other factors that prevent reproduction. The term "species" (pl. species) is the basic unit of classification and a taxonomic rank of an organism, defined by their karyotype, DNA sequence, morphology, behavior, or ecological niche.

[0137] Still further, in some other embodiments, the Bacteroidota bacterium comprises at least one of Bacteroides uniformis, Bacteroides vulgatus, Bacteroides xylanisolvens, Bacteroides caccae, Bacteroides cellulosilyticus, Bacteroides massiliensis, Bacteroides ovatus, Bacteroides stercoris and Bacteroides faecis or any isolate or species thereof.

[0138] In the present disclosure, the genetically engineered bacterium is locked in a desired orientation in at least one genomic locus displaying phase variation. In some embodiments, the genomic locus comprises at least one intergenic region / s and / or intragenic region / s. "Intergenic regions" are a stretch of DNA sequences located between genes, and include promoters, enhancers, and other regulatory elements, origins of replication, transposons and viruses. Non-functional DNA elements include for example pseudogenes and repetitive DNA, both of which are types of junk DNA. "Intergenic regions" are a stretch of DNA sequences located within genes.

[0139] In some embodiments, the genomic locus of the genetically engineered bacterium disclosed herein comprises nucleic acid sequence / s encoding and / or regulating at least one bacterial outersurface molecule. In some other embodiments, the genomic locus comprises nucleic acid sequence / s encoding and / or regulating at least one bacterial internal molecule.

[0140] In some further embodiments, the genomic locus of the genetically engineered bacterium of the present disclosure comprises nucleic acid sequence / s encoding and / or regulating at least one molecule that modifies or regulates the at least one outersurface and / or internal molecule / s.

[0141] ''Outersurface'' molecules refer herein to molecules which reside within the cell surface of an organism and / or at the outside part of the cell surface of an organism. These may include proteins, lipoproteins, glycoproteins, lipooligosaccharides (LOS), lipopolysaccharides (LPS), pili, fimbriae, or polysaccharide capsules. Such structures serve as interfaces between the bacterium and its environment and are critical in mediating host-microbe interactions, immune recognition, colonization, and biofilm formation. ''Internal” molecules refer herein to molecules which reside inside the cell, in the internal area of an organism's cell. The locus determined for phase variations may comprise, in some embodiments, a sequence that modify or regulate the outer or inner membrane, specifically, a sequence that changes directly or indirectly the activity, stability, post translational modifications, of the outer or inner molecule, thereby, defining the term "modify " as used herein.

[0142] In some specific embodiments, the bacterial outersurface molecule comprises capsular polysaccharides (CPSs) and the at least one molecule that modifies or regulates the at least one outersurface and / or internal molecule / s comprises at least one member of UpxY family transcription antiterminator. Bacterial "capsular polysaccharides (CPSs)" are a diverse class of high molecular weight polysaccharides, that confer protective effects to their bearers against a wide range of environmental pressures, most notably against the immune system during infection of their animal hosts, by hiding cell-surface components that might otherwise elicit host immune response. Although capsules are often associated with descriptions of pathogenic bacteria due to the large proportion of encapsulated invasive pathogens, non-pathogenic and commensal bacteria also benefit from the ability to envelope themselves with a capsule. In Gram-negative bacteria, capsular polysaccharides are often attached to the outer membrane at their reducing end through covalently- linked lipids that are inserted into the lipid bilayer of the membrane. This provides a surface layer of water-saturated, high molecular weight polysaccharides that limit desiccation in the face of harsh environmental conditions, block infection by most bacteriophages, and thwart phagocytosis and other host immune responses by physically restricting access to cell surface antigens. These polysaccharide cloaks are likely rational targets for wide-spectrum therapeutic compounds aimed at replacing or supplementing antibiotic treatment of microbial infections, as removal of the capsule exposes bacteria to routine immune clearance pathways mediated frequently by activation of the complement system. Different serotypes of capsular polysaccharides express structural differences in the capsular polysaccharide (CPS). One example is the CPS serotype 3 or CPS3).

[0143] In the context of the present disclosure, the expression of CPSs is under the regulatory control of at least one molecule that modifies or regulates the expression of the outersurface and / or internal molecules.

[0144] In some specific embodiments, the regulatory molecule of CPSs comprises at least one member of the UpxY family of transcription antiterminators. The term “UpxY family transcription antiterminator” refers to a family of bacterial regulatory proteins that modulate gene expression by preventing premature transcription termination, thereby allowing full transcription of downstream genes. UpxY proteins are commonly found in loci encoding CPS biosynthesis and export machinery, particularly in Bacteroides species. These antiterminators recognize and bind specific RNA structures or transcriptional elements, interacting with RNA polymerase or other components of the transcriptional apparatus to override intrinsic or Rho-dependent termination signals. As such, they are essential for coordinated expression of large polysaccharide biosynthetic operons. In Bacteroides, the UpxY-mediated antitermination mechanism enables expression of specific CPS loci in a phase-variable manner, with expression switching dependent on the orientation of promoter inversion and activity of recombinase enzymes. By genetically engineering such bacteria to control or lock the activity or expression of UpxY or its regulatory elements (e.g., promoter orientation, invertase binding sites), the present disclosure provides means to stably modulate CPS expression and, consequently, the immunomodulatory effect of the bacterium on the host subject.

[0145] In some embodiments, the CPSs comprise polysaccharides (PSs), and the PSs comprise polysaccharide A (PSA), "polysaccharides (PSs)" refers to high-molecular- weight carbohydrate polymers composed of repeating monosaccharide units or oligosaccharide sequences linked by glycosidic bonds. These polysaccharides may vary in structure, molecular weight, sugar composition, and branching, and can be either homopolysaccharides (composed of a single type of monosaccharide) or heteropolysaccharides (composed of two or more types of monosaccharides).

[0146] In particular embodiments, the PSs include polysaccharide A (PSA), a well-characterized immunomodulatory polysaccharide known to be expressed by certain bacterial species, such as Bacteroides fragilis. PSA typically comprises a repeating unit structure rich in positively and negatively charged monosaccharide residues, and may be characterized by its zwitterionic nature. The structure of PSA allows it to interact with host immune cells and modulate immune responses, including the activation of antigen-presenting cells and the induction of regulatory T cell populations.

[0147] As indicated above, in some embodiments, the locus in the genetically engineered bacterium of the present disclosure is the UpxY locus. It should be noted that according to some embodiments, the phase variation in the promoter region of the UpxY locus is associated with responsiveness or irresponsiveness of a subject suffering from melanoma to at least one therapeutic compound, and / or therapeutic regimen. In some embodiments, the phase variation in the UpxY locus of the genetically engineered bacterium is associated with responsiveness to at least one immune- checkpoint inhibitor.

[0148] The term "UpxY locus" refers to a specific genetic region typically found within bacterial genomes, particularly in Gram-negative bacteria, that encodes a regulatory protein associated with capsular polysaccharide (CPS) biosynthesis. The UpxY gene (sometimes referred to in the literature as a member of the "Upx" family, e.g., upxY, upxZ) is most commonly characterized as a transcriptional antiterminator or positive regulator that modulates the expression of downstream genes within a capsular polysaccharide biosynthetic operon. As mentioned above, the genetically engineered bacterium of present disclosure is locked in a desired orientation in at least one genomic locus displaying phase variation. The phase variation, in some embodiments, comprises at least one of: DNA inversion, DNA recombination, transposition mechanism, slipped strand mispairings (SSM) and phase variation via differential methylation.

[0149] Thus, in some embodiments, the phase variation comprises DNA inversion.

[0150] DNA inversion is carried out by enzymes that recognize inverted repeat regions and flip the DNA sequence in between them or which reside next to the switch. For example, if a promoter region lies within the sequence flanked by the inverted repeats this leads to shut down of gene expression. In some other embodiments, the phase variation comprises DNA recombination.

[0151] DNA recombination provides a pathway for DNA re-arrangement and subsequent phase variation. Events arising from recombination mechanisms are often due to DNA deletions and thus tend to be in a one way 'ON' to 'OFF' direction. In some other embodiments, the phase variation comprises transcription mechanism.

[0152] Transposition mechanism leads to either insertion or excision of transponsable elements. A transposable element (TE, transposon, or jumping gene) is a nucleic acid sequence in DNA that can change its position (jump) within a genome from one location to another, sometimes creating or reversing mutations and altering the cell's genetic identity and genome size. In some further embodiments, the phase variation comprises slipped strand mispairings (SSM). Slipped strand mispairings (SSM) (also known as replication slippage) occur during the process of DNA synthesis i.e., DNA replication, repair and recombination. During DNA synthesis template and nascent strand transiently separates from each other and then reanneal. In reannealing step, nascent strand on the template strand can be slipped either in forward direction or in backward direction, resulting in "bulge" formation. The bulge is formed either on the template strand due to forward slippage or in the nascent strand due to backward slippage, leading to either contraction or expansion of the repeat tracts which in turn could affect in many ways transcription or translation of the affected contingency gene depending on the position of the repeat tract. Short sequence repeats (SSRs) are homo- or hetero-nucleotide repeats in DNA that are highly prone to insertion / deletion errors due to Slipped-Strand Mispairings (SSMs) during DNA replication. Still further, in some embodiments, the phase variation comprises phase variation via differential methylation. Phase variation via differential methylation is described as epigenetic event as phenotypes are altered but not genotype, therefore maintaining the integrity of genome (e.g., epigenetic variations). In prokaryotes, DNA methylation occurs mainly at the nucleotide adenine although cytosine methylation can also occur. DNA methylation usually occurs at specific target sites and is carried out either by methyltransferases that are part of dedicated Restriction- Modification (RM) systems or by orphan methyltransferases.

[0153] In some embodiments, the phase variation comprises DNA inversion in at least one promoter region of at least one genomic locus in the disclosed genetically engineered bacterium, thereby converting the ON / OFF orientation of the at least one promoter region / s. The term "promoter region" as used herein refers to a nucleic acid sequence located upstream (5' direction) of a gene or operon that functions as a regulatory element directing the initiation of transcription by RNA polymerase. A promoter contains specific DNA motifs recognized and bound by RNA polymerase and, in many cases, associated transcription factors, thereby enabling or regulating the synthesis of RNA from the downstream coding sequence. In some specific and non-limiting embodiments, the phase variations may occur in the UPxY locus as discussed herein, specifically, at any region or part of the UPxY locus of B. ovatus. In some embodiments, the phase variations may occur in the UPxY promoter region. In yet some further embodiments, the phase variations in this locus involve inversions, and result either in the major "OFF" orientation of this promoter, or the "ON" orientation that allows the translation of the UPxY coding region. In some specific embodiments, the UPxY coding region of B. ovatus is as denoted by SEQ ID NO: 27. In some embodiments, the phase variations may occur in the promoter region of the UPxY, as denoted by SEQ ID NO; 26. It should be noted that the B. ovatus UPxY promoter region of SEQ ID NO: 26, is at the "OFF" orientation, and is also referred to herein as the wild type (WT) sequence. Alternatively, the phase variation, specifically inversion may occur in the B. ovatus UPxY promoter region that is in the "ON" orientation. In some embodiments, such promoter region comprises the nucleic acid sequence as denoted by SEQ ID NO: 35.

[0154] Still further, the genetically engineered bacterium of the present disclosure comprises at least one modification in a target nucleic acid sequence that comprises, comprised within, or encodes at least one element that induces directly or indirectly phase variation in the at least one genomic locus. In some specific and non-limiting embodiments, the element comprises at least one DNA invertase. In some embodiments, the invertase is a tyrosine invertase. Still further, in more specific embodiments, the invertase is a tyrosine invertase of B. ovatus. In some particular embodiments, the B. ovatus tyrosine invertase is encoded by a nucleic acid sequence as denoted by SEQ ID NO: 25. In some alternative or additional embodiments, the element may be a nucleic acid sequence that comprises at least one sequence targeted by, and / or recognized by, a sequence that is subjected for inversion by the invertase, or bound by, the at least one tyrosine invertase. More specifically, the inverse recognizes a short DNA segment flanked by inverted repeats (IRs). Specifically, flanked at both, the 5' and 3' ends thereof by IRs. This invertible sequence, may be also referred to herein as a switch region or invertible element, is typically of about 150 to 300 base pairs long in some embodiments, and contains a promoter that controls downstream gene expression. The inverted repeats themselves are usually of about 20 to 35 bp each and are positioned symmetrically at the boundaries of the switch (the promoter). The invertase binds to these inverted repeats, and / or any additional recognition / binding sites, catalyzing DNA inversion between them. When the segment flips orientation, the promoter’s direction reverses, effectively turning transcription of the downstream genes “ON” or “OFF” (also referred to herein as the “ON” or “OFF” orientation). In more specific embodiments, sequences targeted by at least one tyrosine invertase may be any sequence that participates either directly or indirectly in the inversion process, sequence that is subjected for inversion by the invertase. In some embodiments, such sequences may comprise an inverted repeat (IR) sequences that directly participate in the inversion process. As used herein, inverted repeat (IR) sequences are short stretches of DNA in which two sequences on the same strand are identical or nearly identical but arranged in opposite orientations, each being the reverse complement of the other. These sequences typically flank an invertible DNA segment that includes a promoter or other regulatory element controlling the expression of adjacent genes. The invertase enzyme specifically recognizes these IR sequences to catalyze site-specific recombination, resulting in the reversible inversion of the intervening DNA segment. When this segment contains a promoter, its inversion alters the transcriptional orientation with respect to the downstream gene, thereby switching gene expression between an "ON" and "OFF" state. The precise sequence composition, length (commonly 10 to 30 base pairs), and spacing of the IRs are essential determinants of the specificity and efficiency of the invertase-mediated recombination event. In some specific embodiments the IR sequence referred to as the elements participating in the inversion process may be derived from the B. ovatus UPxY promoter region, and may comprise the nucleic acid sequence as denoted by SEQ ID NO: 30 and SEQ ID NO: 31 (in the "OFF" orientation, also referred to herein as the WT sequence), and / or the nucleic acid sequences of SEQ ID NO: 36 and SEQ ID NO: 37 (in the "ON" orientation). Accordingly, the element as used herein, may be either the IR sequences, or any other sequence within the B. ovatus UPxY promoter region recognized or bound by the invertase. Thus, in some alternative or additional embodiments, the element may be a sequence being or comprising an invertase specific binding site. The term “binding site” refers to a specific DNA sequence that is recognized and bound by a protein, such as a recombinase, transcription factor, or other DNA-interacting enzyme. Binding sites often serve as control elements that allow proteins to perform functions like turning genes on or off, cutting or rearranging DNA, or initiating other regulatory processes. The related term “recognition site” is used to describe the sequence that a protein identifies to carry out its function, whether or not it remains bound for an extended time. In the present disclosure, the binding site and recognition site are used here interchangeably.

[0155] An "invertase-specific binding site" is a particular type of recognition site used by DNA invertase enzymes, also called site-specific recombinases. This binding site typically flanks an invertible DNA element (such as a promoter or coding sequence), and serves as a recombination recognition site, enabling the invertase to catalyze site-specific inversion of the intervening DNA segment.

[0156] Invertase enzymes, such as members of the tyrosine recombinase or serine recombinase families, bind to these specific DNA sequences to mediate the inversion of DNA between two oppositely oriented binding sites. The orientation and sequence of these sites are critical for proper recognition and recombination activity.

[0157] In the context of phase-variable gene expression, an invertase- specific binding site typically flanks a switchable promoter or coding region. The invertase-mediated inversion results in ON / OFF or alternative expression states, for example, turning expression of a capsular polysaccharide (CPS) operon ON or OFF.

[0158] In some specific embodiments, the DNA invertase comprises at least one of a tyrosine site-specific recombinase (Tsrs) and a serine site-specific recombinase (Ssr). These enzymes mediate sitespecific recombination at defined DNA sequences, enabling the inversion, excision, or integration of DNA segments in a sequence- specific and orientation-dependent manner. The recombination event is catalyzed without the need for extensive sequence homology, relying instead on the recognition of short, specific recombination sites.

[0159] A "tyrosine site-specific recombinase (Tsrs)" is a type of recombinase enzyme that utilizes a conserved tyrosine residue in its active site to catalyze strand cleavage and rejoining reactions. The recombinase binds to specific DNA sequences, typically arranged as inverted or direct repeats, and facilitates recombination through a stepwise mechanism involving sequential single-strand exchanges. This class of recombinases generally forms a synaptic complex of four subunits, which enables coordinated cleavage and re-ligation of the DNA strands. Tyrosine recombinases do not require ATP or other cofactors, and the recombination they mediate is typically precise and reversible. Notable examples of tyrosine recombinases include, for example Hin from Salmonella enterica, which mediates phase variation of flagellar genes by inverting DNA segments flanked by hix sites. In some embodiments, Tsrs enzymes are used to control the orientation of promoters or gene cassettes, allowing regulated or phase-variable gene expression.

[0160] A "serine site-specific recombinase (Ssr)", in contrast, utilizes a conserved serine residue to form a transient covalent intermediate with the DNA. These recombinases typically function as dimers or tetramers and catalyze simultaneous double-strand breaks at each recombination site, followed by a 180-degree rotation of the DNA segment and re-ligation. This mechanism allows for efficient and often unidirectional recombination. Serine recombinases recognize short, asymmetric DNA sequences and are known for their robustness and high recombination efficiency. Examples include for example Tn3 resolvase from Escherichia coli.

[0161] Still further, in some embodiments, the invertase of the disclosed genetically engineered bacteria may be at least one MPI, specifically “Mutator Phage Invertase” or “Multiple Promoter Invertase”, that are site-specific DNA recombinase (invertase) enzymes that mediates promoter inversion. These invertases belongs to the tyrosine recombinase family, as confirmed by conserved catalytic residues (RHRY motif), and recognize inverted repeats (typically 20-30 bp) that flank the invertible promoter region (about 200-300 bp in length).

[0162] The present disclosure provides genetically engineered bacteria locked in a desired orientation in a locus that displays phase variation. In some specific embodiments, the locus is the UPxY locus. However, any alternative loci may be applicable for the bacteria of the present disclosure. In some embodiments, identification of the desired locus displaying phase variation that may be appropriate for the engineered bacteria of the present disclosure may be identified any method known in the art, for example, as described by Moxon R., et al., Annu. Rev. Genet. (2006); 40:307- 333; Phillips Z.N., et al., Biochem. Soc. Trans. (2019);47: 1131— 1141; West P.T., et al., Curr. Opin. Microbiol. (2022); 69: 102192; Goldberg A., et al., Genome Med. (2014);6: 112.

[0163] The bacterium of the preset disclosure comprises at least one modification in a target nucleic acid sequence. In some embodiments, the target nucleic acid encodes at least one of the DNA invertase. In some specific embodiments, the DNA invertase is the tyrosine invertase. It should be noted that in some embodiments, the tyrosine invertase is located in close proximity to the genomic locus that displays phase variations associated with the immunological state of a subject that carry the bacteria. For example, the UpxY locus, that displays phase variation associated with the responsiveness of the subject to therapeutic compounds (immune checkpoint inhibitors). Still further, in some embodiments, the tyrosine invertase is the B. ovatus. According to some embodiments, the target nucleic acid encoding the invertase, may comprise the nucleic acid sequence as denoted by SEQ ID NO: 25, or any parts or fragments thereof.

[0164] In yet some other alternative embodiments, the target nucleic acid comprises at least one sequence that is subjected for inversion by the invertase, and / or at least one invertase specific binding site, and thus the engineered bacterium of the preset disclosure comprises at least one modification in the invertase binding sites. The modification in the invertase binding sites reduces or eliminates recognition of the sites by the invertase. The binding site / s are located within or in close proximity to the genomic locus displaying phase variation. In some embodiments, the sequences that are subjected for inversion by the invertase, and / or binding sites comprise inverted repeats (IRs) in (or close to) the promoter region of the UpxY locus, that allow recognition of the invertase (also referred to herein as palindromic sequences, invertase binding sites, or invertase recognition sites), that upon binding to its sites inverts the promoter region to ON or OFF orientation. Modification in at least one of these inverted repeat palindromic sequences eliminates recognition of these sites by the invertase, and / or prevents the inversion of the invertible sequence residing between the inverted repeats and thus prevents inversion of the genomic locus (phase variation). In other words, inhibition of the phase variation in the locus results in locked orientation of the genomic locus, that cannot be inverted. The inverted repeats targeted by the invertase, and / or subjected to inversion by the invertase, may reside in the promoter region of the UpxY locus. In some specific embodiments, promoter region of the B. ovatus UpxY locus comprises the nucleic acid sequence as denoted by SEQ ID NO: 26 (the "OFF" orientation, referred to herein as the WT sequence), or alternatively, by SEQ ID NO: 35 (the "ON" orientation). Accordingly, for the "OFF" orientation, the target sequence may comprise or may be comprised within at least one of the "OFF" orientation IR sequences, specifically, the upstream IR sequence as denoted by SEQ ID NO: 30, and / or the downstream sequence as denoted by SEQ ID NO: 31 (for the "OFF" orientation), or alternatively, in a fragment of at least one of these IRs, e.g., a fragment comprising the nucleic acid sequence as denoted by SEQ ID NO: 33. In some embodiments, the inverted repeats may be of the "ON" orientation. Accordingly, the target sequence may comprise or may be comprised within at least one of the "ON" orientation IR sequences, specifically, the upstream IR sequence as denoted by SEQ ID NO: 36, and / or the downstream sequence as denoted by SEQ ID NO: 37 (for the "ON" orientation), or alternatively, in a fragment of at least one of these IRs, e.g., a fragment comprising the nucleic acid sequence as denoted by SEQ ID NO: 39.

[0165] In some embodiments, the modification in the target nucleic acid sequence comprises at least one of deletion, insertion, replacement, inversion, point mutation, rearrangement.

[0166] As used herein, the term “modification” of a target nucleic acid sequence refers to any intentional change introduced into the DNA sequence of a bacterium by genetic engineering techniques. In certain embodiments, the modification comprises at least one of the following: deletion, insertion, replacement, inversion, point mutation, or rearrangement of one or more nucleotides within the target sequence. These modifications may be introduced by molecular cloning, recombineering, gene editing systems (e.g., CRISPR-based genome editing), site-directed mutagenesis, or other suitable genetic manipulation methods.

[0167] In some embodiments, the target sequence is modified by a deletion of one or more nucleotides. "Deletion” refers to the removal of one or more nucleotides from the target nucleic acid sequence. The deleted region may range from a single nucleotide to an entire gene, operon, or regulatory element. Deletions may be used to disrupt gene function, remove regulatory control elements, or eliminate undesired sequences such as recombination sites or mobile genetic elements.

[0168] In some embodiments, the target sequence is modified by an insertion of one or more nucleotides. "Insertion” refers to the addition of one or more nucleotides into the target nucleic acid sequence. Inserted sequences may include foreign genes, promoters, regulatory elements, tags, or synthetic constructs.

[0169] In some embodiments, the target sequence is modified by a replacement of one or more nucleotides. "Replacement” (also referred to as substitution) refers to the removal of a native sequence and its substitution with a different nucleotide sequence. Replacement may involve swapping a coding region, promoter, or other functional element with an alternative version, such as replacing a native gene with a codon-optimized gene, a mutant allele, or a homologous gene from another species.

[0170] In some embodiments, the target sequence is modified by an inversion of several nucleotides. "Inversion" refers to the reversal in orientation of a segment of DNA within the target sequence. The inverted segment is flanked by recognition sites for a site-specific recombinase or invertase and is flipped 180 degrees in orientation. Inversions may be used to control phase-variable gene expression, switch between alternative promoters or coding sequences, or regulate transcriptional direction. In some embodiments, the target sequence is modified by a point mutation at one or more nucleotides. "Point mutation" refers to a change of a single nucleotide in the target sequence, including transitions, transversions, insertions, or deletions of a single base pair. Point mutations may be silent (synonymous), missense (resulting in an amino acid change), nonsense (introducing a premature stop codon), or regulatory (affecting promoter or enhancer function).

[0171] In some embodiments, the target sequence is modified by rearrangement of two or more nucleotides. "Rearrangement” refers to any reorganization of DNA segments within the target nucleic acid sequence, including duplications, translocations, or reordering of genes or regulatory elements. Rearrangement may occur within a locus or between loci and may affect gene expression, regulation, or genome architecture.

[0172] As indicated above, the genetically engineered bacterium of the present disclosure comprises at least one modification, as described herein, in one or more nucleotides of a target sequence. In the context of the present disclosure, “one or more” encompasses modifications in from about 1 to about 1,000 nucleotides, or more. In certain embodiments, the modification involves at least one modification in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,

[0173] 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,

[0174] 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,

[0175] 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100; 0, 50, 100,

[0176] 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more nucleotides within the target sequence. In certain embodiments, the modification is introduced within a nucleic acid sequence that encodes a protein product. The modification may modify at least about 5% to about 100% of the target sequence, such that the alteration encompasses a minor region, a substantial portion, or the entirety of the nucleic acid sequence. For example, the modification may involve changes in from at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or up to 100% of the nucleotides constituting the target sequence. The extent of modification may depend on the desired outcome, including partial modulation of gene expression or complete elimination of the encoded protein activity. Such modifications may include substitution, insertion, deletion, inversion, or rearrangement of one or more nucleotides, or a combination thereof, and may be directed toward any portion of the nucleic acid sequence, including regulatory, coding, or non-coding regions. In certain embodiments, the modification results in a detectable change in the expression, abundance, or function of the protein product encoded by the modified sequence. In some embodiments, the target sequence comprises or is comprised within a sequence encoding the invertase. In some embodiments, the invertase is the tyrosine invertase of B. ovatus. In more specific embodiments, the sequence encoding the B. ovatus tyrosine invertase may comprise the nucleic acid sequence as denoted by SEQ ID NO: 25. In yet some further embodiments, the modification is a deletion modification deleting the entire gene encoding the invertase, thereby preventing and / or reducing the expression and / or the levels of a functional invertase. Accordingly, the absence or reduction in the invertase prevents and / or reduces the inversion in the genomic locus that display phase variation. Thus, in the absence or reduction of inversion, the adjacent genomic locus (also referred to herein as the target locus), is locked in the desired orientation. Specifically, in some embodiments, an adjacent genomic locus is, or comprises the B. ovatus UPxY locus. Accordingly, in some embodiments, the deletion of the invertase gene of SEQ ID NO: 25 or parts thereof, prevents or reduces the expression of a functional invertase and thus, the inversion of the UPxY promoter region. In the absence of inversion, the promoter is locked either the "OFF" orientation, as denoted by SEQ ID NO: 26, or the "ON" orientation as denoted by SEQ ID NO: 35.

[0177] In yet some alternative or additional embodiments, the target sequence comprises or is comprised within a sequence recognized by, targeted by and / or bound by at least one invertase. In some embodiments, the target sequence is at least one of the inverted repeat sequences that participate in inversion of the invertible sequence within the B. ovatus UPxY locus. Accordingly, for the "OFF" orientation, the target sequence may comprise or may be comprised within the upstream IR sequence as denoted by SEQ ID NO: 30, and / or the downstream sequence as denoted by SEQ ID NO: 31 (for the "OFF" orientation), or alternatively, in a fragment of at least one of these IRs, e.g., a fragment comprising the nucleic acid sequence as denoted by SEQ ID NO: 33. In some embodiments, the inverted repeats may be of the "ON" orientation. Accordingly, the target sequence may comprise or may be comprised within the upstream IR sequence as denoted by SEQ ID NO: 36, and / or the downstream sequence as denoted by SEQ ID NO: 37 (for the "ON" orientation), or alternatively, in a fragment of at least one of these IRs, e.g., a fragment comprising the nucleic acid sequence as denoted by SEQ ID NO: 39. In some embodiments, the target sequences within at least one IR sequence of one of the orientations (the desired orientation) is scrambled. Specifically, at least 10 to 30 nucleotides of at least one of the IR sequences of the desired orientation (either the "OFF' or the "ON") undergoes at least one rearrangement event thereby creating a scrambled sequence. In some further embodiments, the deletion and / or point mutation in the target nucleic acid sequence locks the adjacent genomic locus in a desired orientation. In some embodiments, the genomic locus is adjacent to the target nucleic acid sequence. For example, the B. ovatus UPxY genomic locus, is adjacent, or is located in close proximity with the tyrosine invertase gene.

[0178] As used herein, the term "adjacent" , when referring to a genomic locus, denotes a nucleic acid sequence that is positioned next to or in close proximity to another specified sequence, in this case, the target nucleic acid sequence. The adjacent locus may be immediately contiguous (i.e., directly neighboring without intervening sequence) or separated by a short intervening sequence (e.g., noncoding DNA, regulatory elements, or untranslated regions), provided that it remains functionally or structurally linked to the target sequence.

[0179] In certain embodiments, the adjacent genomic locus may include a promoter, gene, operon, recombination site, invertible element, or any other functional nucleic acid sequence whose behavior (e.g., orientation, expression, or accessibility) is influenced by modifications in the target sequence. For example, a deletion or point mutation introduced within or near an invertase binding site, or invertible sequence in the target sequence may prevent further inversion events, thereby "locking" the adjacent genomic locus, such as an invertible promoter or coding region, into a fixed desired orientation. This results in stable, unidirectional gene expression and eliminates phase variability or recombination-dependent switching.

[0180] The term "adjacent" is not limited to a fixed number of base pairs and may encompass sequences that are within a range sufficient to confer direct or regulatory influence between the modified and adjacent regions.

[0181] In some specific embodiments, the genomic locus comprises the UpxY locus. According to some embodiments, the target nucleic acid sequence encodes at least one invertase located in close proximity to the UpxY locus. Accordingly, deletion in or of the target nucleic sequence that encodes the invertase, eliminated the expression of invertase and thus prevents inversion (phase variation) in the adjacent UpxY locus. This locks the promoter of the UpxY locus in a specific desired ON or OFF orientation. Engineered bacterium with the desired orientation is selected. In some non-limiting embodiments, for the preparation of the engineered bacteria having deletion of the tyrosine invertase coding region, the deletion may be performed as described in Example 2. More specifically, in some embodiments where the targeted invertase coding region is adjacent to the UpxY locus, an upstream homology arm (US) may include sequences complementary to the promoter region of the UpxY gene, at the desired orientation (either "ON" or "OFF"). Non-limiting embodiments for such homology arms may comprise the US sequences as denoted by SEQ ID NO: 17 (for the "OFF" orientation) or SEQ ID NO: 18 (for the "ON" orientation). The downstream homology arm (DS) in some embodiments may contain sequences complementary to nucleic acid sequences located downstream to the invertase coding sequence. Non-limiting embodiments for such homology arm (the DS) may comprise the nucleic acid sequence as denoted by SEQ ID NO: 16. Alternatively, the DS arm may be complementary to sequences that are complementary to sequences at the downstream part of the invertase encoding region, enabling deletion of most of the invertase coding sequence, such that the resulting modified bacteria will not express a functional invertase.

[0182] As shown in the Examples (see experimental procedures, Example 1 and Figure 2), use of specific recognition arms (DS and US), allows deletion of the entire invertase gene and selection of a desired orientation for a desired ON or OFF orientation for the UpxY promoter. It should be understood that any genomic locus of interest may be locked by this strategy (deletion of the invertase). It should be understood that the engineered bacteria may include deletion of one or more additional or alternative invertases or recombinases, e.g., MPIs, to prevent or reduce rescue of the inversion process in the absence of the deleted tyrosine invertase.

[0183] In some alternative embodiments, the genomic locus of interest comprises the UpxY locus, and modification is performed in the invertase targeted sequences, or sequences inverted by the invertase, or at the invertase binding and / or / recognition sites of this locus. More specifically, according to these embodiments, the target nucleic acid sequence resides within the UpxY locus, specifically, in the promoter region of the UpxY gene, and comprises specific invertible targeted sequences for at least one invertase, specifically, the inverted repeat sequences (IRs). Accordingly, at least one mutation in the target nucleic sequence that comprises the sequences of at least one inverted repeat, or of any other invertase binding or recognition sites, reduces or prevents recognition and inversion of these mutated (or deleted) sequences by the invertase, thereby reducing or inhibiting inversion of the promoter region of the UpxY locus. This locks the promoter of the UpxY locus in a desired orientation.

[0184] According to this embodiment, the inverted repeats (IR) site (acting as the "invertible sequences" or the invertase recognition sites) should be elected. As also exemplified by the experimental procedures, Example 4 and Figure 10. In some embodiments, the IRs are characterized as having a minimum length of 11 bp with no mismatches, or alternatively, of a length of 13bp inverted repeats with one mismatch. In yet some further embodiments, of a length of 16bp inverted repeats with one mismatch. Still further, in some embodiments, the IRs are characterized as having a minimum length of between about 30 bp or more (with no mismatches) to about 45 base pairs or less. The number of mismatches between the inverted repeats is less than or equal to the aforementioned maximum mismatch threshold. Still further, in some embodiments, the IR sequences of the promoter region of B. ovatus UPxY may comprise between about 20 to 35 nucleotides. In yet some further embodiments, the IR sequences of the promoter region may be in the length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 nucleotides. In some specific embodiments, the IR sequences of the B. ovatus UPxY promoter region may comprise the nucleic acid sequence as denoted by SEQ ID NO: 30 (for the IR located upstream in the 'OFF' orientation), and SEQ ID NO: 31 (for the IR located downstream in the 'OFF' orientation), or of a fragment of said IRs as denoted by SEQ ID NO: 33 (for the IR located downstream in the 'OFF' orientation).

[0185] Still further, in some embodiments the IR sequences of the B. ovatus UPxY promoter region may comprise the nucleic acid sequence as denoted by SEQ ID NO: 36 (for the IR located upstream in the 'ON' orientation), and SEQ ID NO: 37 (for the IR located downstream in the 'ON' orientation), or of a fragment of said IRs. For example, a fragment comprising the nucleic acid sequence as denoted by SEQ ID NO: 39 (for the IR located downstream in the 'ON' orientation).

[0186] Still further, in some embodiments, at least one modification may be performed in at least one nucleotide of at least one IR sequence, to prevent appropriate inversion of the promoter region. More specifically, about 10 to 20 or more nucleotides are modified, specifically, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, may be modified in at least one IR sequence. In yet some further embodiments, at least 50% of the nucleotides in at least one of the two IR sequences (either of the "ON" or "OFF" orientation), may be modified, specifically, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% of the nucleotides in one or two IRs (either of the "ON" or "OFF" orientation), may be modified. Still further, in some embodiments, the modification is scrambling of at least one of the two IR sequences (either of the "ON" or "OFF" orientation), specifically, arranging the nucleotides originally present in each of the sequence in a scrambled order. In some embodiments, the downstream IR sequence is modified. Non-limiting examples for a scrambled IR sequence exemplified by the present disclosure may be the downstream IR sequence of the "OFF" orientation, having the nucleic acid sequence as denoted by SEQ ID NO: 32, where the scrambled sequence (a fragment of the IR) is as denoted by SEQ ID NO: 34. In yet some further embodiments, the downstream IR sequence of the "ON" orientation, having the nucleic acid sequence as denoted by SEQ ID NO: 38, where the scrambled sequence (a fragment of the IR) is as denoted by SEQ ID NO: 40.

[0187] By introducing mutations to the IR region, the invertase cannot recognize the IR and thus, no inversion occurs in the specific genomic locus. The engineered bacteria are locked for one of the phases (ON or OFF orientation). It should be understood that any genomic locus of interest may be locked by this strategy (mutation of the invertase binding sires (IRs)).

[0188] Still further in some additional or alternative embodiments, the genomic locus may be modified. More specifically, according to such embodiment, the target nucleic acid sequence may be a sequence comprising at least in part, the genomic locus that undergoes phase variation. This sequence can be replaced with a sequence having the desired phase variation, for example, with a sequence having the desired orientation. For example, in case that the UpxY locus is the locus of interest, replacement of the entire locus with a nucleic acid sequence having the desired orientation can be performed. The entire sequence (or alternatively, only the promoter region in the desired orientation), can be provided as a synthetic nucleic acid sequence with IR mutations (to avoid, reduce or prevent inversion), or as a recombinant sequence.

[0189] In some embodiments, the desired orientation is an ON orientation of the promoter region of said UpxY locus. In some embodiments, the desired orientation is an OFF orientation of the promoter region of said UpxY locus. A non-limiting example for a scrambled promoter region locked in the "OFF" orientation is as denoted by SEQ ID NO: 21. In another non-limiting embodiment, a scrambled promoter sequence locked at the "ON" orientation is as denoted by SEQ ID NO: 22.

[0190] In some embodiments, the ON orientation of the promoter region of said UpxY is associated with responsiveness of a subject to immunotherapy. As used herein, the term ^immunotherapy” refers to any therapeutic approach, composition, or method that intends to modulate, enhance, suppress, or otherwise alter an immune response for the purpose of preventing, treating, ameliorating, or managing a disease or physiological condition. Immunotherapy may involve the activation or inhibition of innate or adaptive immune components, including but not limited to T cells, B cells, macrophages, dendritic cells, natural killer (NK) cells, regulatory immune cells, antibodies, cytokines, chemokines, or antigen-presenting cells. Immunotherapeutic strategies include, without - 41 - limitation, the administration of immune checkpoint inhibitors, cytokine therapies, monoclonal or polyclonal antibodies, immune cell therapies (e.g., CAR-T cells, TCR-engineered cells), vaccines (including therapeutic vaccines), adjuvants, or microbial-based agents that stimulate or dampen host immune responses. The immunotherapy may be active (stimulating the host's own immune system), passive (administering immune components), adoptive (transfer of ex vivo modified immune cells), or combinatorial, and may be administered alone or in conjunction with other therapeutic modalities such as chemotherapy, radiation, antimicrobial therapy, or gene therapy.

[0191] In some embodiments, the "ON" orientation in the UPxY promoter is associated with responsiveness of melanoma patients to immunotherapy, specifically, using immune-checkpoint inhibitors.

[0192] As indicated above, the present disclosure provides genetically engineered bacterium and / or a population of bacteria comprising said bacterium.

[0193] In some embodiments, the genetically engineered bacterium is B. ovatus bacterium comprising at least one modification at the UPxY locus, and / or the adjacent tyrosine invertase locus.

[0194] In some embodiments, the bacterium of the present disclosure may comprise a deletion of the tyrosine invertase gene as denoted by SEQ ID NO: 25. In some embodiments, such bacterium may comprise a UPxY locus, where the promoter region is at the "ON" orientation, specifically, where the promoter region of the B. ovatus UPxY comprises the nucleic acid sequence as denoted by SEQ ID NO: 35. Accordingly, since the modified bacterium has a deleted tyrosine invertase gene, and thus, does not express tyrosine invertase, it displays a locked "ON" orientation, enabling the expression of the UPxY gene.

[0195] In some additional embodiments, the bacterium of the present disclosure may comprise a deletion of the tyrosine invertase gene as denoted by SEQ ID NO: 25. In some embodiments, such bacterium may comprise a UPxY locus, where the promoter region is at the "OFF" orientation, specifically, where the promoter region of the B. ovatus UPxY comprises the nucleic acid sequence as denoted by SEQ ID NO: 26. Accordingly, since the modified bacterium has a deleted tyrosine invertase gene, and thus, does not express tyrosine invertase, it displays a locked "OFF" orientation, preventing and / or reducing the expression of the UPxY gene.

[0196] In some further embodiments, the bacterium of the present disclosure is a B. ovatus bacterium comprising a modification in at least one IR sequence within the promoter region of the B. ovatus UPxY. In some embodiments, the modification is in the downstream IR sequence of the promoter region. Disturbance of the IR sequence prevents the recognition of the IR by the invertase, prevents inversion of the promoter region, and thus the original orientation of the promoter region (either "OF" or "ON") is locked.

[0197] Thus, in some embodiments, the genetically engineered or modified bacterium comprises an IR sequence as denoted by SEQ ID NO: 32. Since this IR sequence is a modified (scrambled) downstream IR sequence of the B. ovatus UPxY promoter at the "OFF" orientation, it will not allow an inversion of the promoter region. Such bacterium is locked at the "OFF" orientation. In some embodiments such genetically engineered bacterium comprises a B. ovatus UPxY promoter comprising the nucleic acid sequence as denoted by SEQ ID NO: 28. In yet some further embodiments, the present disclosure provides a genetically engineered or modified bacterium comprising modification is two elements, at the IR and deletion of the invertase. In some embodiments the bacterium comprising an IR sequence as denoted by SEQ ID NO: 32, and a deleted tyrosine invertase gene, specifically, deletion in the nucleic acid sequence as denoted by SEQ ID NO: 25. This bacterium does not express the tyrosine invertase gene, and is locked at the OFF orientation of the UPxY promoter. Such bacterium does not express the tyrosine invertase and does not express the UPxY gene product.

[0198] In yet some further embodiments, the genetically engineered or modified bacterium comprises an IR sequence as denoted by SEQ ID NO: 38. Since this IR sequence is a modified (scrambled) downstream IR sequence of the B. ovatus UPxY promoter at the "ON" orientation, it will not allow an inversion of the promoter region. Such bacterium is locked at the "ON" orientation and thus expresses the UPxY gene product. In some embodiments such genetically engineered bacterium comprises a B. ovatus UPxY promoter comprising the nucleic acid sequence as denoted by SEQ ID NO: 29. In yet some further embodiments, the present disclosure provides a genetically engineered or modified bacterium comprising modification in two elements, at the IR and deletion of the invertase. In some embodiments the bacterium comprising an IR sequence as denoted by SEQ ID NO: 38, and a deleted tyrosine invertase gene, specifically, deletion in the nucleic acid sequence as denoted by SEQ ID NO: 25. This bacterium does not express the tyrosine invertase gene and is locked at the ON orientation of the UPxY promoter. Such bacterium expresses the UPxY gene product but does not express the tyrosine invertase.

[0199] Another aspect of the present disclosure relates to a composition comprising a genetically engineered bacterium or a population comprising the bacterium. More specifically, the genetically engineered bacterium is locked in a desired orientation in at least one genomic locus displaying phase variation. The bacterium of the present disclosure comprises, comprised within, at least one modification in a target nucleic acid sequence that comprises or encodes at least one element that induces directly or indirectly phase variation in the at least one genomic locus. The composition further comprising at least one of acceptable carrier / s, diluent / s, excipient / s and additive / s.

[0200] In some embodiments, the composition is a probiotic composition.

[0201] As used herein, the term “probiotic composition” refers to a formulation comprising one or more live microorganisms, when administered in adequate amounts to a host, confer a health benefit. In the context of this disclosure, the probiotic composition includes at least one genetically engineered bacterium that is locked in a desired orientation at a genomic locus undergoing phase variation, thereby stabilizing its gene expression profile and associated phenotype.

[0202] The probiotic composition further comprises one or more acceptable carriers, diluents, excipients, or additives, suitable for maintaining bacterial viability, facilitating delivery, or enhancing formulation stability. Such carriers may include aqueous buffers, cryoprotectants, polysaccharide matrices, encapsulation agents, or lyophilization media. The composition may be formulated for oral, enteric, rectal, mucosal, or topical administration, depending on the intended site of colonization or therapeutic action. The engineered bacteria within the probiotic composition may be designed to colonize specific microbiome niches (e.g., the gut, skin, or urogenital tract), and may provide functions such as immune modulation, competitive exclusion of pathogens, metabolic support, or targeted delivery of therapeutic molecules.

[0203] In some other embodiments, the composition is formulated as an add-on to a solid, semi-solid or liquid food, beverage, food additive, food supplement, medical food, drug and / or a pharmaceutical composition.

[0204] The term “add-on to a solid, semi-solid or liquid food” , as used herein, refers to any formulation or ingredient, including but not limited to live microorganisms, that is incorporated into a food matrix for the purpose of delivering a functional, nutritional, or therapeutic effect. This includes integration into conventional foods such as yogurts, nutrition bars, juices, soups, or smoothies. The add-on may be blended, layered, encapsulated, or otherwise incorporated into the food without substantially altering its basic identity as a food product. More specifically, "solid food” refers to firm, shape-retaining food products (e.g., nutrition bars, baked goods), "semi-solid food" refers to foods with soft, spreadable textures (e.g., yogurts, puddings, sauces) and "liquid food” refers to pourable food matrices (e.g., drinks, broths, smoothies).

[0205] The term “beverage” refers to any liquid preparation intended for oral consumption. This includes but is not limited to water-based drinks, dairy or plant-based beverages, juices, teas, fermented drinks (e.g., kombucha, kefir), or fortified drinks. In the context of the present disclosure, the beverage may serve as a delivery medium for the genetically engineered bacterium, maintaining its viability and enabling gastrointestinal administration.

[0206] The term food additive”, as used herein, refers to any substance added to food for a technological purpose, such as preservation, texture, flavor, color, or stability, and includes both natural and synthetic ingredients. A food additive may also include functional bioactives, such as probiotics, vitamins, or bioengineered microorganisms, provided they meet regulatory safety standards for food inclusion. The term “food supplement” (or dietary supplement) refers to a product intended to supplement the normal diet and which is a concentrated source of nutrients or other substances with nutritional or physiological effect. This includes capsules, powders, tablets, or liquid formulations containing one or more active ingredients such as vitamins, minerals, enzymes, amino acids, plant extracts, or live microorganisms (e.g., probiotics), intended to support general health or specific physiological functions. The term “medical food” , as used herein, refers to a food product that is formulated to be consumed or administered enterally under the supervision of a physician, and which is intended for the specific dietary management of a disease or condition with distinctive nutritional requirements. Medical foods are not conventional foods or supplements and are subject to regulatory frameworks. The term “drug”, as used herein, refers to any substance or combination of substances that is intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease, or that affects the structure or function of the body. Drugs may be delivered orally, topically, parenterally, or via other routes, and may include live biotherapeutic products (LBPs) composed of genetically engineered microorganisms that exert therapeutic effects through immune modulation, metabolic activity, or microbiome remodeling.

[0207] In some further embodiments, the composition is a pharmaceutical composition.

[0208] The term "pharmaceutical compositions" , refers to compositions that comprise a therapeutically effective amount of at least one active agent, i.e. a genetically engineered bacterium or a population comprising the bacterium according to the present disclosure, and optionally, at least one pharmaceutically acceptable carrier. The term 'pharmaceutically acceptable' means approved by a regulatory agency or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term 'carrier' denotes to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Examples of such pharmaceutical carriers are sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Suitable pharmaceutical excipients may include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. A composition can further contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained- release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. In specific embodiments, the compositions of the invention may be formulated in accordance with routine procedures as pharmaceutical compositions adapted for intravenous administration in humans. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. Where the composition is administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[0209] The pharmaceutical compositions of the present disclosure can be administered and dosed in accordance with good medical practice, systemically, for example by parenteral, e.g. intravenous, intraperitoneal or intramuscular injection. In another example, the pharmaceutical composition can be introduced to a site by any suitable route including intravenous, subcutaneous, transcutaneous, topical, intramuscular, intraarticular, subconjunctival, or mucosal, e.g. oral, intranasal, or intraocular administration. Local administration to the area in need of treatment may be achieved by, for example, by local infusion during surgery, topical application, direct injection into the specific organ, etc. More specifically, the compositions of the invention and the compositions used in the methods, described herein after, may be adapted for administration by parenteral, intraperitoneal, transdermal, oral (including buccal or sublingual), rectal, topical (including buccal or sublingual), vaginal, intranasal and any other appropriate routes. Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s). It should be noted that any of the administration modes discussed herein, may be applicable for any of the methods of the invention as described in further aspects of the invention herein after. Compositions and formulations for oral administration may include powders or granules, suspensions or solutions in water or nonaqueous media, capsules, sachets, lozenges (including liquid-filled), chews, multi- and nanoparticulates, gels, solid solution, liposome, films, ovules, sprays or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Pharmaceutical formulations adapted for rectal administration may be presented as suppositories or enemas. Pharmaceutical formulations adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations. Pharmaceutical compositions used to treat subjects in need thereof according to the invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. The compositions may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain stabilizers. The pharmaceutical compositions of the present invention also include, but are not limited to, emulsions and liposome-containing formulations. It should be understood that in addition to the ingredients particularly mentioned above, the formulations may also include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents. The compositions of the invention may also be administered directly to the eye or ear, typically in the form of drops of a micronised suspension or solution in isotonic, pH-adjusted, sterile saline. Other formulations suitable for ocular and aural administration include ointments, biodegradable (e.g. absorbable gel sponges, collagen) and non-biodegradable (e.g. silicone) implants, wafers, lenses and particulate or vesicular systems, such as niosomes or liposomes. A polymer such as crossed- linked polyacrylic acid, polyvinylalcohol, hyaluronic acid, a cellulosic polymer, for example, hydroxypropylmethylcellulose, hydroxy ethylcellulose or methyl cellulose or a heteropolysaccharide polymer, for example, gelan gum, may be incorporated together with a preservative, such as benzalkonium chloride. Such formulations may also be delivered by iontophoresis. Formulations for ocular and aural administration may be formulated to be immediate and / or modified release. Modified release includes delayed, sustained, pulsed, controlled, targeted, and programmed release. In specific embodiments, the unit dosage formulations are those containing a daily dose or sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient. Of particular relevance are formulations of compositions of the invention adapted for use as a nano- or micro-particles. Nanoscale drug delivery systems using liposomes and nanoparticles are emerging technologies for the rational drug delivery, which offers improved pharmacokinetic properties, controlled and sustained release of drugs and, more importantly, lower systemic toxicity. A particularly desired solution allows for externally triggered release of encapsulated compounds. Externally controlled release can be accomplished if drug delivery vehicles, such as liposomes or polyelectrolyte multilayer capsules, incorporate nanoparticle (NP) actuators.

[0210] In case used as a pharmaceutical composition, the disclosed composition may comprise a therapeutically effective amount of the disclosed genetically engineered bacteria. It should be understood that the engineered bacteria may be provided in the disclosed composition either as a single therapy or in combination with other active ingredients.

[0211] As used herein, the term "therapeutically effective amount” means an amount of a compound or composition which is administered to a subject in need thereof, necessary to effect a beneficial change in the severity of a disease or disorder, or prevent such disease, in said subject. This amount should also be within specific pharmacological ranges, to avoid toxic effects by over-dosing. For example, in the present disclosure, a therapeutically effective amount of at least one of the engineered bacterium of the present disclosure, for the treatment of an immune-related disorder would be the amount of these engineered bacteria administered to a subject which would induce a beneficial change in the subject, alleviating, ameliorating, or preventing the recurrence of the immune-related disorder, without causing detrimental side effects, or causing only mild sideeffects. It is understood that the therapeutically effective amount is not an absolute term and depends on subjective circumstances, such as the subject's age, health, weight, and various other statistics, as described in the and specifically determined by the attendant physician or other person skilled in the art after an evaluation of the subject’s conditions and requirements. In some embodiments, an effective amount of the bacteria may involve a amount of between about 106to IO20of the genetically engineered bacteria as disclosed by the present disclosure, per kg of body weight. Specifically, between 106and 1012CFU (colony-forming units) per subject per day. More specifically, about 1O8-1O10CFU / day. Alternatively, the effective amount may be expressed in mg of bacterial biomass or dry weight, typically 0.1-100 mg / day, corresponding roughly to 107-10ncell equivalents.

[0212] The composition further comprising, in some embodiments, at least one probiotic and / or at least one therapeutic compound. It should be thus understood that the present disclosure provides in some embodiments thereof, compositions and methods for combination therapy.

[0213] In some embodiments, the genetically engineered bacterium of the composition is as defined in above. Specifically, in some embodiments, the disclosed composition comprises an effective amount of engineered bacteria or a population thereof. The disclosed bacteria comprise a deletion of the tyrosine invertase gene as denoted by SEQ ID NO: 25, or of any fragment or portion thereof. In yet some further embodiments, such bacteria of the disclosed composition comprise a UPxY locus comprising a promoter region locked at the "OFF" orientation. Such promoter region may comprise the nucleic acid sequence as denoted by SEQ ID NO: 26. In some alternative embodiments, the disclosed bacteria comprise a deletion of the tyrosine invertase gene as denoted by SEQ ID NO: 25, or of any fragment or portion thereof. In yet some further embodiments, such bacteria of the disclosed composition comprise a UPxY locus comprising a promoter region locked at the "ON" orientation. Such promoter region may comprise the nucleic acid sequence as denoted by SEQ ID NO: 35.

[0214] In some embodiments, the genetically engineered or modified bacterium of the disclosed composition comprises an IR sequence as denoted by SEQ ID NO: 32 (locked at the "OFF" orientation). In some embodiments such genetically engineered bacterium comprises a B. ovatus UPxY promoter comprising the nucleic acid sequence as denoted by SEQ ID NO: 28. In some other embodiments the bacterium of the disclosed composition comprising an IR sequence as denoted by SEQ ID NO: 32, and a deleted tyrosine invertase gene SEQ ID NO: 25.

[0215] In yet some further embodiments, the genetically engineered or modified bacterium comprises an IR sequence as denoted by SEQ ID NO: 38 (the "ON" orientation). In some embodiments such genetically engineered bacterium comprises a B. ovatus UPxY promoter comprising the nucleic acid sequence as denoted by SEQ ID NO: 29. In some embodiments the bacterium of the disclosed composition comprises an IR sequence as denoted by SEQ ID NO: 38, and a deleted tyrosine invertase gene, specifically, deletion in the nucleic acid sequence as denoted by SEQ ID NO: 25. This bacterium does not express the tyrosine invertase gene and is locked at the ON orientation of the UPxY promoter. Such bacterium expresses the UPxY gene product but does not express the tyrosine invertase.

[0216] In yet some further embodiments, the disclosed composition comprises a combination of any of the disclosed genetically engineered bacteria, as disclosed by the present invention, e.g., those having a locked OFF orientation by deletion of the tyrosine invertase gene, and / or those having a locked OFF orientation achieved by a scrambled IR repeat sequence and / or those having a locked OFF orientation achieved by a scrambled IR repeat sequence together with a deletion of the tyrosine invertase gene. Alternatively, the combination of the bacteria may include those having a locked ON orientation by deletion of the tyrosine invertase gene, and / or those having a locked ON orientation achieved by a scrambled IR repeat sequence, and / or those having a locked ON orientation achieved by a scrambled IR repeat sequence together with a deletion of the tyrosine invertase gene.

[0217] The present disclosure further provides a combined composition comprising (a) of any genetically engineered bacteria disclosed by the present disclosure, or any combinations thereof; and (b) at least one immune-checkpoint inhibitor, as disclosed by the present disclosure.

[0218] Another aspect of the present disclosure relates to a method for modulating an immune response in a subject in need thereof. The method comprising the step of administrating to the subject at least one genetically engineered bacterium, a population comprising the bacterium, or any composition thereof, wherein the bacterium is locked in a desired orientation in at least one genomic locus displaying phase variation. This bacterium comprises at least one modification in a target nucleic acid sequence that comprises or encodes at least one element that induces directly or indirectly phase variation in the at least one genomic locus.

[0219] It should be understood that the term "modulating" as used herein encompasses any change or modification to any of the immune response components of a subject or any other host and / or to the immunological state of a subject, and / or the immune system of the subject, or any cellular or non-cellular components thereof. More specifically, components of the immune system as used herein refer to cellular components such as macrophages, neutrophils, dendritic cells, natural killer cells, T cells, B cells, and any regulatory cells, specifically, T regulatory cells, physical barriers such as the skin and mucous membranes, complement proteins, signaling molecules produced by immune cells and other cell types, regulating immune responses by mediating cell-to-cell communication, inflammation, immune cell activation, differentiation, migration, and homeostasis. The term modulation of an immune response or the immune system of the subject may also encompass in some embodiments, changes in the genetic loci encoding molecules such as major histocompatibility complex (MHC) proteins, and the like. In yet some further embodiments, such change may include an increase, enhancement or alternatively, decrease in at least one of the immune response components and / or immunological state in relation to their normal and / or baseline level. More specifically, in some embodiments, "modulation" as used herein may further encompasses changing the nature, the extent, the direction of the immune response in the subject, or changing the amount or function of at least one component (either cellular or non-cellular components) of the immune system of the subject. In some embodiments, when referring to the extent or the direction of an immune response, or the level of specific components thereof, modulation as used herein encompasses either reduction or enhancement of the immune response, or components thereof. More specifically, in case the modulation involves enhancement of the immune response (e.g., as reflected for example by enhanced cytokine expression, or immune regulatory cells), such enhancement is useful in case of cancer (e.g., melanoma), and any disease that immune modulation may benefit with. Accordingly, in some embodiments, "increase", "rise", "elevation", "expansion", "augmentation", "enhancement", "amplification", "upregulation", "intensification", as referred to herein, relate to the elevation of the immune response, or any components of the immune response by any one of about 1% to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%, as compared with the immune response with no administration of the engineered bacteria of the present disclosure, specifically those locked at the "ON" orientation of the UPxY locus.

[0220] Alternatively, in some embodiments, for example, where reduction in the immune response is required (for example, in cases of inflammatory and / or autoimmune disorders such as IBD and the like, locking the "OFF" orientation of the UPxY locus may be beneficial. Accordingly, "inhibition", "moderation", “reduction” or "attenuation" as referred to herein, relate to the retardation, restraining or reduction of the immune response, or any components of the immune response by any one of about 1% to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%. Therefore, the term inhibit, or decrease refers to an inhibition of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 folds or more, as compared to the level, extent or nature of the immune response prior to the administration of the engineered bacteria or without the administration of the engineered bacteria of the present disclosure.

[0221] In some embodiments, the subject is suffering of at least one immune-related disorder.

[0222] In some further embodiments, the immune-related disorder is at least one of: a proliferative disorder, an inflammatory disorder, an autoimmune disorder, an immune-deficiency condition and / or an infectious disease.

[0223] In some embodiments, the genetically engineered bacterium is as defined above.

[0224] More specifically, for treating melanoma, the disclosed methods may use at least one engineered B. ovatus bacteria locked at the "ON" orientation of the UPxY locus. In some embodiments, such bacteria may comprise a deletion of the tyrosine invertase gene (as denoted by SEQ ID NO: 25), where the promoter region of the UPxY locus comprises the nucleic acid sequence as denoted by SEQ ID NO: 35. In yet some further embodiments, the disclosed method may use an engineered bacteria comprising a modified UPxY promoter, having a scrambled IR sequence, specifically, a promoter having the downstream IR sequence as denoted by SEQ ID NO: 38. In some embodiments, such bacteria has a UPxY promoter region having the nucleic acid sequence as denoted by SEQ ID NO: 29. In yet some further embodiments, the disclosed methods may also use a bacteria comprising the UPxY promoter region as denoted by SEQ ID NO: 29, together with a deletion of the tyrosine invertase gene as denoted by SEQ ID NO: 25, or any combinations of the engineered bacteria displaying locked "ON" orientation of the UPxY locus.

[0225] In some embodiments, the modulation of an immune response comprises improving responsiveness of the subject to at least one therapeutic compound or therapeutic regimen.

[0226] As used herein, the term “improving responsiveness of a subject to immunotherapy” refers to any measurable enhancement in one or more clinical, biochemical, molecular, or immunological parameters that indicate an increased therapeutic benefit of an immunotherapy, relative to an appropriate control or baseline (for example, the same subject prior to treatment or an untreated control subject). In certain embodiments, the improvement corresponds to an increase of about 5% to about 100%, including but not limited to about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, in at least one parameter indicative of therapeutic responsiveness. Such parameters may include an increase in tumor regression, disease stabilization, or overall survival; an increase in immune effector activity such as T-cell activation, cytotoxic lymphocyte infiltration, cytokine secretion, or antibody production; a reduction in tumor burden or circulating tumor cell count; an improvement in progression-free survival or objective response rate; or a measurable decrease in immunosuppressive cell populations, such as regulatory T cells or myeloid-derived suppressor cells, within the tumor microenvironment or circulation. In some embodiments, improved responsiveness is evidenced by a statistically significant increase in one or more of the foregoing parameters compared to baseline or control, or by the attainment of a clinically meaningful benefit as determined according to recognized medical or regulatory standards.

[0227] In some specific embodiments, the subject is a subject suffering from melanoma, wherein the bacterium is engineered to lock the promoter region of the UpxY locus in an ON orientation, and wherein the subject is treated with at least one immune check point inhibitor. The term “immune checkpoint inhibitors” refers to a class of therapeutic agents that enhance immune system activity by blocking inhibitory signaling pathways, known as immune checkpoints, which normally function to maintain self-tolerance and prevent excessive immune activation. These inhibitors act by interfering with specific receptor-ligand interactions that suppress T-cell activation and effector function, thereby restoring or amplifying antitumor immune responses. In the context of melanoma, immune checkpoint inhibitors have revolutionized therapy by reactivating cytotoxic T lymphocytes that recognize and destroy malignant cells. Clinically established immune checkpoint inhibitors used for the treatment of melanoma include antibodies targeting the cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), such as ipilimumab; antibodies directed against the programmed cell death protein 1 (PD-1) receptor, such as nivolumab and pembrolizumab; and antibodies targeting the PD- 1 ligand (PD-L1) expressed on tumor or immune cells, such as atezolizumab, avelumab, and durvalumab. By blocking these inhibitory pathways, the drugs relieve the suppression of T-cell responses, leading to enhanced tumor cell recognition and killing. Combination regimens, such as ipilimumab administered with nivolumab, have demonstrated improved efficacy in advanced melanoma by concurrently targeting distinct immune regulatory checkpoints.

[0228] It should be understood that the present disclosure further provides at least one genetically engineered bacterium, a population comprising the bacterium, or any composition thereof, as disclosed herein, for use in a method for modulating an immune response in a subject in need thereof. Another aspect of the present disclosure relates to a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one pathologic disorder in a subject. The method comprising the steps of administrating to the subject at least one genetically engineered bacterium, or a population comprising said bacterium, wherein the genetically engineered bacterium is locked in a desired orientation in at least one genomic locus displaying phase variation. This bacterium comprises at least one modification in a target nucleic acid sequence that comprises, comprised within or encodes at least one element that induces directly or indirectly phase variation in the at least one genomic locus.

[0229] In some embodiments, the pathologic disorder is at least one immune-related disorder.

[0230] In some further embodiments, the immune-related disorder is at least one of: a proliferative disorder, an inflammatory disorder, an autoimmune disorder, an immune-deficiency condition and / or an infectious disease.

[0231] In some embodiments, the genetically engineered bacteria are as defined above.

[0232] In some specific embodiments, the subject is suffering from melanoma, and the bacterium is engineered to lock the promoter region of the UpxY locus in an ON orientation.

[0233] In some further embodiments, the subject is further treated with at least one check point inhibitor. The present disclosure further provides in some aspects thereof, the at least one genetically engineered bacterium, a population comprising the bacterium, or any composition thereof, as disclosed herein, for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one pathologic disorder in a subject.

[0234] Another aspect of the present disclosure relates to a method for the preparation of an immunomodulatory composition comprising the following steps: Step (a) includes genetically modifying at least one target nucleic acid sequence in a bacterium. The target sequence comprises or encodes at least one element that induces directly or indirectly phase variation in at least one genomic locus displaying phase variation. The modification results in locking the genomic locus in a desired orientation. Step (b) includes admixing a bacterium locked in a desired orientation of the genomic locus obtained by step (a), with at least one of carrier / s, diluent / s, excipient / s and additive / s.

[0235] The term “genetically modifying”, as used herein in step (a), refers to the process of intentionally altering the genetic material (e.g., DNA) of a bacterium by introducing at least one targeted change in a nucleic acid sequence. This may include, but is not limited to, deletion, insertion, substitution, inversion, or rearrangement of specific nucleotide sequences using techniques such as gene editing systems (e.g., the CRISPR / Cas systems), site-directed mutagenesis, homologous recombination, recombineering, or transposon-based approaches. In the context of the present disclosure, genetic modification is applied to a target sequence that comprises or encodes at least one element responsible for inducing phase variation in a genomic locus. The modification is specifically designed to lock the phase-variable locus in a fixed orientation, thereby stabilizing gene expression.

[0236] As shown by the present Examples, in some specific and non-limiting embodiments, the process of "genetically modifying" in accordance with the present disclosure, involves either deletion of the nucleic acid sequence encoding the tyrosine invertase, and / or modification in the sequences targeted by the tyrosine invertase, within the promoter region of the B. ovatus UPxY gene. The disclosed modification process (also referred to herein as "genetically modifying”), may be performed as described above in connection with other aspects of the present disclosure, and / or, as described by Examples 1 and 2 (deletion of the tyrosine invertase) and Example 4 (for the scrambled IR sequencees).

[0237] The term “admixing” , as used herein in step (b), refers to the act of combining or blending the genetically modified bacterium, locked in the desired genomic orientation, with one or more carriers, diluents, excipients, or additives to form a composition suitable for storage, delivery, or administration. The admixing may be carried out for example in solid, semi-solid, or liquid form and can involve processes such as mixing, suspending, encapsulating, lyophilizing, or emulsifying, depending on the intended formulation. This step ensures that the engineered bacterium is incorporated into a stable and physiologically acceptable immunomodulatory composition.

[0238] As used herein, the term “immunomodulatory composition” refers to a composition capable of modulating, directly or indirectly, an immune response in a subject. The modulation may involve enhancement, suppression, or redirection of one or more aspects of the innate or adaptive immune system, and may result in either a stimulatory or tolerogenic effect, depending on the context of use. The composition further includes one or more carriers, diluents, excipients, or additives suitable for administration, formulation stability, or delivery. The immunomodulatory composition may be administered for prophylactic, therapeutic, or diagnostic purposes and may be designed for use in treating, preventing, or managing immune-related conditions, including but not limited to inflammatory diseases, autoimmune disorders, immune deficiencies, allergies, infections, or cancer. In some embodiments, the phase variation in the genomic locus is associated with at least one physiological state and / or condition in the subject.

[0239] In some other embodiments, the composition is a personalized composition.

[0240] The term “personalized composition” , as used herein, refers to a composition that is formulated, selected, or adjusted based on individual-specific characteristics of a subject. These characteristics may include, but are not limited to, the subject’ s genetic profile, microbiome composition, immune status, physiological condition, disease state, medical history, lifestyle factors, diet, or therapeutic responsiveness. In the context of the present disclosure, a personalized composition may comprise a genetically engineered bacterium selected or modified to align with the unique microbial, immunological, or metabolic profile of the subject, thereby enhancing therapeutic efficacy, safety, or host compatibility.

[0241] In some embodiments, the bacterium is of an autologous source.

[0242] The term “autologous source”, as used herein, refers to a source that is derived from the same individual subject to whom the composition or treatment is ultimately administered. In the context of the present disclosure, a bacterium of autologous source refers to a bacterial strain or isolate that is originally obtained from the microbiome of the same subject, for example, from stool, saliva, skin, or mucosal samples, and subsequently genetically modified and reintroduced into that subject. Autologous sourcing may reduce the risk of immunogenicity or microbiome disruption and may improve colonization compatibility and therapeutic response.

[0243] In some other embodiments, the modification is performed ex vivo.

[0244] The term “ex vivo”, as used herein, refers to procedures or modifications that are performed outside of a living organism, typically in a controlled laboratory environment. In the context of the present disclosure, ex vivo modification refers to the genetic engineering of a bacterium that is conducted outside the subject’s body, such as in vitro culture systems, prior to its formulation into a composition and reintroduction into the subject. This approach allows for precise manipulation, screening, and validation of the modified bacterium before administration, and is particularly applicable to autologous or personalized therapeutic strategies.

[0245] As mentioned above, the phase variation referred to in the present disclosure is associated with at least one physiological state and / or condition in a subject. Still further, the compositions and methods of the present disclosure relate to modulating an immune response in a subject in need thereof or for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one pathologic disorder in a subject. A subject in accordance with the present disclosure may be at least one organism of the biological kingdom Animalia or of the biological kingdom Plantae. Still further, the present disclosure relates to the treatment of subjects or patients in need thereof. By “patient” or “subject in need”, in accordance with all aspects of the preset disclosure, it is meant any organism who may be affected by the above-mentioned conditions, and to whom the therapeutic and prophylactic methods herein described are desired, including humans, domestic and non-domestic mammals such as canine and feline subjects, bovine, simian, equine and rodents, specifically, murine subjects. More specifically, the methods of the present disclosure are intended for mammals. By “mammalian subject” means any mammal for which the proposed therapy is desired, including human, livestock, equine, canine, and feline subjects, most specifically humans.

[0246] In some embodiments, the methods of the present disclosure may be applicable for any organism of the biological kingdom Animalia. In more specific embodiments, such organism may be any unicellular or multicellular invertebrate or vertebrate organism. More specifically, invertebrates, may be organisms of the Phylum Porifera - Sponges, the Phylum Cnidaria - Jellyfish, hydras, sea anemones, corals, the Phylum Ctenophora - Comb jellies, the Phylum Platy helminthes - Flatworms, the Phylum Mollusca - Molluscs, the Phylum Arthropoda - Arthropods, the Phylum Annelida - Segmented worms like earthworm and the Phylum Echinodermata - Echinoderms. Still further, in some embodiments, the present disclosure may be applicable for any vertebrate organism, specifically, any organism derived from any of the vertebrates groups that include Fish, Amphibians, Reptiles, Birds and Mammals (e.g., Marsupials, Primates, Rodents and Cetaceans). In some particular embodiments, the present disclosure may be applicable for a mammal (specifically, at least one of a human, Cattle, rodent, domestic pig (swine, hog), sheep, horse, goat, alpaca, lama and Camels). More specifically, in some embodiments, as indicated herein, the present disclosure may be applicable for a vertebrate organism. Vertebrates comprise all species of animals within the subphylum Vertebrata (chordates with backbones). The animals of the vertebrates group include Fish, Amphibians, Reptiles, Birds and Mammals (e.g., Marsupials, Primates, Rodents and Cetaceans).

[0247] Vertebrates represent the overwhelming majority of the phylum Chordata, with currently about 66,000 species described. Vertebrates include the jawless fish and the jawed vertebrates, which include the cartilaginous fish (sharks, rays, and ratfish) and the bony fish. Still further, in some embodiments, the subject of the of the preset disclosure may be any one of a human or non-human mammal, an avian, an insect, a fish, an amphibian, a reptile, a crustacean, a crab, a lobster, a snail, a clam, an octopus, a starfish, a sea-urchin, jellyfish, and worms.

[0248] In more specific embodiments, the subject of the present disclosure may be a mammal. In yet some further embodiments, such mammalian organisms may include any member of the mammalian nineteen orders, specifically, Order Artiodactyla (even-toed hoofed animals), Order Carnivora (meat-eaters), Order Cetacea (whales and purpoises), Order Chiroptera (bats), Order Dermoptera (colugos or flying lemurs), Order Edentata (toothless mammals), Order Hyracoidae (hyraxes, dassies), Order Insectivora (insect-eaters), Order Lagomorpha (pikas, hares, and rabbits), Order Marsupialia (pouched animals), Order Monotremata (egg-laying mammals), Order Perissodactyla (odd-toed hoofed animals), Order Pholidata, Order Pinnipedia (seals and walruses), Order Primates (primates), Order Proboscidea (elephants), Order Rodentia (gnawing mammals), Order Sirenia (dugongs and manatees), Order Tubulidentata (aardvarks).

[0249] In yet some further embodiments, the present disclosure may be applicable for any organism of the order primates. More specifically, primates are divided into two distinct suborders, the first is the strepsirrhines that includes lemurs, galagos, and lorisids. The second is haplorhines - that includes tarsier, monkey, and ape clades, the last of these including humans. In yet some further embodiments, the present disclosure may be applicable for any organism of the subfamily Homininae, that includes the hylobatidae (gibbons) and the hominidae that includes ponqunae (orangutans) and homininae [gorillini (gorilla) and hominini ((panina(chimpanzees) and hominina (humans))]. Thus, in some embodiments, a subject as disclosed herein relates to a human subject. In some embodiments, the human subject may be of any sex, ethnic group, age or physical or mental condition.

[0250] In some specific embodiment, the present disclosure may be applicable for a mammal that may be at least one of a Cattle, domestic pig (swine, hog), sheep, horse, goat, alpaca, lama and Camels. More specifically, the subject the present disclosure as well as the methods disclosed herein above offer great economic advantage for any industrial or agricultural use of animals, specifically, livestock. Thus, in some specific embodiments, the present disclosure may be applicable for mammalian livestock, specifically those used for meat, milk and leather industries. Livestock are domesticated animals raised in an agricultural setting to produce labor and commodities such as meat, eggs, milk, fur, leather, and wool. The term includes but is not limited to Cattle, sheep, domestic pig (swine, hog), horse, goat, alpaca, lama and Camels. Of particular interest are cattle applicable in the meat and milk industry, as well as in the leather industry. More specifically, in certain embodiments, the subject of the present disclosure may be Cattle, colloquially cows, that are the most common type of large, domesticated ungulates, that belong to the Bovidae family.

[0251] In yet some further embodiments, the organism applicable in the methods of the present disclosure, may be avian organisms. In yet some further specific embodiments, the present disclosure may be suitable for birds. More specifically, domesticated and undomesticated birds are also suitable organisms for the present disclosure.

[0252] Therefore, in certain embodiments, the avian organism of the preset disclosure may be any one of a domesticated and an undomesticated bird. In more specific embodiment, the avian organism may be any one of a poultry or a game bird. In some specific embodiments, the avian organism may be of the order Galliformes which comprise without limitation, chicken, quail, turkey, duck, Gallinacea sp, goose, pheasant and other fowl. The term "avian" relates to any species derived from birds characterized by feathers, toothless beaked jaws, the laying of hard- shelled eggs, a high metabolic rate, a four-chambered heart, and a lightweight but strong skeleton. The term "hen" includes all females of the avian species.

[0253] It is to be understood that the terms "treat”, “treating”, “treatment" or forms thereof, as used herein, mean preventing, ameliorating or delaying the onset of one or more clinical indications of disease activity in a subject having a pathologic disorder. Treatment refers to therapeutic treatment. Those in need of treatment are subjects suffering from pathologic disorders. Specifically, providing a "preventive treatment" (to prevent) or a "prophylactic treatment" is acting in a protective manner, to defend against or prevent something, especially a condition or disease. The term “treatment or prevention” as used herein, refers to the complete range of therapeutically positive effects of administrating to a subject including inhibition, reduction of, alleviation of, and relief from, an immune-related condition and illness, immune-related symptoms or undesired side effects or immune-related disorders. More specifically, treatment or prevention of relapse or recurrence of the disease, includes the prevention or postponement of development of the disease, prevention or postponement of development of symptoms and / or a reduction in the severity of such symptoms that will or are expected to develop. These further include ameliorating existing symptoms, preventing- additional symptoms and ameliorating or preventing the underlying metabolic causes of symptoms. It should be appreciated that the terms "inhibition", "moderation", “reduction”, "decrease" or "attenuation" as referred to herein, relate to the retardation, restraining or reduction of a process by any one of about 1% to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%, 100% or more. With regards to the above, it is to be understood that, where provided, percentage values such as, for example, 10%, 50%, 120%, 500%, etc., are interchangeable with "fold change" values, i.e., 0.1, 0.5, 1.2, 5, etc., respectively. The term "amelioration" as referred to herein, relates to a decrease in the symptoms, and improvement in a subject's condition brought about by the methods according to the present disclosure, wherein said improvement may be manifested in the forms of inhibition of pathologic processes associated with the immune-related disorders described herein, a significant reduction in their magnitude, or an improvement in a diseased subject physiological state. The term "inhibit" and all variations of this term is intended to encompass the restriction or prohibition of the progress and exacerbation of pathologic symptoms or a pathologic process progress, said pathologic process symptoms or process are associated with.

[0254] The term "eliminate" relates to the substantial eradication or removal of the pathologic symptoms and possibly pathologic etiology, optionally, according to the methods of the present disclosure described herein. The terms "delay", "delaying the onset", "retard" and all variations thereof are intended to encompass the slowing of the progress and / or exacerbation of a disorder associated with the immune-related disorders and their symptoms slowing their progress, further exacerbation or development, so as to appear later than in the absence of the treatment according to the present disclosure. It should be noted that the terms "disease", "disorder", "condition" and "illness", are equally used herein. It should be appreciated that any of the methods described by the present disclosure may be applicable for treating and / or ameliorating any of the disorders disclosed herein or any condition associated therewith. It is understood that the interchangeably used terms "associated", “linked” and "related", when referring to pathologies herein, mean diseases, disorders, conditions, or any pathologies which at least one of: share causalities, co-exist at a higher than coincidental frequency, or where at least one disease, disorder condition or pathology causes the second disease, disorder, condition or pathology. More specifically, as used herein, “disease”, “disorder”, “condition”, “pathology” and the like, as they relate to a subject's health, are used interchangeably and have meanings ascribed to each and all of such terms. It is to be understood that the definitions provided herein for each of the terms used throughout the present disclosure are intended to apply uniformly to all aspects, embodiments, and examples described herein. Unless expressly indicated otherwise, each definition shall be construed as being relevant and applicable to each of the disclosed aspects, compositions, methods, and uses of the invention.

[0255] All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0256] The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range. Thus, as used herein the term "about" refers to ± 10 %. The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to". This term encompasses the terms "consisting of" and "consisting essentially of". The phrase "consisting essentially of" means that the composition or method may include additional ingredients and / or steps, and / or parts, but only if the additional ingredients and / or steps do not materially alter the basic and novel characteristics of the claimed composition or method. Throughout this specification and the Examples and claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0257] It should be noted that various embodiments of this invention may 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 sub ranges 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 sub ranges 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, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging / ranges between" a first indicate number and a second indicate number and "ranging / ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between. As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0258] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0259] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0260] Disclosed and described, it is to be understood that this invention is not limited to the particular examples, methods steps, and compositions disclosed herein as such methods steps and compositions may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

[0261] It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise.

[0262] EXAMPLES

[0263] Experimental Procedures

[0264] Computational analysis

[0265] The PhaseFinder algorithem was used to asses DNA inversions in commensal bacteria as described in Jiang et al. ( doi: 10.1126 / science.aau5238\ using the reference genome as described in Carraso et. Al. ( doi: 10.1016 / j.chom.2024.02.003).

[0266] Bacterial engineering - 'deletion method'

[0267] [Garcfa-Bayona, L. & Comstock, L. E. mBio 10 (2019). https: / / doi.org: 10.1128 / mBio.01762-19] Outline:

[0268] • Plasmid linearization - step la in Figure 2B

[0269] • Design and amplify down and up stream sections - step lb in Figure 2B

[0270] • Gibson (add the down and up stream sections and create circular plasmid) - step 2 in Figure 2B

[0271] • Transformation (electroporation / heat shock) - step 3, left side in Figure 2B

[0272] • Mating E. coli and target bacteria - step 3, right side in Figure 2B

[0273] • Remove E. coli - step 4 in Figure 2B

[0274] • Remove target bacteria with the wrong insertion - step 5 in Figure 2B

[0275] • Colonies selection - step 6 in Figure 2B

[0276] • Final checks - step 7 in Figure 2B

[0277] Requirements:

[0278] • E. co li SI 7kpir

[0279] • The target bacteria need to be Erythromycin resistant

[0280] • The target bacteria need to be Gentamycin sensitive

[0281] Workflow:

[0282] Plasmid linearization (step la in Fig. 2B):

[0283] A glycerol stock of a pEGB13 plasmid ( AddGene Plasmid #126618) prepared according to the manufacturer's instructions was linearized using the following primers of Table 1: A PCR was run directly on a colony

[0284] PCR reaction: o lOul K enzyme 2x ready mix o 0.5ul from pLGB 13-linR stock of lOuM o 0.5ul from pLGB 13-linF stock of lOuM o 5ul plasmid template o 4ul DNase RNase free DDW

[0285] PCR program: o 95°C 3min o (98°C 20 sec, 60°C 15 sec, 72°C 3min) *32 o 72°C 5min o 4°C indefinitely

[0286] The DNA was cleaned (for example using AMPure beads), send for sequencing and stored in - 20°C.

[0287] Amplification of down and upstream sections (step lb in Fig. 2B)

[0288] Specific 500-1000 bases upstream (US) and downstream (DS) to the target gene for deletion (in this case tyrosine invertase gene) were designed. For preparation of the US region, sequences containing a portion of the promoter region of UPxY gene of B. ovatus were used, wherein the promoter region was positioned either in the 'OFF' orientation or in the "ON orientation. More specifically, for preparing US region in an 'OFF' orientation, the primers of SEQ ID NO: 11 and 12 were used. For preparing a US region having the 'ON' orientation, the primers of SEQ ID NO: 11 and 13 were used. The DS region for the recombination plasmid used to delete the invertase gene, was prepared using primers of SEQ ID NO: 14 and 15, that amplify a region residing upstream of the invertase gene. Accordingly, the use of the above DS and US recognition arms in the deletion plasmid result in deletion of the entire invertase sequence and election of a desired 'ON' or 'OFF' orientation for the UpxY promoter.

[0289] PCR amplification was utilized to create the US and DS sequences using the target bacteria DNA as template (NCBI accession number NZ_CP012938.1 for B. ovatus), and the primers as discussed herein (Table 2). The resulting DS sequence is denoted by the nucleic acid sequence of SEQ NO: 16, the US of the 'OFF' orientation comprises the nucleic acid sequence as denoted by SEQ ID NO: 17, and the US of the 'ON' orientation comprises the nucleic acid sequence as denoted by SEQ ID NO: 18.

[0290] Table 2 Primers of the DS and US

[0291] *Note that for primers of SEQ ID NO: 14 and 15, when looking at the DNA sequence from the 5' end in Fig. 2 A, the order is: the UPxY gene, the invertase, and the target sequence.

[0292] PCR reaction: o lOul K enzyme 2x ready mix o 0.5ul from R primer stock of lOuM o 0.5ul from F primer stock of lOuM o lul of target bacterial template o 8ul DNase RNase free DDW

[0293] PCR program: o 95°C 3min o (98°C 20 sec, 68°C 15 sec, 72°C 30sec) *30 o 72°C Imin o 4°C indefinitely The DNA was cleaned (for example using AMPure beads), send for sequencing and stored in - 20°C.

[0294] Gibson (add the down and up stream and create circular plasmid) (step 2 in Fig. 2B)

[0295] Gibson reaction: o 15ul Gibson mix o lul DS (~10ng / ul) o lul US (~10ng / ul) o 3ul linear plasmid (~10ng / ul)

[0296] The reaction was placed at 50°C for 1 hour

[0297] The Gibson output was checked using the following primers of Table 3:

[0298] PCR program to test Gibson: o lOul K enzyme 2x ready mix o lul from R primer stock of lOuM o lul from F primer stock of lOuM o lul of Gibson output o 7ul DNase RNase free DDW

[0299] PCR program: o 95°C 3min o (98°C 20 sec, 60°C 15 sec, 72°C Imin) *30 o 72°C 3.5min o 4°C indefinitely

[0300] The Gibson output should be -2000 bases. If the Gibson did not work, the output should be only -150 bases.

[0301] Transformation (step 3, left side in Fig. 2B)

[0302] Electroporation'.

[0303] 1. Prepare competent cells for electroporation as described below.

[0304] 2. In a clean chilled Eppendorf, mix gently 49ul of competent cells and lul of DNA. - Il ¬i. Transfer the mixed sample into a chilled cuvette, and electroporate using BioRad machine set on 1.5Kv and ~15uF.

[0305] 4. Add 1ml of LB 37°C and incubate at 37°C for at least 2 hours.

[0306] 5. Transfer lOOul to LB+AMP plates and spread evenly on the plate.

[0307] 6. Incubate at 37°C for overnight (o / n).

[0308] Competent prep for electroporation:

[0309] 1. Shake 2mL E. coli at 37°C overnight.

[0310] 2. Add 200uL of the E. coli to 20mL of LB in 50ml falcon (twice), incubate under shaking at 37°C for 1.5 hours (OD600 of 0.6).

[0311] 3. Combine both batches and centrifuge for 10 min at 4000g 4°C.

[0312] 4. Dispose supernatant and resuspend in 10ml cold DDW on ice.

[0313] 5. Centrifuge for 10 min at 4000g 4°C.

[0314] 6. Dispose supernatants and resuspend in 10ml cold 10% glycerol in DDW on ice.

[0315] 7. Centrifuge for 10 min at 4000g 4°C.

[0316] 8. Dispose supernatants and resuspend in 10ml cold 10% glycerol in DDW on ice.

[0317] 9. Centrifuge for 10 min at 4000g 4°C.

[0318] 10. Dispose supernatants and resuspend in 1ml cold 10% glycerol in DDW on ice.

[0319] 11. Aliquot and store at -80°C until use.

[0320] Colony checking

[0321] To confirm the insertion of the plasmid and verify its identity, the same primers used in the Gibson PCR test were employed herein.

[0322] 3-4 bacterial colonies were chosen, and each grown in liquid LB+AMP for ~5h (until growth was seen), lul from the grown bacteria was added into lOOul of DDW and used as the DNA template of this colony.

[0323] PCR program to test Gibson: o 5ul K enzyme 2x ready mix o 0.5ul from R primer stock of lOuM o 0.5ul from F primer stock of lOuM o lul of DNA template o 3ul DNase RNase free DDW - 1 -

[0324] PCR program: o 95°C 3min o (98°C 20 sec, 60°C 15 sec, 72°C Imin) *30 o 72°C 2.5min o 4°C indefinitely

[0325] A gel was run to confirm the presence of a band of the correct size.

[0326] Mating E. coli and target bacteria (step 3, right side in Fig. 2B)

[0327] 1. Dilute 1: 10 of B. ovatus ATCC 8483 over-night (o / n) starter in 20ml Brain-Heart Infusion Medium (BHIS) and incubate at 37°C anaerobically until reaching 0.3-0.5 OD.

[0328] 2. Dilute 1:50 of E. coli Zpirl7 from starter (grown at 37°C for o / n) into LB+AMP and shake at 37°C aerobically until reaching 0.3-0.5 OD (~4h).

[0329] 3. Wash donor culture (E. coli) in 20ml BHI (centrifuge 5min, 4000 rpm).

[0330] 4. Combine 20 ml of donor bacteria (E. coli) and 20 ml of recipient bacteria (B. Ovatus ATCC 8483).

[0331] 5. Centrifuge for 5min at 4,500x g and discard supernatant

[0332] 6. Resuspend in BHIS 1 ml of the combined mixture and transfer into an Eppendorf tube.

[0333] 7. Centrifuge for 5min at 4,500x g and discard the supernatant

[0334] 8. Drop the remaining at the center of BHIS plate and let dry.

[0335] 9. Flip the plate and incubate aerobically 37°C for overnight.

[0336] Remove E. coli + Remove target bacteria with the wrong insertion (step 4 in Fig. 2B)

[0337] Elimination of E. coli and target bacteria lacking the plasmid is achieved using Gentamycin and Erythromycin (GE) on BHIS plates. Gentamycin removes E. coli, while Erythromycin selects for plasmid integration in Bacteroides.

[0338] 1. Prepare Gentamycin Erythromycin (GE) plates with 200ug / ml Gentamycin and 15ug / ml Erythromycin.

[0339] 2. Using a swab take a little bit of the central dot from a mating colony and streak on the entire plate evenly.

[0340] 3. Using a swab take a large portion of the central dot and streak on the entire plate evenly - do this on total of 5 plates and use as much of the mating central dot as possible

[0341] 4. Incubate for at least 48h at 37°C, anaerobically 5. If colonies have grown, streak 4-8 colonies on GE plates.

[0342] 6. Incubate at 37°C for 48h anaerobically.

[0343] Remove wrong insertion (step 5 in Fig. 2B)

[0344] In this step, the grown colonies are allowed to grow further to ensure recovery, giving the bacteria an opportunity to discard the plasmid (following the deletion). Afterward, selection is performed to identify bacteria that contain the correct insert or no insert at all.

[0345] 1. Grow 4-8 colonies in 3ml liquid BHIS each, for o / n at 37°C anaerobically.

[0346] 2. Dilute 10’1, 10'2, 10'3in Eppendorf tubes, then plate lOOul of the non-diluted and each dilution on aTCioo plates (O.lug / ml anhydrotetracyclin (aTC) plates in BHIS), spread the lOOul well using hockey stick spreader.

[0347] 3. Incubate at 37°C for 48h anaerobically.

[0348] 4. Choose 8 colonies and plate on aTCioo plates.

[0349] 5. Incubate at 37°C for 48h anaerobically.

[0350] Colonies selection and ensuring correct insert (step 6 in Fig. 2B)

[0351] This step ensures selection of colonies that have lost the gene without reverting to the wild type. A PCR using the DS R and the US F primer is conducted - if the gene was deleted, a smaller PCR product should be detected.

[0352] Final checks (step 7 in Fig. 2B)

[0353] An additional PCR and full sequencing are performed to confirm that the deletion is in the desired region. qPCR mutation test

[0354] This test was done to verify the 'ON' / 'OFF' orientation of the promoter region of UPxY gene of B. ovatus.

[0355] Table 4: Primers: * The promoter region of UPxY gene of B. ovatus in the 'OFF' orientation

[0356] ** The promoter region of UPxY gene of B. ovatus in the 'ON' orientation *** Down stream gene.

[0357] Reaction mix: o lOul SG (from @Luna) o lul F o lul R o lul sample o 7ul water

[0358] PCR program: o 95°C 20 sec o (95°C 1 sec, 60°C 20 sec) *40 o 95°C Isec o 60°C 20 sec o 95°C Isec

[0359] No reaction was observed with bacteria different than B. ovatus or using feces sample without B. ovatus, demonstrating the specificity of primers.

[0360] Bacterial engineering - 'scrambled method'

[0361] Bacterial engineering using the 'scrambled method' was performed in the promoter of the UPxY gene, similarly to the 'deletion method' described above and illustrated in Figure 2B, except for the following modifications: To scramble the gene regions, standard amplification was not used as in the deletion approach done in step lb of Fig. 2B. Instead, the inventors designed and ordered synthetic DNA sequences, denoted as SEQ ID NO: 21 and 22, corresponding to the 'OFF' and 'ON' orientations, respectively. These sequences were incorporated as the inserted regions in the plasmid construct.

[0362] Additionally, for the final verification step (Step 7 in Fig. 2B), a quantitative PCR (qPCR) was employed using the following primers: Table 5: primers

[0363] * The promoter region of UPxY gene of B. ovatus in the 'OFF' orientation with the reverse in the scrambled area

[0364] ** The promoter region of UPxY gene of B. ovatus in the 'ON' orientation with the reverse in the scrambled area

[0365] RNA expression of the UPxY gene

[0366] Trizol reagent was used to extract RNA from B. ovatus and its mutants according to the manufacturer's instructions (Thermo). Briefly, the bacteria were grown in liquid media (BHIS) to log phase (0.5-0.8 OD600), subjected to centrifugation (10,000 xG for 5 minutes) and the RNA was extracted from the resuspended pellet. DNAse was used to remove DNA (NEB). Then cDNA was generated using Quantbio kit according to the manufacturer's instructions. qPCR was performed on the cDNA using the below primers. To quantify UPxY gene expression, gyrA used as a reference gene (where its expression level is not affected by the engineering) using the primers mentioned in table 6 Following qPCR, delta Ct was calculated for each mutant (delta Ct = UpxYct value - gyrAct value), then delta delta Ct was calculated for the change in gene expression between the mutants (Gene expression between the mutants = On mutant delta Ct value - Off mutant delta Ct value)

[0367] Table 6: Primers:

[0368] Kinetics test -

[0369] To test the mutant bacterial growth rate, an overnight (o / n) starter was used, and the bacteria were grown in basal and BHIS media for 24 hours at 37°C. The similarity in the following parameters were checked for all mutants: delay to start the log phase, slop and OD of stationary phase (Fig. 3).

[0370] Also tested was the OD to CFU of the mutants and WT by sampling bacterial media every hour and plating in different concentration to find the CFU. The resulting curves are presented in Fig.

[0371] 8.

[0372] Biofilm formation test

[0373] A crystal violate (CV) protocol was used to test biofilm formation in basal and YCFA media [Xu, T. el al. Characterization of Mixed-Species Biofilms Formed by Four Gut Microbiota. Microorganisms 10, 2332 (2022)].

[0374] The bacteria were grown in basal media or YCFA media for O / N and the cells were washed so only the biofilm is left in the well. The biofilm was then fixed and rewashed. 0.5% CV was then added, washed and let dry. Then the color was eluted, and its intensity measured (Fig. 4). The measured intensity of each mutant was normalized to mean of WT.

[0375] Splenocyte effect in vitro

[0376] Freshly harvested spleens from 6-10 weeks mice were used. The immune cells were isolated by lysing the blood cells using Ammonium-Chloride-Potassium (ACK) lysis buffer for 1 minute. The cells were then washed twice and counted. lxlO 6 cells in 1ml RPMI were seeded in each well and treated as follows:

[0377] Treatment with only anti-CD3, LPS, BHIS (controls).

[0378] Treatment with anti-CD3, LPS, BHIS and addition of fixed bacteria in log phase (wild type, mutants in 'ON' orientation or mutants in 'OFF' orientation).

[0379] Fixing of the bacteria was done in 4% paraformaldehyde (PFA) for Ih followed by washing. Fixed bacteria were added at a ratio of 100: 1 (100 fixed bacteria to 1 immune cell). At these conditions, the immune cells will multiply while the bacteria are fixed.

[0380] After 24h of treatment, 200ul of the media was removed to test for cytokines and replaced with fresh media. After 4 days (from start) media was removed again for cytokine test and the cells growth was stopped.

[0381] TNF-a, IL-6 and IFN-g were tested after 24-hours treatment using the following kits: ELISA MAX™ Standard Set Mouse TNF-alpha (BLG-430901)

[0382] Mouse IL-6 ELISA MAX™ Standard (BLG-431303)

[0383] ELISA MAX™ Standard Set Mouse IFN-g (BLG-430801)

[0384] IL- 17 and IL- 10 were tested after 4 days treatment using the following kits:

[0385] - Mouse IL-17A ELISA MAX™ Standard (BLG-432501)

[0386] - Mouse IL-10 ELISA MAX™ Standard (BLG-431411)

[0387] Monocolonization of germ-free (GF) mice

[0388] Germ-free (GF) mice were monocolonized once at 4 weeks with lOOul of o / n grown bacteria. The mice were then kept in sterile conditions for 14 days and sacrificed (after verifying they were not contaminated with other bacteria). Immune phenotype was tested using FACS (Fig. 6), and the CFU of the bacteria was tested in the mice feces, cecum, and small intestine (Fig. 7 and 9).

[0389] Immune phenotype using FACS

[0390] The tumor draining lymph node was extracted after sacrificing the mice. The lymph node was then cleaned from the fat tissue surrounding it and a 40um strainer was used to obtain single cells. Cells were stained for viability and immune markers using the antibodies indicated in the table below. Cells were stained for extracellular markers and then for intracellular markers using foxp3 staining buffers according to the manufacturer's instructions (eBio science). Following staining and washing steps, cells were analyzed using symphony s2 [Becton, Dickinson - (BD)] flow cytometry machine. Acquired data was analyzed using FlowJO software (BD).

[0391] The following antibody panel was used:

[0392] Table 7:

[0393] Colony Forming Units (CFU) test

[0394] Bacteria were serially diluted from 10A-4 to 10A-8 in growth media (BHIS) and lOul of each dilution was seeded on BHIS-agar plate. Following 24h incubation at 37°C under anaerobic conditions the number of colonies were counted in each dilution, and the average number of bacteria per ml was calculated.

[0395] CFU-OD curve

[0396] At hourly intervals, samples were collected to measure both optical density (OD) at 600 nm and colony-forming units (CFU). The corresponding OD readings were recorded for each time point. The CFU test was performed as described above per each time point. Based on the collected data, a correlation formula was generated to accurately represent the bacterial growth curve. This formula enables estimation of bacterial concentration (CFU / mL) from OD measurements alone.

[0397] Cancer model

[0398] Six weeks old mice were administered orally (200ul of bacteria or media only for the SPF) for 3 consecutive days. After a week from the first gavage, YUMM1.7 (500,000 cells / mouse) cell line was injected to the flank. Priore to injection, cells were propagated in DMEM media. After 5 days from tumor injection mice treated with anti-PDl (250ug / mouse, IP injection, twice, two days interval) as well as oral gavage with the bacteria (200ul cultured bacteria). At day 10 from tumor injection, mice were sacrificed, tumor weights were recorded and draining lymph nodes were analyzed using flow cytometry immune characterization. We used flowJO software to analyze and gate the cells by immune populations.

[0399] EXAMPLE 1

[0400] Design and preparation of "locked" mutants

[0401] The inventors used published databases of patients before commencing cancer immunotherapy and correlated the bacterial composition and genome phase state to immunotherapy response status.

[0402] Genome DNA inversion state is defined as the orientation of various regions along the bacterial genome potentially indicating bacterial functionality. A computational analysis revealed that Bacteroides Ovatus UPxY gene promoter phase, turns significantly 'ON' in responders, in comparison to non-responders (Fig. 1). Following this finding, the inventors sought to engineer the bacteria to become “locked” in each of the DNA inversions orientations. To do so, the inventors used a previously published plasmid to eliminate the bacteria’s ability to inverse this promotor area (pLGB13 plasmid published by Comstock et al., [mBio. (2019 Aug 13); 10(4):e01762-19. doi: 10.1128 / mBio.01762-19.]. Graphical illustration of the process and plasmid design is presented in Fig. 2A-2B. Editing the bacterial gene produces a mutant strain where the UPxY gene is either permanently turned 'ON' or 'OFF', resulting in the gene being either constantly expressed or never expressed. Manipulation of the bacterial genomic region to lock the UPxY gene in a permanently 'ON' or 'OFF' orientation was achieved either by knocking out the tyrosine recombinase gene (named as 'deletion method') or by scrambling the recombinase binding sites within the promoter region of the UPxY gene (named as 'scrambled methos'), as demonstrated in Fig. 10.

[0403] It should be noted that the experiments shown in Figures 3-9 were performed using mutants generated by the 'deletion method', whereas those presented in Figures 11-16 were conducted using mutants generated by the 'scrambling method'. EXAMPLE 2

[0404] In vitro and ex vivo characterization of engineering B. ovatus generated by knocking out the tyrosine recombinase gene

[0405] Fig. 3 shows that the locked orientation (either 'ON' or 'OFF') (using the 'deletion method'), does not affect the growth of the mutants.

[0406] The various mutants were next characterized in vitro, and the effect of the mutated bacteria on mammalian splenocytes was examined ex vivo. Testing the phenotypic effect of the introduced mutations showed significant reduction in biofilm formation for the locked 'ON' mutants (Fig. 4). Introducing the mutant bacteria to immune cells in vitro demonstrated an effect on IL-6, IL- 10 and TNF-a cytokine secretion following immune stimulation, showing that the locked 'OFF' mutant cause less immune stimulation (Fig. 5). These results establish the feasibility of using the mutants for immune modulation.

[0407] EXAMPLE 3

[0408] In vivo characterization of engineering B. ovatus generated by knocking out the tyrosine recombinase gene

[0409] The inventors next evaluated in vivo by monocolonizing germ free (GF) mice with the various mutants. The inventors observed that locked 'OFF' and locked 'ON' mutants activate the immune system differently (Fig. 6). The locked 'OFF' induced more regulatory T cell (Treg) (more CD4+ foxp3+) and more PDL-1+ expressing cells (Fig. 6B), both suggest less immune activation and that can lead to better conditions to tumor growth and worse conditions for response as well as better immune state for immune mediated diseases such as IBD and autoimmune diseases.

[0410] The bacteria colonize the cecum and colon (feces) more than the small intestine (SI) and there is no difference between the 'ON' and 'OFF' mutants (Fig. 7, 9).

[0411] No changes in bacterial growth rates were observed (Fig. 3, 8).

[0412] EXAMPLE 4

[0413] In vitro and ex vivo characterization of engineering B. ovatus generated using scrambled invertase recognition sequences of the UPxY promoter

[0414] To assess the effect of B. ovatus genome modification on gene expression, we followed gene expression using reverse transcriptase real time qPCR. The engineered bacteria for locked “OFF” mutants show lower UPxY RNA expression compared to the WT or locked “ON” mutant, and found to be similar in both methods of engineering (Fig. 11). Engineered bacteria for “ON” mutant show 4-8 fold higher UPxY RNA expression of the mutants created by both methods, "scrambled" and "deletion " (Fig. 11). The results demonstrate the agreement between bacterial genome modifications and UPxY RNA expression.

[0415] EXAMPLE 5

[0416] In vivo Effect of engineering B. ovatus generated using scrambled invertase recognition sequences on tumor growth and immune recruitment

[0417] To investigate the effect of B. ovatus on tumor model, C57BL / 6 SPF mice were colonized with the engineered bacterial mutants, followed by subcutaneous injection of the YUMM1.7 melanoma cell line into the flanks to induce tumor formation (Fig. 12). Tumor size and immune cell population in the tumor draining lymph node were monitored over time. A significant reduction in tumor weight was observed in mice colonized with the B. ovatus strain engineered to lock the target locus in the 'ON' orientation, compared to those colonized with the 'OFF' orientation strain (p = 0.046; Fig. 13).

[0418] Immune profiling of the draining lymph nodes by flow cytometry revealed a significant enrichment of CDl lb+cells (Fig. 14) and macrophages (Fig. 15) in mice colonized with the 'ON' strain, relative to both the 'OFF' mutant and the 'SPF' control group. Additionally, mice colonized with the 'OFF' strain exhibited a reduction in CD4+natural killer (NK) cells (Fig. 16).

[0419] These findings identify immune and tumor-related changes associated with the orientation of the engineered locus and support the conclusion that supplementation with appropriately engineered B. ovatus strains may enhance antitumor immune responses and improve the efficacy of immunotherapy .

Claims

CLAIMS:

1. A genetically engineered bacterium, locked in a desired orientation in at least one genomic locus displaying phase variation, or a population comprising said bacterium, wherein said bacterium comprises at least one modification in a target nucleic acid sequence that comprises, comprised within, or encodes at least one element that induces directly or indirectly phase variation in said at least one genomic locus.

2. The genetically engineered bacterium according to claim 1, wherein said phase variation in said genomic locus is associated with at least one physiological state and / or condition in a subject.

3. The genetically engineered bacterium according to claim 2, wherein said physiological state and / or condition comprises at least one of: an immunological state and / or condition, a metabolic state, diet, behavioral / mental state and / or condition in said subject.

4. The genetically engineered bacterium according to claim 3, wherein said immunological state and / or condition comprises and / or reflects at least one immune-related disorder in said subject and / or an immune-response of said subject to said immune-related disorder.

5. The genetically engineered bacterium according to claim 4, wherein said immune-related disorder comprises at least one of: a proliferative disorder, an inflammatory disorder, an infectious disease, an autoimmune disorder, an immune-deficiency condition, a neurodegenerative and / or cognitive and / or mental disorder, a metabolic disorder, and a condition involving at least one wound in at least one tissue and / or organ of said subject.

6. The genetically engineered bacterium according to claim 5, wherein said proliferative disorder is at least one malignant neoplastic disorder.

7. The genetically engineered bacterium according to claim 6, wherein said malignant neoplastic disorder comprises melanoma.

8. The genetically engineered bacterium according to claim 5, wherein said inflammatory disorder comprises inflammatory bowel disease (IBD).

9. The genetically engineered bacterium according to any one of claims 3 to 8, wherein said immune-response of said subject comprises responsiveness of the subject to at least one therapeutic compound and / or therapeutic regimen.

10. The genetically engineered bacterium according to any one of claims 1 to 9, wherein said bacterium belongs to at least one bacterial species residing within at least one microbiome community of said subject.

11. The genetically engineered bacterium according to claim 10, wherein said microbiome is a gut microbiome.

12. The genetically engineered bacterium according to any one of claims 1 to 11, wherein said bacteria comprise at least one bacterium of at least one phylum selected from Bacteroidota, Verrucomicrobiota, proteobacteria, actinobacteria, firmicutes and Tenericutes.

13. The genetically engineered bacterium according to claim 12, wherein said Bacteroidota bacterium is of the genus of at least one of: Bacteroides, Bacteroidia, Bacteroidales, Bacteroidaceae and Phocaeicola.

14. The genetically engineered bacterium according to any one of claims 1 to 13, wherein said bacteria comprises Bacteroides ovatus or any isolate or species thereof.

15. The genetically engineered bacterium according to any one of claims 1 to 14, wherein said genomic locus comprises at least one intergenic region / s and / or intragenic region / s.

16. The genetically engineered bacterium according to any one of claims 1 to 15, wherein said genomic locus comprises nucleic acid sequence / s encoding and / or regulating at least one bacterial outersurface molecule and / or bacterial internal molecule and / or at least one molecule that modifies or regulates said at least one outersurface and / or internal molecule / s.

17. The genetically engineered bacterium according to claim 16, wherein said bacterial outersurface molecule comprises capsular polysaccharides (CPS) and wherein said at least molecule that modifies or regulates said at least one outersurface and / or internal molecule / s comprises at least one member of UpxY family transcription antiterminator.

18. The genetically engineered bacterium according to any one of claims 1 to 17, wherein said locus is the UpxY locus, and wherein phase variation in the promoter region of said UpxY locus is associated with responsiveness or irresponsiveness of a subject suffering from melanoma to at least one immune-checkpoint inhibitor.

19. The genetically engineered bacterium according to any one of claims 1 to 18, wherein said phase variation comprises at least one of: DNA inversion, DNA recombination, transposition mechanism, slipped strand mispairings (SSM) and phase variation via differential methylation.

20. The genetically engineered bacterium according to any one of claims 1 to 19, wherein said phase variation comprises DNA inversion in at least one promoter region of at least one genomic locus, thereby converting the ON / OFF orientation of said at least one promoter region / s.

21. The genetically engineered bacterium according to any one of claims 1 to 20, wherein said element that induces directly or indirectly phase variation in said at least one genomic locus comprises at least one of: (i) at least one DNA invertase; and / or (ii) at least one nucleic acid sequence targeted by, subjected to inversion, and / or recognized by at least one DNA invertase.

22. The genetically engineered bacterium according to claim 21, wherein said DNA invertase comprises at least one of a tyrosine site-specific recombinase (Tsrs) and a serine site-specific recombinase (Ssr).

23. The genetically engineered bacterium according to any one of claims 1 to 22, wherein said target nucleic acid sequence:(i) encodes at least one of said DNA invertase; or(ii) comprises or comprised within at least one nucleic acid sequence targeted by, subjected to inversion, and / or recognized by at least one DNA invertase.

24. The genetically engineered bacterium according to any one of claims 1 to 20, wherein said modification comprises at least one of deletion, insertion, replacement, inversion, point mutation, rearrangement in said target nucleic acid sequence.

25. The genetically engineered bacterium according to claim 24, wherein deletion and / or point mutation in the target nucleic acid sequence locks the adjacent genomic locus in a desired orientation.

26. The genetically engineered bacterium according to any one of claims 1 to 25, wherein said genomic locus comprises the UpxY locus, wherein said target nucleic acid sequence encodes or comprised within a sequence encoding at least one invertase located in close proximity to said UpxY locus, and wherein deletion in said target nucleic sequence that encodes or comprised within a sequence encoding said invertase, locks the promoter of said UpxY locus in a desired orientation.

27. The genetically engineered bacterium according to any one of claims 1 to 26, wherein said genomic locus comprises the UpxY locus, wherein said target nucleic acid sequence resides within the UpxY locus and comprises and / or is comprised within a nucleic acid sequence targeted by, subjected to inversion, and / or recognized by at least one DNA invertase, and wherein at least one mutation in said target nucleic sequence, locks the promoter of said UpxY locus in a desired orientation.

28. The genetically engineered bacterium according to any one of claims 26 and 27, wherein said desired orientation is an ON orientation of the promoter region of said UpxY locus.

29. The genetically engineered bacterium according to any one of claims 1 to 28, wherein ON orientation of the promoter region of said UpxY is associated with responsiveness of a subject to immunotherapy .

30. A composition comprising a genetically engineered bacterium, locked in a desired orientation in at least one genomic locus displaying phase variation, or a population comprising said bacterium, wherein said bacterium comprises at least one modification in a target nucleic acid sequence that comprises, comprised within or encodes at least one element that induces directly or indirectly phase variation in said at least one genomic locus, said composition further comprising at least one of acceptable carrier / s, diluent / s, excipient / s and additive / s.

31. The composition according to claim 30, wherein said composition is a probiotic composition.

32. The composition according to claim 30, wherein said composition is a pharmaceutical composition.

33. The composition according to any one of claims 30 to 32, wherein said genetically engineered bacterium is as defined in any one of claims 1 to 29.

34. A method for modulating an immune response in a subject in need thereof, the method comprising the step of administrating to said subject at least one genetically engineered bacterium, locked in a desired orientation in at least one genomic locus displaying phase variation, a population comprising said bacterium, or any composition thereof, wherein said bacterium comprises at least one modification in a target nucleic acid sequence that comprises, comprised within or encodes at least one element that induces directly or indirectly phase variation in said at least one genomic locus.

35. The method according to claim 34, wherein said subject is suffering from at least one immune-related disorder.

36. The method according to claim 35, wherein said immune-related disorder is at least one of: a proliferative disorder, an inflammatory disorder, an autoimmune disorder, an immune- deficiency condition and / or an infectious disease.

37. The method according to any one of claims 34 to 36, wherein said genetically engineered bacteria is as defined in any one of claims 1 to 29.

38. The method according to any one of claims 34 to 37, wherein said modulation of an immune response comprises improving responsiveness of said subject to at least one therapeutic compound or therapeutic regimen.

39. The method according to claim 38, wherein said subject is a subject suffering from melanoma, wherein said bacterium is engineered to lock the promoter region of the UpxY locus in an ON orientation, and wherein the subject is treated with at least one immune check point inhibitor.

40. A method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one pathologic disorder in a subject, the method comprising the steps of administrating to said subject at least one genetically engineered bacterium, locked in a desired orientation in at least one genomic locus displaying phase variation, or a population comprising said bacterium, wherein said bacterium comprises at least one modification in a target nucleic acid sequence that comprises, comprised within, or encodes at least one element that induces directly or indirectly phase variation in said at least one genomic locus.

41. The method according to claim 40, wherein said pathologic disorder is at least one immune-related disorder.

42. The method according to claim 41, wherein said immune-related disorder is at least one of: a proliferative disorder, an inflammatory disorder, an autoimmune disorder, an immune- deficiency condition and / or an infectious disease.

43. The method according to claim 42, wherein said genetically engineered bacteria is as defined in any one of claims 1 to 29.

44. The method according to any one of claims 40 to 43, wherein said subject is suffering from melanoma, and wherein said bacterium is engineered to lock the promoter region of the UpxY locus in an ON orientation.

45. The method according to claim 44, wherein said subject is further treated with at least one check point inhibitor.

46. A method for the preparation of an immunomodulatory composition comprising the step of:(a) genetically modifying at least one target nucleic acid sequence in a bacterium, said target sequence comprises, is comprised within, or encodes at least one element that induces directly or indirectly phase variation in at least one genomic locus displaying phase variation, wherein said modification results in locking the genomic locus in a desired orientation;(b) admixing a bacterium locked in a desired orientation of said genomic locus obtained by step (a), with at least one of carrier / s, diluent / s, excipient / s and additive / s.

47. The method according to claim 46, wherein said phase variation in said genomic locus is associated with at least one physiological state and / or condition in said subject.

48. The method according to any one of claims 46 and 47, wherein said bacterium is of an autologous source.

49. The method according to claim 48, wherein said modification is performed ex vivo.

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