EP4486898A2 - Genetically modified bacterium including a lysis gene - Google Patents

Abstract

A genetically modified bacterium comprising a lysis gene. The lysis gene includes a nucleic acid encoding for a protein, the expression of which results in lysis of the bacterium, wherein the nucleic acid expresses the protein in response to exposure to radiation. The bacterium may be genetically modified via the insertion of a plasmid that includes a nucleic acid encoding the lysis gene, a nucleic acid encoding a first promoter for initiating transcription of the lysis gene, a nucleic acid encoding a biotherapeutic, and a nucleic acid encoding a second promoter for initiating transcription of the biotherapeutic.

Description

GENETICALLY MODIFIED BACTERIUM INCLUDING A LYSIS GENE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is an international (PCT) application that claims priority to, and the benefit of the filing date of, U.S. Patent Application Serial No. 63/315,512, filed on March 1, 2022, the disclosure of which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] Aspects of the present invention relate generally to systems and methods for delivery of therapeutics into a subject for treatment of various diseases and conditions.

BACKGROUND OF THE INVENTION

[0003] This section is intended to introduce the reader to various aspects of art that may be related to various aspects of the present invention, which are described and/or claimed below. This discussion is believed to be helpful in providing the reader with background information to facilitate a better understanding of various aspects of the present invention. Accordingly, it should be understood that these statements are to be read in this light, and not as admissions of prior art.

[0004] Programmed microbiome-based strategies for treatment of various diseases and conditions have shown promise in addressing several problems related to targeted delivery of therapeutics to a disease site (such as the delivery of therapeutic proteins to tumor tissue). One particular example of a microbiome-based strategy is the use of bacteria as a vessel for targeted cancer therapy. Bacteria can reduce issues seen with conventional cancer treatments (such as chemotherapy - which relies on passive diffusion of a drug, and can cause substantial unwanted side effects). Bacteria, however, can be modified to produce therapeutic proteins, and then deliver these proteins (their “cargo”) directly to a tumor site.

[0005] However, concerns related to local control of cargo release (e.g., release of therapeutic proteins) from the engineered bacteria and overall safety in vivo remain. Improving local control of cargo release might have potential to enhance therapeutic efficacy and survival outcomes. Ability to control a local population of the bacteria (once introduced into a subject) can also address concerns related to uncontrolled growth of bacteria, which can lead to infection and sepsis. Currently, many strategies are under investigation to improve local tumor control. However, an effective solution to these issues has not yet been obtained. SUMMARY OF THE INVENTION

[0006] Certain exemplary aspects of the invention are set forth below. It should be understood that these aspects are presented merely to provide the reader with a brief summary of certain forms the invention might take and that these aspects are not intended to limit the scope of the invention. Indeed, the invention may encompass a variety of aspects that may not be explicitly set forth below.

[0007] As described above, certain drawbacks exist when using bacteria as a vessel for delivery of a therapeutic to the site of a disease or condition. Aspects of the present invention, however, address these concerns by providing strategies that combine existing therapeutic modalities such as ionizing radiation (IR) with microbiome therapy in order to gain from the synergistic potential between the two modalities. This coupling is particularly promising as genes that encode for therapeutic proteins can be synced with genes that are sensitive to IR and can be programmed in the microbiome to act synchronously.

[0008] And so, one aspect of the present invention is directed to a genetically modified bacterium comprising a lysis gene. The lysis gene includes a nucleic acid encoding for a protein, the expression of which results in lysis of the bacterium. Further, the nucleic acid expresses the protein in response to exposure to radiation.

[0009] Another aspect of the present invention is directed to a plasmid for insertion into a bacterium, the plasmid comprising a nucleic acid encoding a lysis gene, a nucleic acid encoding a first promoter, a nucleic acid encoding a biotherapeutic molecule, and a nucleic acid encoding a second promoter. In this aspect of the present invention, the nucleic acid encoding a lysis gene includes a sequence encoding a protein, the expression of which results in lysis of a bacterium, that has had the plasmid inserted thereinto. Further, the nucleic acid expresses the protein in response to exposure to radiation. The plasmid of this aspect of the present invention may be used to genetically modify a bacterium into a bacterium including a lysis gene that expresses an encoded protein upon exposure to radiation (such as the bacterium of the first aspect of the invention listed above).

[0010] Various embodiments of the present invention, then, are directed to on-site biotherapeutic delivery systems for treatment of various diseases, conditions, etc. (e.g., cancer). In one particular embodiment, the present inventors programmed a probiotic bacterium - E. coli Nissle 1917 - that delivers a biotherapeutic in a controlled manner via an engineered plasmid: RecRadePOP (3884bp). The RecRadePOP plasmid sequentially harbors RecA-Lysis- RecA(ALexA)-PD-Ll-TEV-Strep-Flag-geneblock that works in tandem. RecA is a repressible RecA promoter, which the present inventors have previously shown responds to radiation, which in turn drives the expression of the downstream fused gene/protein. “Lysis” is a lytic gene, and the present inventors have previously reported the expression of Lysis gene in response to radiation in vitro and in vivo, which leads to bacterial cell lysis. Next, is a modified RecA promoter “RecA(ALexA)” - i.e., a RecA promoter that has the LexA repressor binding site deleted, making this a constitutive promoter, which reportedly expresses 600 times more protein than an inducible pBAD promoter. A biotherapeutic molecule (e.g., an anti-PD-Ll nanobody) is then fused downstream of the constitutive RecA (ALexA) promoter. Tn an embodiment using an anti-PD-Ll nanobody, the nanobody peptide is attached with a TEV cleavage site from Tobacco Etch Virus for peptide and protein tag separation, as the TEV site is, in turn, fused with Flag and Strep II tags for detection and purification. The RecRadePOP plasmid is designed in such a way that following protein expression, RecA-Lysis and RecA (ALexA)-PD-Ll peptides are isolated by means of introducing rmB T1 and T7T1 Terminators. The design is meant to constitutively express the anti-PD-Ll nanobody that will only be delivered when the bacterial cell lysis occurs, while the RecA promoter is meant to expresses the protein of the Lysis gene only after radiation exposure (because radiation activates the RecA promoter driving the expression of Lysis protein). It will be recognized that other biotherapeutics may be substituted for the anti-PD-Ll nanobody discussed above.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and, together with the general description of the invention given above, and the detailed description given below, serve to explain the principles of the invention. Similar reference numerals are used to indicate similar features throughout the various figures of the drawings.

[0012] FIG 1 is a schematic showing a strategy to utilize a RecRadePOP plasmid in conjunction with IR.

[0013] FIG. 2 is a schematic showing the construction of a genetic circuit for controlled lysis. The figure shows a hijack of natural phenomenon by (1) a RecA promoter and (2)a $x 174 E gene from bacteriophage (a Lysis gene). Basal expression of RecA improves when induced about 20-fold (SOS response). Basal expression is useful for recombination, but high expression is preferable for SOS response. The promoter being used in this figure is that of the RecA gene of E.coli. [0014] FIG. 3 is a schematic showing an embodiment of the experimental design including an engineered bacterium having both the RecA promoter and the Lysis gene present (A control has either or both of the promoter or Lysis gene absent, which results in no cell lysis of bacterial cells following radiation.

[0015] FIGS. 4 A, 4B, and 4C are photographs showing the design construction of the RecA- Lysis-ePOP plasmid. FIG. 4A shows PCR amplification of the RecA-Lysis gene for cloning into an ePOP plasmid. FIG. 4B shows restriction digestion of a pCN56 vector and the amplified ‘RecA-lysis’ insert for cloning. FIG. 4C shows a restriction digest with SalI_KpnI.

[0016] FIG. 5 is a photograph of PCR products showing preliminary confirmation of cloning via screening with Colony PCR. The circled bands of ~650bp are expected results, while the circled bands having a size between ~1.0kB and ~1.5kB were unexpected.

[0017] FIG. 6 is a photograph of PCR products showing preliminary confirmation of cloning via screening with Colony PCR. The expected product size is ~500bp, and so the circled bands were subjected to Sanger Sequencing.

[0018] FIG. 7 shows final confirmation of the nucleotide sequence obtained via Sanger Sequencing for one of the particular colonies (Colony 14R) previously screened with Colony PCR.

[0019] FIG. 8 shows final confirmation of the nucleotide sequence obtained via Sanger Sequencing for one of the particular colonies (Colony 14F) previously screened with Colony PCR.

[0020] FIG. 9 shows conventional NCBI-BLAST searches (for the nucleotide sequences obtained for Colony 14R and Colony 14F) to determine regions of similarity (percent identity) between those sequences and that for the insert.

[0021] FIG. 10 is a plasmid map for the RecA-Lysis-ePOP plasmid.

[0022] FIG. 11 is a table showing the ratio of 4Gy exposed bacteria to unexposed bacteria to be used in a LIVE/DEAD™ Assay (via a LIVE/DEAD™ assay kit available from Invitrogen). [0023] FIG. 12 is an image of plate reader results. 100 pl each of the exposed (4Gy) and unexposed bacterial cultures from the mix of FIG. 11 were added with 100 pl of the bacterial LIVE/DEAD™ assay solution and fluorescence was measured immediately at 485, 520 and 485, 640 nm using FLUOstar (Polarstar®, BMG) plate reader.

[0024] FIGS. 1 A-F are a series of graphs showing fluorescence at 485nm, 520nm at various radiation doses (unexposed, 0.5Gy, l.OGy, 2.0Gy, 4.0Gy, 8.0Gy, 16.0Gy, and 25.0Gy) for various populations of bacteria. FIG. 13A shows fluorescence of RecA-Lysis-ePOP-EcN for the various radiation doses. FIG. 13B shows fluorescence of RecA-Lysis-ePOP-DH5a for the various radiation doses. FIG. 13C shows fluorescence of ePOP-EcN for the various radiation doses. FIG. 13D shows fluorescence of ePOP-DH5a for the various radiation doses. FIG. 13E shows fluorescence of EcN for the various radiation doses. FIG. 13F shows fluorescence of DH5a for the various radiation doses. Thus, FIGS. 13A and 13B show the results for bacteria being including an ePOP plasmid that includes the RecA promoter and the lysis gene (13 A being the probiotic Nissle 1917 strain of E. coli, and 13B being the DH5a strain of E. coll). FIGS. 13C-13F thus are controls as none of those include the promoter or lysis gene.

[0025] FIGS. 14A-F are a series of graphs showing fluorescence at 485nm, 520nm at a 4Gy radiation dose for various ratios of exposed to unexposed populations of bacteria (ratios of 100;0, 90:10, 50:50, 10:90, and 0:100). FIG. 14A shows fluorescence of RecA-Lysis-ePOP- EcN for the various ratios of exposure versus unexposure. FIG. 14B shows fluorescence of RecA-Lysis-ePOP-DH5a for the various ratios of exposure versus unexposure. FIG. 14C shows fluorescence of ePOP-EcN for the various ratios of exposure versus unexposure. FIG. 14D shows fluorescence of ePOP-DH5a for the various ratios of exposure versus unexposure. FIG. 14E shows fluorescence of EcN for the various ratios of exposure versus unexposure. FIG. 14F shows fluorescence of DH5a for the various ratios of exposure versus unexposure. Thus, FIGS. 14A and 14B show the results for bacteria being including an ePOP plasmid that includes the RecA promoter and the lysis gene (14A being the probiotic Nissle 1917 strain of E. coli, and 14B being the DH5a strain of E. coli). FIGS. 14C-14F thus are controls as none of those include the promoter or lysis gene.

[0026] FIG. 15 is a table showing the number of bacteria surviving after different radiation doses, versus when they were not exposed or unexposed.

[0027] FIGS. 16A-D are graphs based on the data of the table of FIG. 15.

[0028] FIG. 17 is a table of various mixtures of live and dead cells mixed in different ratios.

[0029] FIGS. 18A and 18B are a pair of graphs showing bioluminescence using pAK-GFP- Luxl-EcN (the pAKgfpLuxl plasmid includes an insert of bacterial luciferase).

[0030] FIG. 19 is a table of various mixtures of live and dead cells mixed in different ratios.

[0031] FIG. 20 is a graph showing bioluminescence of RecA-Lysis-ePOP+pAK-GFP-Luxl- EcN at various rations of live to dead cells.

[0032] FIGS. 21A-B, 22A-B, 23A-B, 24A-B, and 25 are images describing a study of qualitative bioluminescence measurements using an In Vivo Imaging System (FVIS).

[0033] FIGS. 26A-D are graphs showing an estimation of bacterial cell lysis following exposure of tumors / thigh muscles to 2.0 Gy dose of radiations. E. coli Nissle 1917 (EcN (harbouring RecA-Lysis-ePOP and pAKgfpLuxl plasmids) was injected in tumors generated using MC38 cell line and thigh muscles in a C57BL/6J mice. Tumors and thigh muscles were in an experimental (test) group were irradiated with 2.0 Gy dose of radiations and imaged using IVIS in vivo Imaging system (Perkin Elmer). ROI values from Tumor (FIG. 26A) and Thigh Muscles (FIG. 26B) were plotted as shown using Graph Pad Prism (8.1). Tumors and Thigh muscles were aseptically extracted and Cfu counts were performed with serial dilutions for Tumors (FIG. 26C) and Thigh Muscles (FIG. 26D), and plotted as shown. Significance was determined by performing unpaired t-tests with SD (Cfu -Colony Forming Units, SD- Standard Deviation).

[0034] FIGS. 27A and 27B show High Fidelity PCR amplification for RecA-Lysis- RecA(ALexA)-PD-Ll geneblock.

[0035] FIGS. 28 A and 28B show High Fidelity PCR amplification for RecA-Lysis- RecA(ALexA)-PD-Ll geneblock.

[0036] FIG. 29 is a restriction digest for cloning PD-L1 into ePOP plasmid.

[0037] FIG. 30 is a schematic showing RecA-Lysis-RecA(ALexA)-PD-Ll-TEV-Strep-Flag- geneblock.

[0038] FIG. 31 is a schematic showing the construction of a RecRadePOP plasmid.

[0039] FIG. 32 shows a Colony PCR for the confirmation of RecA-lysis-Rec-PD-Ll-ePOP/cm cloning. In FIG. 32, “A” denotes cloning using ePOP-Seq_F and Rec gBlock_R primers, and “B” denotes cloning using Rec gBlock_F and Rec gBlock_Rl primers. Further, in FIG. 32, circles represent the positive colonies selected after screening and gel electrophoresis for final confirmation with Sanger Sequencing.

[0040] FIG. 33 shows Colony PCR for the confirmation of RecA-lysis-Rec-PD-Ll-ePOP/cm cloning using Rec gBlock_F and Rec gBlock_Rl primers. Further, in FIG. 33, circles represent the positive colonies selected after screening and gel electrophoresis for final confirmation with Sanger Sequencing.

[0041] FIG. 34 is an SDS-PAGE Western blot analysis of intracellular proteins (cell fraction). [0042] FIG. 35 is an SDS-PAGE Western blot analysis of extracellular proteins (supernatant). [0043] FIG. 36 is a table showing exposed Radiation doses for various bacterial strains and OD600 before and after radiation exposure.

[0044] FIG. 37 is a graph showing RecA-Lysis-RecA(ALexA)-PD-Ll-ePO: OD600 before and after radiation exposure at various levels of radiation exposure (plotting data from the table in FIG. 36). [0045] FIG. 38 shows an SDS-PAGE Western blot analysis of extracellular proteins (cell fraction) in RecA-Lysis-RecA(ALexA)-PD-Ll-ePOP-EcN 1917 at various levels of radiation exposure.

[0046] FIGS. 39 and 40 are images from a survival study using MC38 cell line and C57BL/6J mice for testing efficacy of anti-PD-Ll Nanobody generated using RecA-Lysis-RecA(ALexA)- PD-L1 ePOP construct in bacteria.

[0047] FIGS. 41A-C are graphs showing bioluminescence from the survival study of FIGS. 39 and 40. FIG. 41 A shows bioluminescence change on Day 1, FIG. 41B shows bioluminescence change on Day 3, and FIG. 41C shows bioluminescence change on Day 5.

[0048] FIG. 42 is schematic of the survival study using MC38 cell line and C57BL/6 mice for testing efficacy of anti-PD-Ll Nanobody generated using RecA-Lysis-RecA(ALexA)-PD-Ll ePOP construct in bacteria.

[0049] FIG. 43 is a graph showing tumor growth over time for various bacterial strains subjected to radiation (or unexposed) in mice of the survival study.

[0050] FIG. 44 is a graph showing the results of a survival study for the in vivo analysis of PD- L1 nanobody blockade using C57BL6 mice and MC38 tumors.

[0051] FIG. 45 is a graph showing the survival proportions of mice in the survival study using MC38 cell line and C57BL/6 mice.

[0052] FIG. 46 is a table showing the survival proportions of mice in the survival study using MC38 cell line and C57BL/6 mice (table of data plotted in graph of FIG. 45).

[0053] FIG. 47 is a map of RecRadePOP-PD-Ll.

[0054] FIGS. 48 and 49 are schematics showing an assay that was performed to determine if the PD-L1 nanobodies were capable of inhibiting the PD-1 and PD-L1 interaction.

[0055] FIG. 50 is a graph plotting fold induction (A.U.) versus Log 10 (Control) pg/ml for each of anti-PD-Ll nanobody, anti-PD-Ll antibody, anti-PD-1 antibody, and E. coli Nissle 1917 resulting from the assay shown in the schematic of FIG. 48.

[0056] FIG. 51A is a schematic showing process of the study with bacterial injection into C57BL6/J mice.

[0057] FIG. 5 IB is a schematic of a study that evaluated the efficiency of anti-PD-Ll nanobody expression and its effect on improving targeted therapeutic outcomes in vivo (on a total of seven treatment groups using rigorous control groups assessed in parallel.

[0058] FIG. 51C is a graph showing tumor growth over time for various bacterial strains subjected to radiation (or unexposed) in mice of the survival study using MOC1 cell line and C57BL/6 mice. [0059] FIG. 5 ID is a graph showing the survival proportions of mice in the survival study using MOC1 cell line and C57BL/6 mice.

DETAILED DESCRIPTION OF THE INVENTION

[0060] One or more specific embodiments of the present invention will be described below. In an effort to provide a concise description of these embodiments, all features of an actual implementation may not be described in the specification. It should be appreciated that in the development of any such actual implementation, as in any engineering or design project, numerous implementation-specific decisions must be made to achieve the developers’ specific goals, such as compliance with system-related and business-related constraints, which may vary from one implementation to another. Moreover, it should be appreciated that such a development effort might be complex and time consuming, but would nevertheless be a routine undertaking of design, fabrication, and manufacture for those of ordinary skill having the benefit of this disclosure.

[0061] One skilled in the art will recognize that the various embodiments may be practiced without one or more of the specific details described herein, or with other replacement and/or additional methods, materials, or components. In other instances, well-known structures, materials, or operations are not shown or described in detail herein to avoid obscuring aspects of various embodiments of the invention. Similarly, for purposes of explanation, specific numbers, materials, and configurations are set forth herein in order to provide a thorough understanding of the invention. Furthermore, it is understood that the various embodiments shown in the figures are illustrative representations and are not necessarily drawn to scale.

[0062] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure, material, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention, but does not denote that they are present in every embodiment. Thus, the appearances of the phrases “in an embodiment” or “in another embodiment” in various places throughout this specification are not necessarily referring to the same embodiment of the invention. Further, “a component” may be representative of one or more components and, thus, may be used herein to mean “at least one.”

[0063] As described above, certain drawbacks exist when using bacteria as a vessel for delivery of a therapeutic to the site of a disease or condition. Aspects of the present invention, however, address these concerns by providing strategies that combine existing therapeutic modalities such as ionizing radiation (IR) with microbiome therapy in order to gain from the synergistic potential between the two modalities. This coupling is particularly promising as genes that encode for therapeutic proteins can be synced with genes that are sensitive to IR and can be programmed in the microbiome to act synchronously.

[0064] As described above, one aspect of the present invention is directed to a genetically modified bacterium comprising a lysis gene. The lysis gene includes a nucleic acid encoding for a protein, the expression of which results in lysis of the bacterium. Further, the nucleic acid expresses the protein in response to exposure to radiation.

[0065] Another aspect of the present invention is directed to a plasmid for insertion into a bacterium, the plasmid comprising a nucleic acid encoding a lysis gene, a nucleic acid encoding a first promoter, a nucleic acid encoding a biotherapeutic molecule, and a nucleic acid encoding a second promoter. In this aspect of the present invention, the nucleic acid encoding a lysis gene includes a sequence encoding a protein, the expression of which results in lysis of a bacterium, that has had the plasmid inserted thereinto. Further, the nucleic acid expresses the protein in response to exposure to radiation. The plasmid of this aspect of the present invention may be used to genetically modify a bacterium into a bacterium including a lysis gene that expresses an encoded protein upon exposure to radiation (such as the bacterium of the first aspect of the invention listed above).

[0066] Aspects of the present invention, then, are directed to an engineered probiotic (EP) bacterium that can be used as a system for exporting therapeutics. The EP bacterium is capable of synthesizing a multitude of genetically-encoded, unique, therapeutic molecules including, but not limited to, cytokines, anti-inflammatory and anti- cancer nanobodies (such as anti-PD- Ll, anti-EGFR, anti-TNFa, anti-INF y and GM-CSF, IL-2, etc.) - many of which have a cytotoxic effect on cells (such as tumor cells). To this end, one embodiment of the invention uses a probiotic Escherichia coli Nissle 1917 (EcN) bacterium to synthesize and deliver therapeutic proteins (such as those listed above) directly to a disease site (e.g., tumor). In order to increase the specificity of the cytotoxic protein delivery, and to achieve temporal control of protein expression, the genetic construct of the EP bacterium may include a prokaryotic radioinduced promoter to control gene expression. In one particular embodiment, this promoter may be the promoter of a RecA gene, which belongs to the SOS-repair system of bacteria. As will be described in greater detail below, the RecA promoter can be induced with IR using a lysis gene. In one embodiment, the lysis gene may be obtained from <|>xl74 bacteriophages. Activation of the promoter in turn drives the expression of the fused lysis gene, which leads to bacterial cell lysis followed by release of the encoded therapeutic protein(s). [0067] In one specific embodiment, the present inventors accomplished this by custom designing a high copy number-ColEl backbone-RecRadePOP plasmid construct. The RecRadePOP (3884 bp) plasmid was designed in-silico using DNA2.0, and it sequentially harbors genetic elements “RecA-Lysis-rmB-T7Te-RecA(ALexA)-PD-Ll-TEV-Strep-Flag- tag” which work in tandem. Further, T7Te and rmB terminators were incorporated to isolate the peptides following translation. A schematic showing the RecRadePOP plasmid can be seen in FIG. 1.

[0068] Further aspects of the present invention concern the expression of the Lysis gene in response to radiation in vitro and in vivo. As will be shown below in greater detail below (in the Detailed Description and in the Examples herein), the present inventors obtained significant bacterial cell lysis, even at radiation doses as low as 2.0-4.0 Gy. The ability to achieve significant cell lysis at these low doses is advantageous for clinical settings since most studies use radiation doses of at least 20-25 Gy for induction of gene expression. Further, one embodiment of the genetic construct incorporates a strong constitutive promoter [the promoter being RecA (ALexA)]. In this embodiment, the LexA repressor binding site has been ablated to enable constitutive expression, making it ~650x stronger than the constitutive AraBAD promoter. Next in the design, to evaluate the protein expression driven by this constitutive RecA (ALexA) promoter, the present inventors chose an anti-PD-Ll nanobody as a biotherapeutic molecule and fused it in downstream of the RecA (ALexA) promoter. As shown herein, the nanobody peptide is further fused at the carboxy terminus with a TEV cleavage site from Tobacco Etch Virus for peptide separation, followed by Flag and Strep II tags for detection and purification. This design serves a plug-and -play system. And because it is decorated with restriction endoglucases enabling modularity, it allows the testing of a permutation and combination of nanobody (such as anti-PD-Ll and anti-EGFR) and small therapeutic biomolecules (such as GM-CSF, IL-2, TNFa, and INFy). In certain embodiments herein, the present inventors report a dual-promoter system, where RecA and RecA (ALexA) promoters work in coordination. This design is meant to constitutively express, for example, the anti-PD-Ll nanobody driven by modified RecA (ALexA) promoter. However, the therapeutic anti-PD-Ll nanobody will only be released when the bacteria are exposed to radiations of appropriate doses - thereby resulting in cell lysis and nanobody release.

[0069] As described above, one aspect of the present invention is directed to a genetically modified bacterium comprising a lysis gene including a nucleic acid encoding for a protein, the expression of which results in lysis of the bacterium. Further, the design of the genetically modified bacterium is such that the protein is expressed only in response to some stimuli - such as exposure to radiation. In this manner, the lysis of the bacterium can be controlled (and thus the release of any biotherapeutic material produced by the bacterium can be controlled as well. [0070] In order to accomplish the above, the genome of the bacterium (as modified) may, in certain embodiments, include a plasmid, wherein the nucleic acid sequence of the plasmid includes the lysis gene. The source of the lysis gene may be from any suitable organism, so long as it may be included in a plasmid to be successfully inserted into the genome of the bacterium. In one particular embodiment, the lysis gene is obtained from a $xl74 bacteriophage. And, in particular embodiments where the lysis gene is obtained from a $xl74 bacteriophage, the nucleic acid of the lysis gene has a sequence of [SEQ ID NO: 3] - (sequences are included in a separate section at the end of this document)

[0071] In order to ensure lysis of the bacterium (to release any biotherapeutic molecule or material therein), the plasmid may further include a nucleic acid sequence encoding a first promoter that is operable to initiate transcription of the nucleic acid sequence of the lysis gene. As described above, the design of the genetically modified bacterium may be such that the protein is expressed only in response to some stimuli - such as exposure to radiation. And so, in certain embodiments of the present invention, the first promoter is an inducible promoter. In particular embodiments, the first promoter is inducible in response to exposure to radiation. In such embodiments, the dose level of exposure may be of any amount that results in transcription of the first promoter and thus the lysis gene. In various embodiments, this radiation does may be at least 0.5Gy, at least l.OGy, at least 2.0Gy, at least 4.0Gy, at least 8.0Gy, at least 16.0Gy, at least 25.0Gy, between 0.5Gy and 25.0Gy, between 0.5Gy and 16.0Gy, between 0.5Gy and 8.0Gy, between 0.5Gy and 4.0Gy, between 0.5Gy and 2.0Gy, between 0.5Gy and l.OGy, between l.OGy and 25.0Gy, between l.OGy and 16.0Gy, between l.OGy and 8.0Gy, between l.OGy and 4.0Gy, between l.OGy and 2.0Gy, between 2.0Gy and 25.0Gy, between 2.0Gy and 16.0Gy, between 2.0Gy and 8.0Gy, and between 2.0Gy and 4.0Gy. [0072] In certain embodiments, the first promoter is the promoter of a RecA gene. And, in certain embodiments where the first promoter is the promoter of a RecA gene, the first promoter has a sequence of [SEQ ID NO: 2],

[0073] As described above, the genetically modified bacterium may be designed to express a biotherapeutic molecule or molecules (which are then subject to release once the bacterium is lysed, in order to deliver the biotherapeutic molecule or molecules for treatment of a disease or condition). And so, the plasmid inserted into the genome - and thus the genome of the modified bacterium, may include at least one gene including a nucleic acid encoding for a biotherapeutic molecule. In certain embodiments, the biotherapeutic molecule is chosen from anti-PD-Ll, anti-EGFR, GM-CSF, IL-2, TNFa, and INFy. In alternate embodiments, the genome of the genetically modified bacterium may include more than one gene encoding for biotherapeutic molecules (such that the bacterium may produce more than one biotherapeutic molecule).

[0074] In order to ensure production of any biotherapeutic molecule or molecules therein, the plasmid - and thus the modified genome of the bacterium - may further include a nucleic acid sequence encoding a second promoter that is operable to initiate transcription of the nucleic acid sequence encoding the biotherapeutic molecule. In certain embodiments, the second promoter is constitutively expressed. And so, in certain embodiments, the second promoter may be a modified promoter of a RecA gene. More specifically, in some embodiments where the second promoter is a modified promoter of a RecA gene, that promoter may be modified by deleting a LexA repressor binding site of the promoter of the RecA gene. And, in certain embodiments where the ssecond promoter is the modified promoter of a RecA gene, the second promoter has a sequence of [SEQ ID NO: 6]. Thus, in embodiments where the genetically modified bacterium has an inducible first promoter for the lysis gene and a constitutively expressed second promoter for the biotherapeutic molecule(s), the genetically modified bacterium can be designed in a manner that is continuously expresses the biotherapeutic molecules, however, it only expresses the protein to lyse the bacterium once exposed to a sufficient dose of radiation to induce the first promoter.

[0075] Additionally, the genetically modified bacterium may further include a TEV cleavage site from Tobacco Etch Virus, wherein the cleavage site is associated with the nucleic acid encoding the biotherapeutic molecule. The genetically modified bacterium may further include one or both of a Flag tag and a Strep II tag.

[0076] Further, the the bacterium that is genetically modified may be E. coli, and in more specific embodiments, may be E. coli Nissle 1917.

[0077] As described above, another aspect of the present invention is directed to a plasmid for insertion into a bacterium, the plasmid comprising a nucleic acid encoding a lysis gene, a nucleic acid encoding a first promoter, a nucleic acid encoding a biotherapeutic molecule, and a nucleic acid encoding a second promoter. In this aspect of the present invention, the nucleic acid encoding a lysis gene includes a sequence encoding a protein, the expression of which results in lysis of a bacterium, that has had the plasmid inserted thereinto. Further, the nucleic acid expresses the protein in response to exposure to radiation. The plasmid of this aspect of the present invention may be used to genetically modify a bacterium into a bacterium including a lysis gene that expresses an encoded protein upon exposure to radiation (such as the bacterium of the first aspect of the invention listed above). [0078] As noted, the plasmid includes the lysis gene. The source of the lysis gene may be from any suitable organism, so long as it may be included in a plasmid to be successfully inserted into the genome of the bacterium. In one particular embodiment, the lysis gene is obtained from a $xl74 bacteriophage. And, in particular embodiments where the lysis gene is obtained from a $xl 74 bacteriophage, the nucleic acid of the lysis gene has a sequence of [SEQ ID NO: 3].

[0079] As described above, the plasmid may further include a nucleic acid sequence encoding a first promoter that is operable to initiate transcription of the nucleic acid sequence of the lysis gene. In certain embodiments of the present invention, the first promoter is an inducible promoter. In particular embodiments, the first promoter is inducible in response to exposure to radiation. In such embodiments, the dose level of exposure may be of any amount that results in transcription of the first promoter and thus the lysis gene. In various embodiments, this radiation does may be at least 0.5Gy, at least l.OGy, at least 2.0Gy, at least 4.0Gy, at least 8.0Gy, at least 16.0Gy, at least 25.0Gy, between 0.5Gy and 25.0Gy, between 0.5Gy and 16.0Gy, between 0.5Gy and 8.0Gy, between 0.5Gy and 4.0Gy, between 0.5Gy and 2.0Gy, between 0.5Gy and l.OGy, between l.OGy and 25.0Gy, between l.OGy and 16.0Gy, between l.OGy and 8.0Gy, between l.OGy and 4.0Gy, between l.OGy and 2.0Gy, between 2.0Gy and 25.0Gy, between 2.0Gy and 16.0Gy, between 2.0Gy and 8.0Gy, and between 2.0Gy and 4.0Gy. [0080] In certain embodiments, the first promoter is the promoter of a RecA gene. And, in certain embodiments where the first promoter is the promoter of a RecA gene, the first promoter has a sequence of [SEQ ID NO: 2],

[0081] As described above, the plasmid may include at least one gene including a nucleic acid encoding for a biotherapeutic molecule. In certain embodiments, the biotherapeutic molecule is chosen from anti-PD-Ll, anti-EGFR, GM-CSF, IL-2, TNFa, and INFy. In alternate embodiments, the genome of the genetically modified bacterium may include more than one gene encoding for biotherapeutic molecules (such that the bacterium may produce more than one biotherapeutic molecule).

[0082] In order to ensure production of any biotherapeutic molecule or molecules therein, the plasmid may further include a nucleic acid sequence encoding a second promoter that is operable to initiate transcription of the nucleic acid sequence encoding the biotherapeutic molecule. In certain embodiments, the second promoter is constitutively expressed. And so, in certain embodiments, the second promoter may be a modified promoter of a RecA gene. More specifically, in some embodiments where the second promoter is a modified promoter of a RecA gene, that promoter may be modified by deleting a LexA repressor binding site of the promoter of the RecA gene. And, in certain embodiments where the second promoter is the modified promoter of a RecA gene, the second promoter has a sequence of [SEQ ID NO: 6].

[0083] Additionally, the plasmid may further include a TEV cleavage site from Tobacco Etch Virus, wherein the cleavage site is associated with the nucleic acid encoding the biotherapeutic molecule. The genetically modified bacterium may further include one or both of a Flag tag and a Strep II tag.

EXAMPLE

[0084] The following Example describes the design, construction, and validation of the programmed lysis of microbiome in accordance with the various aspects of the present invention described herein.

[0085] Design Construction of the RecA-Lysis-ePOP Plasmid for Insertion into Bacteria [0086] Referring now to FIGS. 4 A- 10, construction of bacteria including the genetic construct, and the functionality described above, is disclosed. In that regard, plasmid construction begins with PCR amplification of the gene (i.e., of the RecA-Lysis gene for cloning into an ePOP plasmid), followed by purification of the PCR product [see FIG. 4A, which shows PCR amplification of the RecA-Lysis gene (High-Fidelity PCR for RecA-Lysis gene using gBlock) for cloning into the ePOP plasmid; as can be seen an amplified product appears near the 500 bp marker of the GeneRuler ladder, and appears in all Lanes 2-10].

[0087] The product was then digested using restriction endonucleases along with a suitable plasmid backbone. In that regard, FIG. 4B shows restriction digestion of a pCN56 vector and the amplified RecA-Lysis insert for cloning. In Lanes 2-4, pCN56 plasmid was digested with Sall-Kpnl restriction endonuclease (obtained from ThermoFisher). Lane 5 was empty. Lanes 6-11 had the RecA-Lysis PCR product from FIG. 4A digested with Sall-Kpnl restriction endonuclease. And, FIG. 4C shows restriction digestion of an ePOP plasmid and the amplified RecA-Lysis insert for cloning. In Lanes 2-5, ePOP plasmid was digested with Sall-Kpnl restriction endonuclease. Lane 6 was empty. Lanes 7-10 had the RecA-Lysis PCR product from FIG. 4A digested with Sall-Kpnl restriction endonuclease.

[0088] These restriction digested PCR products and plasmid backbone were cleaned using a Monarch plasmid and gel extraction kit (from New England Biolabs - “NEB”). Following ligation, insert and plasmid were transformed into chemically competent NEB DH5a cells. Successful cloning was confirmed with Colony PCR (see FIGS. 5 and 6), and Sanger Sequencing. FIG. is a photograph of PCR products showing preliminary confirmation of cloning via screening with Colony PCR. The circled bands of ~650bp are expected results, while the circled bands having a size between ~1.OkB and ~1 ,5kB were unexpected. And FIG. 6 is a photograph of PCR products showing preliminary confirmation of cloning via screening with Colony PCR.

[0089] The final step in this portion of the study was confirmation of the gene via Sanger Sequencing. To that end, with NCBI online tools, successful gene sequence following cloning was aligned with the theoretical gene sequence. As shown in FIGS. 7 and 8, 100% match with the theoretical sequence as shown with red color output. The table of FIG. 9 shows the high percent sequence coverage of 83% and 72%, where forward and reverse primers were used for sequencing. A map of the created RecA-Lysis-ePOP plasmid is then shown in FIG. 10.

[0090] Radiation Exposure Study: Testing the Construct for Phenotype and Expression [0091] Following the final confirmation of the ‘RecA-Lysis-ePOP’ plasmid with Sanger sequencing, the RecA-Lysis-ePOP plasmid was transformed into Escherichia coli Nissle 1917 cells (EcN) and Escherichia coli DH5a cells (cells were acquired from NEB). A radiation exposure study was then conducted, to test the construct for phenotype/expression (of the genes of the inserted plasmid).

[0092] Additionally, for the study, empty-ePOP plasmids were also transformed into Escherichia coli Nissle cells (EcN) and Escherichia coli DH5a cells (acquired from NEB) to serve as negative controls. Untransformed EcN and DH5a cells were also used as additional negative controls. Thus, the following six bacterial cultures were prepared and then grown overnight at 37°C and 180 rpm.

Culture 1: RecA_Lysis-ePOP-EcN

Culture 2: RecA_lysis-ePOP-DH5a

Culture 3: Empty-ePOP-EcN

Culture 4: Empty-ePOP-DH5a

Culture 5: EcN

Culture 6: DH5a

[0093] The following day, 1% inoculum was added into the fresh medium (10 ml) and grown until OD600 reached 0.8-0.9 (approx. 3.00 hr). 200 pl each from these cultures in triplicates were then added into each of 8 separate 96-well plates and irradiated with the following radiation doses:

Plate 1: Unexposed/ unirradiated

Plate 2: 0.5 Gy Plate 3: 1.0 Gy

Plate 4: 2.0 Gy

Plate 5: 4.0 Gy Plate 6: 8.0 Gy Plate 7: 16.0 Gy Plate 8: 25 Gy [0094] Following irradiation, the various populations were tested to determine live cells versus dead cells in each population and at each tested dose level of radiation. In doing so, various ratios of the populations were exposed to radiation (for the different doses of radiation). For example, FIG. 11, which shows a study design and exposure schematic: the ratios of bacteria exposed at 40Gy versus unexposed bacteria is shown. To perform the assay, 18 ml DI water + 54 pl component A+ 54 pl component B was used for a 96 well plate. And 3 ml DI water + 9ul component A +9 ul component B was used for a LIVE/DEAD™ Assay (via the LIVE/DEAD™ Cell Imaging Kit available from Invitrogen). As is known to those skilled in the art, this is an assay that allows discrimination between live and dead cells via two probes that measure indicators of cytotoxicity and cell viability: intracellular esterase activity and plasma membrane integrity. And so the components of the kit include cell-permeable dye for the staining of live cells, and cell-impermeable dye for staining of dead and dying cells. As a result, the live cell component will produce green fluorescence at certain wavelengths, and dead cell components will produce red fluorescence at certain wavelengths.

[0095] With reference to FIG. 12, 100 pl each of the exposed (4 Gy) and unexposed bacterial cultures from the above mix were added with 100 pl of the bacterial LIVE/DEAD™ assay solution and fluorescence was measured immediately at 485, 520 and 485, 640 nm using FLUOstar (Polarstar®, BMG) plate reader. In particular, FIG. 12 is a schematic showing the exposed to unexposed ratio of cells in each population for Plate 5, above, which was exposed to 4.0GY IR. (The other plates - Plates 1-4 and 6-8 would be prepared similarly and the exposed cells in each well were exposed to the particular does shown above (i.e., 0 for Plate 1, 0.5Gy for Plate 2, l.OGy for Plate 3, etc.).

[0096] Referring now to FIGS. 13A-F, FIG. 13A shows results for the group where the engineered plasmid was transformed into E. coli Nissle 1917 (EcN) bacteria and exposed to various doses of radiation from 0.5 Gy to 25.0 Gy. Here in FIGS. 13A-F, one can see significant bacterial cell lysis following radiation. A similar result was seen when the present inventors transformed a different type of E.coli called E.coli DH5a (see FIGS. 14A-F). As shown in FIGS. 14A and 14B, the plasmid construct RecA-Lysis-ePOP was transformed into two different bacterial cell types (EcN and E. coli DH5a). Similarly, plasmids without the Lysis gene were also transformed into these two cell types (and wild types of these cells were used as controls). All these bacteria were then also used for a LIVE/DEAD™ assay and significant cell lysis was observed. As can be seen in those graphs, significant bacterial cell lysis was observed when using 0.5Gy, l.OGy, and 2.0Gy doses. However, bacterial lysis did not occur with higher radiation doses; this might suggest recombination occurring at high doses.

[0097] Referring now to FIGS. 15 and 16A-D, in order to confirm the bacterial cell lysis and quantitate it, the present inventors performed an analysis to obtain colony forming units (cfu/ml). Then these engineered bacteria (bacteria containing RecA-Lysis-ePOP plasmid) and bacteria containing control plasmid (i.e., where lysis gene is absent) were exposed to different radiation doses and cfu counts were analyzed by plating bacteria on an agar plate. As can be seen, the bacteria containing plasmid (RecA-Lysis-ePOP) were significantly lysed (FIGS. 16A and 16B), however no significant cell lysis in bacteria containing control plasmid (FIGS. 16 C and 16 D) was observed.

[0098] When the plasmid without lysis gene was transformed into either EcN or E.coli DH5a, the present inventors did not see any significant cell lysis (FIGS. 15 and 16A-D) . Neither was any significant cell lysis seen when wild type EcN or E.coli DH5awere exposed to the various radiation doses. This suggests that the cell lysis occurs only when both the lysis gene and the radiation responsive promoter (RecA) are present.

[0099] Estimation of in vitro Bioluminescence Prior to in vivo Studies

[0100] Next, a study was conducted to understand whether bacterial cell lysis could lead to diminished bioluminescence by using Escherichia coli Nissle (EcN) transformed with a pAK- GFP-Luxl plasmid harboring a bioluminescent Lux gene. Escherichia coli Nissle (EcN) bacteria were chemically transformed with an ampicillin resistant, pAK-GFP-Luxl plasmid.

[0101] And so, 24 hrs. prior to the experiment, EcN-pAKgfpLuxl: bacteria were grown overnight for 16-24 hr and 1% of these bacteria were inoculated into fresh LB medium, supplemented with 100 mg/ml Ampicillin and grown approximately 2.5-3.0 hr, or until optical density (OD) 600 reached -0.8-0.9.

[0102] Following the OD600 measurement, 5 ml cells each were distributed into two 50 ml falcon tubes. One set was heat inactivated at 60 °C for 15 min, with intermittent vertexing.

[0103] FIG. 17 shows a table of live and dead cells that were mixed in the indicated ratios. These cultures were placed into a transparent 96-well plate and read for bioluminescence using a citation machine (BioTek Instruments, Inc.) and data were exported in Excel and plotted using Graph Pad Prism 8. All values were expressed as mean +/-standard error mean (SEM+SD). Statistical analysis was performed with one-way analysis of Variance (ANOVA). p-values < 0.05 were considered statistically significant when compared to the control.

[0104] The positive transformant of EcN (RecA-Lysis-ePOP+pAKgfpLuxl-EcN) grew on both Chloramphenicol and Ampicillin. Because RecA-Lysis-ePOP has a Chloramphenicol cassette, and because pAKgfpLuxl has Ampicillin cassette, this suggested EcN got transformed with both plasmids.

[0105] EcN-AKgfpLux 1 was already growing as it had been inoculated 24 hrs previously on the previous day. Then, fresh cultures of both EcN containing only AKgfpLuxl (as a positive control), and EcN containing both AKgfpLuxl and RecA-Lysis-ePOP (as a test sample) were grown. (The positive control has higher inoculum than the test sample, since it was growing from the previous day).

[0106] Referring now to FIGS. 18A and 18B, the present inventors had observed that if one places the positive control and test samples in close proximity, positive control contributes to the bioluminescence of the Test sample (though this contribution was not statistically significant unless it was more than 20%). The present inventors found that: (1) when bioluminescence was measured as previously using a multimode plate reader (BioNteK), it indeed showed that the EcN was successfully transformed with Lux and RecA plasmids; (2) the difference in bioluminescence can be attributed to the number of cells present in the culture when tested for the bioluminescence; and (3) samples should be placed away from each other in a 96-well plate while measuring the bioluminescence in order to reflect the true values.

[0107] Following the confirmation of the successful transformation of EcN with both RecA and Lux plasmids as shown in FIGS. 18A and 18B, the new EcN strain (harboring both RecA and Lux plasmids) was subjected to bioluminescent measurements by using known ratios of live and dead cells. The various ratios of live cells to dead cells that were used is shown in FIG. 19, and the observed bioluminescence of each of those ratios is shown in FIG. 20. This data shows that bioluminescence indeed decreases as cell lysis occurs. (However, it is noted that, when one looks at the 100% live cells (100:0 ratio) and 80% live cells (80:20 ratio) in FIG. 20, there is no significant difference in results - suggesting that one may needs at least something more than 20% cell lysis in order to find statistically significant cell lysis as measured using bioluminescence.)

[0108] Tumor Generation and In Vivo Cell Lysis Following Radiation Exposure [0109] An MC38 cell line derived from C57BL6 murine colon cancer cells was grown in lx DMEM medium supplemented with 10% Fetal Bovine Serum, 2 mM Glutamine, 0.1 mM Non- essential amino acids, 1 mM Sodium pyruvate, 10 mM HEPES, 50 ug/ml Gentamicin sulphate, and PenStrep until confluency in a T175 flask.

[0110] Next, ~1.0 - 1.5xl0⁶ cells were subcutaneously injected in to 6 C57BL/6 mice and tumors were allowed to grow for 14-17 days. Tumors sizes were monitored, and once a visible tumor appeared, the tumor volume was measured using a Vernier Caliper.

[0111] The 6 mice were divided into 2 separate cages (3 mice per cage). Once tumor volumes reached 700-1000mm³, a fresh culture of IxlO⁷ Escherichia coli Nissle (EcN) harboring both (1) RecA-Lysis-ePOP and (2) pAKgfpLux were injected into all the six mice. (In particular, the bacteria were injected directly into the tumor and the left thigh muscles of each of the 6 mice.) All the mice (in two separate cages) were taken to the Vontz Centre for Molecular Studies (University of Cincinnati) where one group (3 mice) were irradiated with 2.0 Gy dose pf radiations with following specification: 220 KVp, 13 mA with Field size of 1.0 cm using square or Circular collimator/ cone.

[0112] Following radiations, mice were brought back to the Medical Sciences Building (University of Cincinnati) and imaged using an In Vivo Imaging System (IVIS - available from Perkin Elmer) - as shown in FIGS. 21A-25. In particular, three unexposed mice (tumors) were originally imaged. As can be seen in FIG. 21 A, three mice were imaged by positioning the mice as lying on their left leg, so that the signal from tumors above the right leg could be imaged. All three of the mice showed an ROI of IxlO⁷ and above. However, Mouse #2 and Mouse #3 each had an ROI value that was noticeably higher than Mouse #1. Thus, as they could be suppressing signals coming from Mouse #1, Mouse #2 and Mouse #3 were removed, and the remaining mouse (Mouse #1) was imaged. This is shown in FIG. 22B, which shows Mouse #1 having ROI value of IxlO⁷ and above.

[0113] Next, as shown in FIGS. 22A-B, in order to image the signal from the left thigh muscle, the mice were then positioned lying on their right leg, and the image is shown in of FIG. 22A. All three mice were shown as having an ROI of IxlO⁷ and above. However, Mouse #2 had an ROI value that was noticeably higher than Mice #1 and #3. Thus, as it could be suppressing signals coming from Mouse #1 and Mouse #3, Mouse #2 was removed, and the remaining two mice (Mice #s 1 and 3) were imaged. This is shown in FIG. 22B, which shows both Mouse #1 and Mouse #3 having ROI values of IxlO⁷ and above.

[0114] Referring now to FIGS. 23A-B, in order to avoid the possibility of reinforcement of signal due to tumor and thigh, the mouse (Mouse #1) was placed on its back (see FIG. 23 A). When this was done, no signal was obtained from the tumor; rather signal was obtained from the thigh (ROI value of IxlO⁷ and above). In order to get signal from both tumor and thigh, the mouse was placed on its belly and imaged (see FIG. 23B). In this position, distinct signals from both tumor and thigh were captured. As a result, the inventors adopted this method for the remining groups tested (note: ROI values obtained in FIG. 23B were IxlO⁷ for tumor and IxlO⁶ for the thigh muscle).

[0115] And so, referring now to FIGS. 24A-B, Mouse #2 and Mouse #3 were imaged in this same manner (with mice positioned on their bellies). In FIG. 24A, Mouse #2 had an ROI of IxlO⁷ and above, but Mouse #3 did not show any signal. And so, Mouse #2 was removed (because it might be suppressing signal from Mouse #3), and Mouse #3 was then imaged alone. Once imaged alone (as shown in FIG. 24B), Mouse #3 showed an ROI of IxlO⁶ for both the tumor and the thigh muscle.

[0116] Finally, three mice of a second group were imaged in this same manner. The three mice of this group were exposed to a radiation dose of 2.0Gy (in both the tumors and the thigh muscles). As shown in FIG. 25, the ROI values were 10⁶ for all the tumors and thigh muscles in all three mice. This suggests that the irradiated group has had its ROI values decreased from IxlO⁷ and above (e.g., 10⁸, 10⁹) to 10⁶, suggesting bacterial cell lysis following irradiation with a 2.0 Gy dose.

[0117] Thus, by comparing ROI values of the unexposed group (FIGS. 21A-24B) and the exposed group (FIG. 25), the present inventors have demonstrated that the RecA-Lysis-ePOP plasmid gets induced following radiation exposure by means of driving lysis gene expression, which in turn lyses the bacterial cells. And so, the present inventors have demonstrated that RecA-Lysis-ePOP is functional both in vitro and in vivo.

[0118] FIGS. 26A-D shows an estimation of bacterial cell lysis following exposure of tumors / thigh muscles to 2.0 Gy dose of radiations. E. coli Nissle (harbouring RecA-Lysis-ePOP and pAKgfpLuxl plasmids) was injected in tumors generated using MC38 cell line and thigh muscles in a C57BL/6 mice. Tumors and thigh muscles were in an experimental (test) group were irradiated with 2.0 Gy dose of radiations and imaged using IVIS in vivo Imaging system (Perkin Elmer). ROI values from tumor (FIG. 26 A) and thigh muscles (FIG. 26B) were plotted as shown using Graph Pad Prism (8.1). Tumors and thigh muscles were aseptically extracted and Cfu counts were performed with serial dilutions for tumors (FIG. 26C) and thigh muscles (FIG. 26D), and plotted as shown. Significance was determined by performing unpaired t-tests with SD (Cfu -Colony Forming Units, SD- Standard Deviation). [0119] Engineering a modular plasmid construct for the on-site delivery of biotherapeutics for cancer treatment using probiotic E. coli Nissle 1917

[0120] In order to develop an on-site biotherapeutic delivery system for cancer treatment, the present inventors programmed a probiotic bacterium E. coli Nissle 1917, that delivers a biotherapeutic in a controlled manner by means of an engineered plasmid- RecRadePOP. The RecRadePOP (3884 bp) plasmid sequentially harbors RecA-Lysis-RecA(ALexA)-PD-Ll- TEV-Strep-Flag-geneblock that works in tandem (as shown in schematic of FIG. 30). Here the present inventors used a repressible RecA promoter, which, as previously shown, responds to radiation, and that in turn drives the expression of downstream fused gene/protein. The present inventors have previously reported the expression of lysis gene in response to radiation in vitro and in vivo, that leads to bacterial cell lysis. Now the present inventors are using a modified RecA (ALexA) promoter that has a LexA repressor binding site deleted, making this a constitutive promoter, which reportedly expresses 600 times more protein than inducible pBAD promoter. Following the design, the present inventors fused an anti-PD-Ll nanobody as a biotherapeutic molecule downstream of the constitutive RecA (ALexA) promoter. As shown in the design of FIG. 30, nanobody peptide is attached with TEV cleavage site from Tobacco Etch Virus for peptide and protein tag separation, as TEV site is in turn fused with Flag and Strep II tag for detection and purification. Additionally, the construct is designed in such a way that following protein expression, RecA-Lysis and RecA (ALexA)-PD-Ll peptides are isolated by means of introducing rmB T1 and T7T1 Terminators. The design is meant to constitutively express the anti-PD-Ll nanobody that will only be delivered when the bacterial cell lysis occurs, while RecA promoter is meant to expresses the Lysis protein only after radiation exposure as radiations activate the RecA promoter driving the expression of Lysis protein.

[0121] Molecular cloning of nanobodies and small biomolecules into a RecRadePOP plasmid [0122] PCR amplification for Rec-Lysis-RecA-PD-Ll gBlock: For the construction of the RecRadePOP [RecA-Lysis-RecA (ALexA)-PD-Ll-ePOP] plasmid, a gene block was ordered from a GeneArtSynthesis (Thermo Fisher Scientific). It was received as a lyophilized 200 ng PCR product. Upon arrival, it was reconstituted in 50 pL of IxTAE buffer and used as a template in a PCR reaction.

[0123] FIGS. 27A and 27B show High Fidelity PCR amplification for the RecA-Lysis- RecA(ALexA)-PD-Ll geneblock. The RecA-Lysis-RecA (ALexA)-PD-Ll gene block was PCR amplified using primers as follows: (1) Rec gBlock_Fl - Cgtactcgagcaacaatttctacaaaacacttga [SEQ ID NO: 18]; (2) Rec gBLock_Rl (reverse complement) - actgcggccgcttatctagatttctcaaactgcgg [SEQ ID NO: 19]; (3) Rec gBlock_F2 - cgtactcgagcaacaatttctacaaaaca [SEQ ID NO: 20]; (4) Rec gBlock_R2 -

Actgcggccgcttatctagatttct [SEQ ID NO: 21] (reverse complement); and (5) Seq F for Rec-x- strpFlag (forward for sequencing, binds prior to nanobody upstream in constitutive RecA promoter) - Aggaggtgtactagcatatgat [SEQ ID NO: 22], Lane 1 included a 1 kb DNA ladder (GeneRuler), while Lanes 2-12 were run with PCR reaction performed using geneBlock as a template in PCR reaction. Amplified products of the reactions were then run on 1% Agarose gel supplemented with traces of SYBR® safe DNA gel strain (Invitrogen) and picture was taken using Safe Imager™ 2.0 Blue-Light Transilluminator (Thermo Fisher Scientific).

[0124] FIGS. 28A and 28B show High Fidelity PCR amplification for the RecA-Lysis- RecA(ALexA)-PD-Ll geneblock. The RecA-Lysis-RecA (ALexA)-PD-Ll gene block was PCR amplified using the primers described above. Lane 1 included a 1 kb DNA ladder (GeneRuler), while Lanes 2-12 were run with PCR reaction performed using geneBlock as a template in the PCR reaction. Amplified products of the reactions were then run on 1% Agarose gel supplemented with traces of SYBR® safe DNA gel strain (Invitrogen) and picture was taken using Safe Imager™ 2.0 Blue-Light Transilluminator (Thermo Fisher Scientific).

[0125] Amplified PCR product for RecA-Lysis-RecA (ALexA)-PD-Ll-ePOP was digested along with ePOP plasmid as shown in FIG. 28A by using PspXI and Notl restriction enzymes, for placing the RecA-Lysis-RecA (ALexA)-PD-Ll-ePOP) gene sequence in the ePOP plasmid. This resulted in a newly constructed plasmid named RecRadePOP as shown (see FIG. 31), and it has TEV site, and a Flag tag followed by a Strp Tag for detection and purification. Following restriction digest, digest reactions were run on 1% Agarose gel as shown in FIG. 29. These were gel extracted, cleaned, and ligated overnight.

[0126] FIG. 29 shows a restriction digest for cloning PD-L1 into the ePOP plasmid. RecA- Lysis-RecA(ALexA)-PD-Ll gene block was PCR amplified and 1 pg of PCR product along with 1 pg of ePOP plasmid were digested using PxpXL Notl and Xhol-Notl. Lane 1 included a 1 kb DNA ladder (GeneRuler), while Lanes 2-3 were run with PCR for Geneblock and Lanes 4-6 were ePOP plasmid digested with PspXI-Notl. Similarly, Lanes 7-8 were loaded with PCR products for Geenblock and Lanes 9-12 were loaded with ePOP plasmid digested with XhoL Notl. Restriction digests were run on 1% Agarose gel supplemented with traces of SYBR safe DNA gel strain (Invitrogen) and picture was taken using Safe Imager™ 2.0 Blue-Light Transilluminator (Thermo Fisher Scientific).

[0127] Following overnight ligation, 3.5 pL of ligation mixtures was chemically transformed into DH5a competent cells (NEB) and spread on chloramphenicol selection plates. Plates were incubated at 37°C overnight. About 40-colonies were randomly screened for successful cloning by performing colony PCR on the selected colonies, as shown in FIG. 32.

[0128] Referring now to FIG. 33, diagnostic PCR reactions were performed by screening ~36 colonies for the confirmation of RecA-Lysis-Rec-PD-Ll cloning into ePOP plasmid, using DreamTag DNA polymerase (Thermo Fisher Scientific). PCR reactions were run on 1% Agarose, supplemented with traces of SYBR® safe DNA gel stain and visualized by using Invitrogen Safe Imager 2.0 Blue-Light (Thermo Fisher Scientific), while the picture was taken using iPhone. Samples were in Lanes 2-1 , Lane 20 was run with positive control, while Lane 1 loaded with 1 kB DNA ladder (GeneRuler).

[0129] Transformation

[0130] Following the final confirmation of successful molecular cloning, the constructs were ready for testing the expression of the Lysis gene and the PD-L1 nanobody.

[0131] Following confirmation of the successful cloning of RecA-Lysis-RecA (ALexA)-PD- L1 into ePOP plasmid between, PsPXI-Notl with Sanger sequencing and NCBI’s BLAST analysis, the plasmids extracted from Colony 10 and Colony 18 were transformed into chemically competent E. coli Nissle 1917 cells. These were then spread on antibiotic selection plates following overnight incubation at 37 °C.

[0132] SDS-PAGE Western blot analysis

[0133] Single positive clones were selected from each of Colony 10 and Colony 18 and grown in 3 ml LB broth, supplemented with 25 mg/ml of Chloramphenicol, along with Wild Type E. coli Nissle 1917 at 37°C and 200 rpm. Following 24 hr incubation, 1% of these cultures were inoculated into 10 ml of fresh LB supplemented with appropriate antibiotic, as previously described and similarly grown at 37°C and 200 rpm grew until OD600 reaches 0.9- 1.0 (approx. 3.00 hr).

[0134] These cultures were then centrifuged at 4°C for 15 min at 3500 xg and cells were resuspended in 2 ml of the media. These cells were redistributed in a 500 pL Eppendorf tube, and a 1.5 ml Eppendorf tube.

[0135] Both these tubes containing resuspended cells were centrifuged at 4 °C for 15 min at 13,000xg. For the 500 pL cells containing Eppendorfs, supernatant was discarded and labelled as Cell Fraction. In case of the Eppendorfs that contained 1.5 ml re-suspended cells, cell pellet was discarded and 1350 pL supernatant was collected in another Eppendorf and placed on ice. To these Eppendorfs, 150 pL of Ice-cold Tri-Chloro Acetic Acid (TCA) was added incubated on ice for 30 min, following which these were centrifuged at 4°C for 15 min at 3500 xg. The supernatant was discarded, and the pellet was washed with 950 pL of cold- Acetone and centrifuged at 4°C for 15 min at 3500 xg. Following centrifuge, the entire content of the tube was decanted and air dried, prior to adding 2x SDS- loading buffer. The CELL FRACTION and SUPERNATANT containing tubes were added with 2x Loading buffer according to the OD600 by using following equations: (1) Loading Buffer for cell pellet: OD600 x 200/2, and (2) Loading buffer for cell supernatant: OD600xl500x 50.

[0136] Both of these cell fraction and cell supernatants were then heated at 95°C for 10-15 min using a heat block with intermittent shaking.

[0137] Meanwhile, 20 % SDS-PAGE gels were prepared and placed in a SDS-running gel tank (BioRad) containing lx SDS running buffer. The cell fraction and cell supernatants were loaded on the gel and run at 95°C for 2.5 - 3.0 hr. The gels were then transformed to Nitrocellulose paper by using Trans-blot Turbo transfer system (BioRad). Blots were then blocked using 5% milk in 0.1%, lx TBS Tween buffer overnight at 4°C. These were then washed 3x using 0.1%, lx TBS Tween and added with lx TBS buffer. 1:500 HRP-conjugated Flag-Tag Antibody was added and incubated 4.00 hr at room temperature.

[0138] These blots were then washed again 3x times with 0.1 %, IX TBS Tween buffer, and transferred into a suitable box and added with equal volumes of Chemiluminescent Dark and Light substrates (Super Signal, West Pico Plus, Thermo Fisher Scientific) and incubated for 5 min in dark at room temperature (RT). These blots were then developed using ChemiDoc Imager (BioRad). The results are shown in FIG. 34 (for intercellular proteins - cell fractions) and FIG. 35 (for extracellular proteins - cell supernatant). These are discussed further in the section below.

[0139] SDS-PAGE -Western blot analysis: Detection ofPD-Ll nanobody

[0140] As mentioned above, FIG. 34 shows the results of a SDS-PAGE Western blot analysis of intracellular proteins (cell fraction). Cell fraction extracted from the cells expressing RecRadePOP (RecA-Lysis-RecA(ALexA)-PD-Ll) plasmid in E. coli Nissle 1917 was run on a SDS-PAGE gel, with 2x SDS-loading buffer. Lane 1 was loaded with a pre-stained EZ Rec protein Ladder (EZ-Run™ Pre-stained Rec Protein Ladder, Fisher Bioreagents). Lanes 2-5 were run with the RecA-lysis-ePOP plasmid while Lanes 6-10 were run with E. coli Nissle 1917 expressing RecRadePOP (RecA-Lysis-RecA(ALexA)-PD-Ll) plasmid in increasing volume of 5-25 pL, as shown. Blots were developed using ChemiDoc Imaging system (BioRad). As can be seen in FIG. 34, a 17.5kDa band appears only for the cell fraction taken from cells expressing the RecRadePOP plasmid [i.e., those including the PD-L1 gene: RecA- Lysis-RecA(ALexA)-PD-Ll], And that band appears in each of Lanes 6-10. No bands appear in any of the lanes where PD-L1 gene was absent (i.e., in the lanes from cells only having RecA-Lysis-ePOP plasmid).

[0141] Next, FIG. 35 shows the results of a SDS-PAGE Western blot analysis of extracellular proteins (cell supernatant). Proteins from cell supernatant precipitated using a TC A- Acetone method extracted from cells expressing RecRadePOP [RecA-Lysis-RecA(ALexA)-PD-Ll] plasmid in E. coli Nissle 1917 were run on a SDS-PAGE gel, with 2x SDS-loading buffer. Lane 1 was loaded with pre-stained EZ Rec protein Ladder (EZ-Run™ Pre-stained Rec Protein Ladder, Fisher Bioreagents), Lanes 2-3 were loaded with E. coli Nissle 1917 expressing RecRadePOP [RecA-Lysis-RecA(ALexA)-PD-Ll] plasmid extracted and transformed from Sanger sequenced Colony 10, Lanes 4-5 from Colony 14, and Lanes 6-7 from Colony 18. Lanes 8-9 were loaded with wild type (WT) E. coli Nissle 1917, as a control, and Lane 10 was left empty. Blots were developed on ChemiDoc Imaging system (BioRad). No bands appear.

[0142] Absorbance measurement (QD600 nm)

[0143] A single positive clone from Colony 10 of RecRadePOP (3884bp) [RecA-Lysis-RecA (ALexA)-PD-Ll ePOP] was grown in 3 ml LB broth, supplemented with 25 mg/ml of Chloramphenicol, along with Wild Type E. coli Nissle 1917 and RecA-Lysis-ePOP as a negative control at 37 °C and 200 rpm. Following 24 hr incubation, 1 % of these cultures were inoculated into 10 Falcons containing 10 ml of fresh LB broth supplemented with appropriate antibiotic, as previously and similarly grown at 37 °C and 200 rpm grew until OD600 reaches 0.9- 1.0 (approx. 3.00 hr). Following incubation, OD600 were normalized to OD =1.0 Unit and exposed to radiation doses as shown in the Table of FIG. 36. Positive control was prepared by means of heat killing RecRadePOP (3884bp) [RecA-Lysis-RecA (ALexA)-PD-Ll ePOP] bearing cells at 75°C for 10 min. while unexposed cells harboring RecRadePOP (3884bp) [RecA-Lysis-RecA (ALexA)-PD-Ll ePOP] plasmid were used as a negative control along with wild-type E. coli Nissle \ Y1 and E. coli Nissle \ Y1 harboring RecA-Lysis-ePOP plasmid (No PD-L1 nanobody) - also as shown in FIG. 36. FIG. 37 then shows a plot of the OD600 (absorbance) measurements from the table of FIG. 36 before and after radiation exposure for each of the bacterial strains and radiation exposure doses..

[0144] Determination of cargo release following radiation exposure: Detection of PD-L1 nanobody following radiation exposure

[0145] FIG. 38 shows a study regarding the detection of PD-L1 nanobody following radiation exposure. In particular, the figure shows an SDS-PAGE Western blot analysis of extracellular proteins (cell fraction) in RecA-Lysis-RecA(ALexA)-PD-Ll-ePOP-EcN 1917 at various levels of radiation exposure. In that regard, RecA-Lysis-RecA(ALexA)-PD-Ll-ePOP-E. coli Nissle 1917 was exposed to varying levels of radiation at 0.5Gy, l.OGy, 2.0Gy, 4.0Gy, and 8.0Gy. As can be seen in Fig. 38, PD-L1 was observed to be present in cell supernatant (26kDa fragment) only if exposed to radiations. The PD-L1 band appeared at all radiation dose levels, and is also seen in positive control lane. PD-L1 is absent in negative control, and in lanes for RecA-Lysis-ePOP EcN Nissle 1917 and wild type EcN 1917. This demonstrates release of PD-L1 nanobodies following radiation exposure of the cell (resulting in lysis of the cell) - because PD-L1 (1) was detected in cell supernatant following radiation exposure, and (2) was detected in cell supernatant only if exposed to radiation.

[0146] And so, the present inventors have successfully cloned RecA-Lysis-RecA(ALexA)-PD- Ll-TEV-Strep-Flag-geneblock into ePOP to get a RecRadePOP plasmid (3884bp). And, the present inventors have demonstrated the successful expression of PD-L1 antibody in test sample but not in the control sample of E. coli Nissle 1917.

[0147] Survival study using MC38 cell line and C57BL/6 mice for testing efficacy ofanti-PDL- 1 Nanobody generated using RecA-Lysis-RecA(dLexA)-PD-Ll ePOP construct in bacteria [0148] FIGS. 39 and 40 show a survival study using an MC38 cell line and C57BL/6 mice for testing efficacy of anti-PD-Ll Nanobody generated using RecA-Lysis-RecA(ALexA)-PD-Ll ePOP construct in bacteria. Experimental design (Three fraction study): 8 Gy dose MC38 cells and C57 BL/6 mice.

[0149] FIGS. 39 and 40 show the mice injected intratumorally with control bacteria or bacteria expressing engineered plasmids. After radiation exposure, these mice were imaged using a Brucker Imaging system pre- and post-radiation exposure. The bioluminescence data output was plotted and reported in FIGS. 41A-C. In that regard, FIG. 41 A shows imaging pre and post imaging, FIG. 41B on Day 3, and FIG. 41C on day 5. Altogether, FIGS. 41A-C essentially show a significant %fold decrease in bioluminescence in the lysis group that was irradiated consistent with lysis. Post-radiation the present inventors observed the population of bacteria to increase prior to the next RT fraction after 2 days, suggesting repopulation of bacteria occurring intratumorally. We tracked the bacterial numbers over 5 days and observed the population of bacteria to increase near baseline following initial decrease post-RT.

[0150] FIG. 42 shows the schematic diagram of experimental design along with different groups used for comparison. Bacteria were injected intratumorally and exposed to the radiation doses.

[0151] MC38 mice bearing tumors were injected with engineered bacteria or control bacteria and exposed to the radiation. Survival was monitored over a period of time. The survival were plotted and as we can see, the mice injected with engineered bacteria survive significantly longer than the several groups tested. Referring to FIG. 43, tumor growth progression was found to be comparatively slower in radiation treated group, that released anti-PD-Ll nanobody upon induction of RecA promoter followed by bacterial cell lysis. This combination of radiation dose and anti-PD-Ll nanobody in this group demonstrated the slowest tumor growth progression with compared to the other six groups tested, with a significant increase in median survival to up to 35 days

[0152] FIG. 44 then shows a line plot for different groups showing mean tumor volume over the course of study (n = 4). EcN bearing a lysis gene and rest of the control groups comparison was performed using Wilcoxon matched-pairs signed rank paired t-test, p-value summary = 0.0257 * or ** (0.0016), data represented as mean. FIG. 45 then reports percent of each population surviving over time. (Data on survival can be seen in the table of FIG. 46.)

[0153] We evaluated the efficiency of anti-PD-Ll nanobody expression and its effect on improving targeted therapeutic outcomes in vivo. A total of seven treatment groups using rigorous control groups were assessed in parallel, as shown in Fig. 42 represents the mean tumor volume progression with time. MC38 mouse colon cancer tumors grow at a faster rate, and as anticipated, mice in control group (saline) reached their experimental endpoint (tumor size of 1000 mm3) quickly with a median survival of 11 days. Tumor growth progression was found to be comparatively slower in radiation treated group, that released anti-PD-Ll nanobody upon induction of RecA promoter followed by bacterial cell lysis. This combination of radiation dose and anti-PD-Ll nanobody in this group demonstrated the slowest tumor growth progression with compared to the other six groups tested, with a significant increase in median survival to up to 35 days (Lytic EcN (PD-L1)+ 0.0 Gy vs Lytic EcN (PD-L1) + 5.0 Gy : *p =0.0225, Non-lytic EcN (PD-L1)+ 0.0 Gy vs Lytic EcN (PD-L1) + 5.0 Gy: *p=0.0260, Non- Lytic EcN (PD-L1) + 5.0 Gy vs Lytic EcN (PD-L1) + 5.0 Gy: *p=0.0133, Lytic EcN + 5.0 Gy vs Lytic EcN (PD-L1) + 5.0 Gy: *p = 0.0128, Saline + 5.0 Gy vs Lytic EcN (PD-L1) + 5.0 Gy: **p=0.0.0016, Saline + 0.0 Gy vs Lytic EcN (PD-L1) + 5.0 Gy: **p=0.0055).

[0154] PD-1/PD-L1 blockade Nanobody in vitro assay

[0155] Following confirmation of anti-PD-Ll nanobody expression, the present inventors investigated if the nanobodies were capable of inhibiting the PD-1 and PD-L1 interaction. PD- 1 effector cells expressing NFAT-RE-Luciferase and PD-L1 antigen presenting cells (PD-L1- aAPC/CHO-Kl cells) were chosen for this study. An assay was performed using anti-PD-Ll antibody (9.89 mg/ml) and anti-PD-1 antibody (2 mg/ml) along with protein extract isolated from anti-PD-Ll nanobody expressing bacteria and control bacteria. A schematic of the assay is shown in FIGS. 48 and 49.

[0156] Using the fold induction equation, the present inventors found that anti-PD-Ll nanobodies had inhibited the PD-1/PD-L1 interaction almost same as 45 ng/pl anti-PD-1 antibody. The inventors observed a decreasing trend of PD-1/PD-L1 inhibition with decreasing nanobody concentration that was consistent with decreasing concentrations of anti PD-1 antibody, from 45 - 10 ng/pl (see FIG. 50).

[0157] Survival study using MOCJ cell line (head & neck cancer} and C57BL/6 mice for testing efficacy of anti-PD-Ll Nanobody generated using RecA-Lysis-RecA(ALexA}-PD-Ll ePOP construct in bacteria

[0158] The present inventors then evaluated the efficiency of anti-PD-Ll nanobody expression and its effect on improving targeted therapeutic outcomes in vivo. A total of seven treatment groups using rigorous control groups were assessed in parallel, as shown in the schematic of FIGS. 51A and 51B. FIGS. 51C and 51D represent the mean tumor volume progression with time. MOC1 mouse cancer tumors grow at a faster rate, and as anticipated, mice in control group (saline) reached their experimental endpoint (tumor size of 1000 mm³) quickly with a median survival of 35 days. Tumor growth progression was found to be comparatively slower in radiation treated group, that released anti-PD-Ll nanobody upon induction of RecA promoter followed by bacterial cell lysis. This combination of radiation dose and anti-PD-Ll nanobody in this group demonstrated the slowest tumor growth progression with compared to the other six groups tested, with a significant increase in median survival to up to 77 days. [(Lytic EcN (PD-L1) + 0.0 Gy vs Lytic EcN (PD-L1) + 5.0 Gy : **p =0.0013, Non-Lytic EcN (PD-L1) + 5.0 Gy vs Lytic EcN (PD-L1) + 5.0 Gy: ***p=0.0008, Lytic EcN + 5.0 Gy vs Lytic EcN (PD-L1) + 5.0 Gy: **p = 0.0013, Saline + 5.0 Gy vs Lytic EcN (PD-L1) + 5.0 Gy: ***p<0.0001, Saline + 0.0 Gy vs Lytic EcN (PD-L1) + 5.0 Gy: **p<0.0001)]

SEQUENCES

[0159] The following are sequences for the RecA-Lysis-terminator-RecA(ALexA)-PD-Ll- ePOP construct (RecRadePOP).

[0160] Complete plasmid sequence:

[0161] gacgtcagtcctttgattctaataaattggatttttgtcacactattgtatcgctgggaatacaattacttaacataagcacctgta ggatcgtacaggtttacgcaagaaaatggtttgttatagtcgaataaacgcaagggaggttggtatggtacgctggactttgtgggatac cctcgctttcctgctcctgttgagtttattgctgccgtcattgcttattatgttcatcccgtcaacattcaaacggcctgtctcatcatggaagg cgctgaatttacggaaaacattattaatggcgtcgagcgtccggttaaagccgctgaattgttcgcgtttaccttgcgtgtacgcgcagg aaacactgacgttcttactgacgcagaagaaaacgtgcgtcaaaaattacgtgcggaaggagtgagacgtctgtgtggaattgtgagc ggataacaatttcacacagggccctcggacaccgaggagaatgtcaagaggcgaacacacaacgtcttggagcgccagaggagga acgagctaaaacggagcttttttgccctgcgtgaccagatcccggagttggaaaacaatgaaaaggcccccaaggtagttatccttaaa aaagccacagcatacatcctgtccgtccaagcagaggagcaaaagctcatttctgaagaggacttgttgcggaaacgacgagaacag ttgaaacacaaacttgaacagctacggaactcttgtgcgtaaggaaaagtaaggaaaacgattccttctaacagaaatgtcctgagcaa tcacctatgaactgtcgACTCGAGCaacaatttctacaaaacacttgatactgtatgagcatacagtataattgcttcaacagaaca tattgactatccggtattacccggcatgacaggagtaaaaatggctatcgacgaaaacaaacagaaagcgttggcggcagcactggg ccagattgagaaacaatttggtaaaggctccatcatgtaataaggtggttctggtggtggttctggtAGGAGGTGTACTAGa tggtacgctggactttgtgggataccctcgctttcctgctcctgttgagtttattgctgccgtcattgcttattatgttcatcccgtcaacattca aacggcctgtctcatcatggaaggcgctgaatttacggaaaacattattaatggcgtcgagcgtccggttaaagccgctgaattgttcgc gtttaccttgcgtgtacgcgcaggaaacactgacgttcttactgacgcagaagaaaacgtgcgtcaaaaattacgtgcggaaggagtg aACGCGTccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctct actagagtcacactggctcaccttcgggtgggcctttctgcgtttataCTTGATAAGGTCCACGTAGCTGCTAT AATTGCTTCAACAGAACATATTGACTATCCGGTATTACCCGGCAGGAGGTGTACT AGCATATGATGCAGGTCCAATTGCAAGAATCGGGCGGCGGTTTGGTCCAGCCAG GTGGCTCATTACGTTTAAGTTGTGCAGCATCCGGCAAGATGTCCAGCCGTCGTTG TATGGCATGGTTCCGCCAGGCTCCCGGTAAAGAGCGTGAACGTGTAGCGAAGTT GTTGACAACCTCTGGAAGCACCTACCTTGCCGATAGCGTGAAAGGGCGTTTCACC ATTTCGCAGAACAACGCAAAATCGACAGTCTACCTGCAAATGAACTCATTAAAG CCAGAGGATACCGCTATGTATTATTGTGCCGCCGATTCCTTTGAAGACCCCACTT GCACGCTTGTCACTTCGTCGGGTGCTTTCCAGTATTGGGGACAGGGCACCCAAGT GACCGTGTCTaAAATGCATGAAAACCTGTACTTCCAAGGTGACTACAAGGACGAT GACGATAAGTGGAGCCATCCGCAGTTTGAGAAATCTAGATAAgcggccgcttaattaattaat ctagaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgtttatctgttgttgtcggtgaacgctctcctgagtagg acaaatccgccgccctagacctaggcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaat caggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgttttt ccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagatacc aggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaag cgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgtt cagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactg gtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagt atttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcg gtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcag tggaacgaaaactcacgttaagggattttggtcatgactagtgcttggattctcaccaataaaaaacgcccggcggcaaccgagcgttc tgaacaaatccagatggagttctgaggtcattactggatctatcaacaggagtccaagcgagctcgatatcaaattacgccccgccctg ccactcatcgcagtactgttgtaattcattaagcattctgccgacatggaagccatcacagacggcatgatgaacctgaatcgccagcg gcatcagcaccttgtcgccttgcgtataatatttgcccatggtgaaaacgggggcgaagaagttgtccatattggccacgtttaaatcaa aactggtgaaactcacccagggattggctgagacgaaaaacatattctcaataaaccctttagggaaataggccaggttttcaccgtaa cacgccacatcttgcgaatatatgtgtagaaactgccggaaatcgtcgtggtattcactccagagcgatgaaaacgtttcagtttgctcat ggaaaacggtgtaacaagggtgaacactatcccatatcaccagctcaccgtctttcattgccatacgaaattccggatgagcattcatca ggcgggcaagaatgtgaataaaggccggataaaacttgtgcttatttttctttacggtctttaaaaaggccgtaatatccagctgaacggt ctggttataggtacattgagcaactgactgaaatgcctcaaaatgttctttacgatgccattgggatatatcaacggtggtatatccagtga tttttttctccattttagcttccttagctcctgaaaatctcgataactcaaaaaatacgcccggtagtgatcttatttcattatggtgaaagttgg aacctcttacgtgccgatcaacgtctcattttcgccagatatc [SEQ ID NO:1]

[0162] RecA promoter:

[0163] Aacaatttctacaaaacacttgatactgtatgagcatacagtataattgcttcaacagaacatattgactatccggtattacccg gcatgacaggagtaaaaatggctatcgacgaaaacaaacagaaagcgttggcggcagcactgggccagattgagaaacaatttggt aaaggctccatcatgtaataa [SEQ ID NO: 2]

[0164] Lysis gene ($x!74):

[0165] Atggtacgctggactttgtgggataccctcgctttcctgctcctgttgagtttattgctgccgtcattgcttattatgttcatcccg tcaacattcaaacggcctgtctcatcatggaaggcgctgaatttacggaaaacattattaatggcgtcgagcgtccggttaaagccgctg aattgttcgcgtttaccttgcgtgtacgcgcaggaaacactgacgttcttactgacgcagaagaaaacgtgcgtcaaaaattacgtgcgg aaggagtga [SEQ ID NO: 3]

[0166] rmB terminator:

[0167] Caaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctc [SEQ ID

NO: 4]

[0168] T7Te terminator:

[0169] Ggctcaccttcgggtgggcctttctgcg [SEQ ID NO: 5]

[0170] Rec(ALexA) constitutive promoter:

[0171] CTTGATAAGGTCCACGTAGCTGCTATAATTGCTTCAACAGAACATATTGAC TATCCGGTATTACCCGGC [SEQ ID NO: 6]

[0172] Anti-PD-Ll nanobody:

[0173] ATGCAGGTCCAATTGCAAGAATCGGGCGGCGGTTTGGTCCAGCCAGGTGG CTCATTACGTTTAAGTTGTGCAGCATCCGGCAAGATGTCCAGCCGTCGTTGTATG

GCATGGTTCCGCCAGGCTCCCGGTAAAGAGCGTGAACGTGTAGCGAAGTTGTTG ACAACCTCTGGAAGCACCTACCTTGCCGATAGCGTGAAAGGGCGTTTCACCATTT

CGCAGAACAACGCAAAATCGACAGTCTACCTGCAAATGAACTCATTAAAGCCAG AGGATACCGCTATGTATTATTGTGCCGCCGATTCCTTTGAAGACCCCACTTGCAC GCTTGTCACTTCGTCGGGTGCTTTCCAGTATTGGGGACAGGGCACCCAAGTGACC

GTGTCTaAA [SEQ ID NO: 7]

[0174] TEV cleavage site:

[0175] GAAAACCTGTACTTCCAAGGT [SEQ ID NO: 8]

[0176] Flag Tag:

[0177] GACTACAAGGACGATGACGATAAG [SEQ ID NO: 9]

[0178] Strep n tag:

[0179] TGGAGCCATCCGCAGTTTGAGAAA [SEQ ID NO: 10]

[0180]

[0181] Lac operator: 443- 459 17 bp

[0182] bound moiety = lac repressor encoded by lacl

[0183] The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or iso-propyl-P-D-thiogalactopyranoside (IPTG) [0184] >Myc: 661-690, 30bp- CDS

[0185] Myc (human c-Myc proto-oncogene) epitope tag

[0186] Translation: EQKLISEEDL [SEQ ID NO: 11]

[0187] 10 amino acids= 1.2 kDa

[0188] RecA promoter: 826-1017, 192 bp: promoter

[0189] Linker: 1018-1041, 24 bp

[0190] RBS: 1042- 1055, 14 bp

[0191] Lysis gene: 1056-1331, 276 bp- CDS

[0192] Translation:

MVRWTLWDTLAFLLLLSLLLPSLLIMFIPSTFKRPVSSWKALNLRKTLLMASSVRLKP

LNCSRLPCVYAQETLTFLLTQKKTCVKNYVRKE* [SEQ ID NO: 12]

[0193] 91 AMINO ACIDS = 10.6 kDa

[0194] rmB T1 terminator: 1346- 1417, 72 bp: terminator

[0195] transcription terminator T1 from the E. coli rmB gene

[0196] T7Te terminator: 1433-1460, 28 bp: terminator

[0197] Phage T7 early transcription terminator

[0198] RecA(ALexA): 1467-1535, 69 bp- promoter

[0199] Constitutive active E. coli RecA promoter lacking the LexA binding site (Brent and Ptashne, 1981).

[0200] Anti-PD-Ll nanobody: 1556-1942, 387 bp- CDS [0201] MQVQLQESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVA

KLLTTSGSTYLADSVKGRFTISQNNAKSTVYLQMNSLKPEDTAMYYCAADSFEDPTC

TLVTSSGAFQYWGQGTQVTVSK* [SEQ ID NO: 13]

[0202] 129 amino acids= 14.0 kDa

[0203] TEV cleavage site: 1949-1969, 21 bp- CDS

[0204] Tobacco EtchVirus (TEV) protease recognition and cleavage site

[0205] Translation: ENLYFQG [SEQ ID NO: 14]

[0206] 7 amino acids: 870.0 Da

[0207] Flag tag: 1970- 1993, 24 bp- CDS

[0208] FLAG® epitope tag, followed by an enterokinase cleavage site

[0209] Translation: DYKDDDDL [SEQ ID NO: 15]

[0210] 8 amino acids=1.0 kDa

[0211] Strep H tag: 1994-2017, 24 bp- CDS

[0212] Peptide that binds Strep-Tectin®, an engineered form of streptavidin

[0213] Translation: WSHPQFEK [SEQ ID NO: 16]

[0214] 8 amino acids= 1.1 kDa

[0215] rmB T1 terminator: 2058-2144, 87 bp- terminator

[0216] E. coli: rmB gene

[0217] Transcription terminator T1 from the E. coli rmB gene

[0218] ori: 2308- 2896, 589 bp- Rep origin

[0219] direction- LEFT

[0220] High copy number EolEl/pMBl/pBR332/pUC origin of replication

[0221] Lambda tO terminator: 2984- 3078, 95 bp- terminator

[0222] Transcription terminator from phage lambda

[0223] CmR: 3099- 3758, 660 bp- CDS

[0224] Gene- cat- chloramphenicol acetyltransferase- confers resistance to chloramphenicol

[0225] Translation:

[0226] MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFLKTVKKNK

HKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSSLWSEY

HDDFRQFLHIYSQDVACYGENLAYFPKGFTENMFFVSANPWVSFTSFDLNVANMDN

FFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA

* [SEQ ID NO: 17]

[0227] 219 AMINO ACIDS = 25.7 kDa

[0228] Cat promoter: 3759- 3861, 103 bp- promoter [0229] Promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase

[0230] The embodiments of the present invention recited herein are intended to be merely exemplary and those skilled in the art will be able to make numerous variations and modifications to it without departing from the spirit of the present invention. Notwithstanding the above, certain variations and modifications, while producing less than optimal results, may still produce satisfactory results. All such variations and modifications are intended to be within the scope of the present invention as defined by the claims appended hereto

WHAT TS CLAIMED TS:

Claims

1. A genetically modified bacterium comprising a lysis gene, the lysis gene including a nucleic acid encoding for a protein, the expression of which results in lysis of the bacterium, wherein the nucleic acid expresses the protein in response to exposure to radiation.

2. The genetically modified bacterium of claim 1, wherein the genome of the bacterium includes a plasmid, the nucleic acid sequence of the plasmid including the lysis gene.

3. The genetically modified bacterium of claim 2, wherein the lysis gene is obtained from a $xl74 bacteriophage.

4. The genetically modified bacterium of claim 3, wherein the nucleic acid of the lysis gene has a sequence of [SEQ ID NO: 3].

5. The genetically modified bacterium of claim 2, wherein the plasmid further comprises a nucleic acid sequence encoding a first promoter that is operable to initiate transcription of the nucleic acid sequence of the lysis gene.

6. The genetically modified bacterium of claim 5, wherein the first promoter is inducible.

7. The genetically modified bacterium of claim 6, wherein the first promoter is inducible in response to exposure to radiation.

8. The genetically modified bacterium of claim 7, wherein the first promoter is inducible in response to exposure ot radiation at a dose of 2.0 - 4.0Gy.

9. The genetically modified bacterium of claim 5, wherein the first promoter is the promoter of a RecA gene.

10. The genetically modified bacterium of claim 9, wherein the first promoter has a sequence of [SEQ ID NO: 2].

11. The genetically modified bacterium of claim 5, wherein the plasmid further comprises a gene including a nucleic acid encoding for a biotherapeutic molecule.

12. The genetically modified bacterium of claim 11, wherein the biotherapeutic molecule is chosen from anti-PD-Ll, anti-EGFR, GM-CSF, IL-2, TNFa, and INFy.

13. The genetically modified bacterium of claim 11, further comprising a TEV cleavage site from Tobacco Etch Virus associated with the nucleic acid encoding the biotherapeutic molecule.

14. The genetically modified bacterium of claim 1 , further comprising a Flag tag.

15. The genetically modified bacterium of claim 14, further comprising a Strep II tag.

16. The genetically modified bacterium of claim 11, wherein the plasmid further comprises a nucleic acid sequence encoding a second promoter that is operable to initiate transcription of the nucleic acid sequence encoding the biotherapeutic molecule.

17. The genetically modified bacterium of claim 16, wherein the second promoter is constitutively expressed.

18. The genetically modified bacterium of claim 17, wherein the second promoter is a modified promoter of a RecA gene.

19. The genetically modified bacterium of claim 18, wherein the second promoter is modified by deleting a LexA repressor binding site of the promoter of the RecA gene.

20. The genetically modified bacterium of claim 19. wherein the second promoter has a sequence of [SEQ ID NO: 6],

21. The genetically modified bacterium of claim 1, wherein the bacterium that is modified is E. coli.

22. The genetically modified bacterium of claim 1, wherein the bacterium that is modified is E. coli Nissle 1917.

23. A plasmid for insertion into a bacterium, the plasmid comprising a nucleic acid encoding a lysis gene, a nucleic acid encoding a first promoter, a nucleic acid encoding a biotherapeutic molecule, and a nucleic acid encoding a second promoter, wherein the nucleic acid encoding a lysis gene includes a sequence encoding a protein, the expression of which results in lysis of a bacterium, that has had the plasmid inserted thereinto, wherein the nucleic acid expresses the protein in response to exposure to radiation.

24. The plasmid of claim 23, wherein the lysis gene is obtained from a $xl74 bacteriophage.

25. The plasmid of claim 24, wherein the nucleic acid of the lysis gene has a sequence of [SEQ ID NO: 3].

26. The plasmid of claim 23, wherein the first promoter is operable to initiate transcription of the nucleic acid sequence of the lysis gene.

27. The plasmid of claim 26, wherein the first promoter is inducible.

28. The plasmid of claim 27, wherein the first promoter is inducible in response to exposure to radiation.

29. The plasmid of claim 28, wherein the first promoter is inducible in response to exposure to radiation at a dose of 2.0 - 4.0Gy.

30. The plasmid of claim 26, wherein the first promoter is the promoter of a RecA gene.

31. The plasmid of claim 30, wherein the first promoter has a sequence of [SEQ ID NO: 2].

32. The plasmid of claim 23, wherein the biotherapeutic molecule is chosen from anti-PD- Ll, anti-EGFR, GM-CSF, IL-2, TNFa, and INFy.

33. The plasmid of claim 23, wherein the second promoter is operable to initiate transcription of the nucleic acid sequence encoding the biotherapeutic molecule.

34. The plasmid of claim 33, wherein the second promoter is constitutively expressed.

35. The plasmid of claim 34, wherein the second promoter is a modified promoter of a RecA gene.

36. The plasmid of claim 35, wherein the second promoter is modified by deleting a LexA repressor binding site of the promoter of the RecA gene.

37. The plasmid of claim 36. wherein the second promoter has a sequence of [SEQ ID NO: 6].

38. The plasmid of claim 23, further comprising a TEV cleavage site from Tobacco Etch Virus associated with the nucleic acid encoding the biotherapeutic molecule.

39. The plasmid of claim 38, further comprising a Flag tag.

40. The plasmid of claim 39, further comprising a Strep II tag.

41. The plasmid of claim 23, having a sequence of [SEQ ID NO: 1].