Bacterial cells designed to invade and autonomously lyse for intracellular delivery of therapeutic proteins

Abstract

Provided herein is a bacterial delivery platform that exploits a Salmonella -specific mechanism to deliver protein-based drugs intracellularly.

Description

[Technical Field]

[0001] government support This invention was made with government support under Grant No. R43 CA233136 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0002] Priority This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 147,506, filed February 9, 2021, the benefit of which is hereby claimed and incorporated by reference in its entirety. [Background technology]

[0003] Background of the Invention Cancer is generally characterized by uncontrolled and invasive cell growth. These cells may spread to other parts of the body (metastasis). Traditional anti-cancer therapies, consisting of surgical resection, radiation therapy, and chemotherapy, may be effective for some cancers / patients, but are ineffective for many cancer sufferers. Therefore, further medical treatment is required.

[0004] The role of bacteria as anticancer agents has been recognized for over 100 years, and numerous bacterial genera, including Clostridium, Bifidobacterium, and Salmonella, have been shown to preferentially accumulate in tumor tissue and induce regression.

[0005] The use of Salmonella typhimurium to treat solid tumors began with the development of a non-pathogenic strain, VNP20009. Well tolerated in mice and humans, this strain has been shown to preferentially accumulate (>2000-fold) within tumors across the liver, spleen, lungs, heart, and skin, delay tumor growth by 38-79%, and prolong survival in tumor-bearing mice. In early clinical trials, S. typhimurium was found to be well tolerated at high doses and capable of effectively colonizing human tumors. Summary of the Invention

[0006] Summary of the Invention Engineered, non-pathogenic Salmonella selectively colonize tumors 1000 times more frequently than any other organ, invade cytoplasmically into cancer cells, and deliver therapeutics, making the bacteria an ideal delivery vehicle for cancer therapy. Herein, we demonstrate that controlling the activity of flhDC and subsequent flagellar expression in engineered Salmonella enables selective intracellular protein delivery in tumor cells in vivo and in vitro. Expression of flhDC / flagellum is controlled to enable both tumor colonization and cancer cell invasion for the purpose of intracellular protein and therapeutic delivery. Flagella are required for cellular invasion into cancer cells in vitro and in vivo. However, flagellar expression of Salmonella in the bloodstream and / or systemic circulation results in rapid clearance and significantly reduces tumor colonization. As a result, an inducible version of flhDC was engineered into an engineered strain of Salmonella lacking the native version of the transcription factor (alternatively, the endogenous promoter for flhDC can be replaced with an inducible promoter). The inducible system allowed for tight expression control of flhDC within the therapeutic strain. Salmonella lacking the ability to express flhDC colonized tumors with greater selectivity than the parental control strain. Inducing expression of flhDC by "remote control" administration, for example, by arabinose in an arabinose-inducible system, within the engineered Salmonella within the tumor allowed intracellular invasion and protein delivery into tumor cells.

[0007] Described herein are methods for controlling Salmonella containing flagellum expression through external means (e.g., small-molecule inducible gene circuits or inducible expression systems) in such a way that engineered strains of Salmonella do not systemically express flagellin. Once the bacteria have colonized the tumor to optimal levels, a "remote control" / inducible strategy is then employed in which small molecules are used to activate expression of a recombinant and / or inducible version of the motility regulator flhDC, thereby inducing expression of the flagellum and type 3 secretion system.

[0008] Another embodiment provides for the deletion of the SseJ gene in Salmonella delivery strains, which restricts the location of Salmonella to Salmonella-containing vacuoles (SCVs) and increases the delivery capacity of the strain, with or without the aforementioned control of delivery.

[0009] One embodiment provides a bacterial cell comprising: a) inducible expression of a flagellum; and b) a lysis gene or lysis cassette operably linked to a Salmonella promoter induced within the cell. In one embodiment, the bacterial cell is an intratumoral bacterial cell. In another embodiment, the bacterial cell is a Clostridium, Bifidobacterium, Escherichia coli, or Salmonella cell. In one embodiment, the bacterial cell is a Salmonella cell. In one embodiment, the lysis cassette is lysine E from phage phiX174, a lysis cassette of phage iEPS5, or a lysis cassette from lambda phage. In another embodiment, the Salmonella promoter induced in the cell is a promoter for one of the genes in the Salmonella pathogenicity island 2 type III secretion system (SPI2-T3SS) selected from the group SpiC / SsaB, SseF, SseG, SseI, SseJ, SseK1, SseK2, SifA, SifB, PipB, PipB2, SopD2, GogB, SseL, SteC, SspH1, SspH2, or SirP.

[0010] In one embodiment, the cell does not contain endogenous flhDC expression. In another embodiment, the cell contains an exogenous inducible promoter operably linked to an endogenous or exogenous flhDC gene. In one embodiment, the exogenous inducible promoter is operably linked to an endogenous flhDC gene. In another embodiment, the exogenous inducible promoter is operably linked to an exogenous flhDC gene. In an embodiment, the exogenous inducible promoter comprises the arabinose-inducible promoter PBAD (L-arabinose), LacI (IPTG), salR, or nahR (acetylsalicylic acid (ASA)).

[0011] In one aspect, the bacterial cell comprises a deletion of SseJ or has reduced expression of SseJ. One embodiment provides a cell comprising a plasmid expressing a peptide, in one embodiment, the peptide is a therapeutic peptide such as NIPP1 or activated caspase 3.

[0012] One aspect provides a composition comprising a population of cells described herein and a pharmaceutically acceptable carrier. Another embodiment provides a method for selectively colonizing cancer cells, such as tumor and / or tumor-associated cells, comprising administering a population of bacterial cells described herein to a subject in need thereof. In one embodiment, the tumor-associated cells are tumor cells or intratumoral immune cells, cancer cells, or intratumoral stromal cells. Another embodiment provides a method for treating cancer, comprising administering to a subject in need thereof an effective amount of a population of bacterial cells described herein to treat the cancer. A further embodiment provides a method for inhibiting tumor growth / proliferation or reducing tumor volume / size, comprising administering to a subject in need thereof an effective amount of a population of bacterial cells described herein to suppress tumor growth or reduce tumor volume. Another embodiment provides a method for treating metastasis, reducing the formation / number of metastases, or inhibiting the spread of metastases, comprising administering to a subject in need thereof an effective amount of a population of bacterial cells described herein to treat metastasis, reduce the formation / number of metastases, or inhibit the spread of metastases. In one embodiment, the tumor, tumor-associated cell, cancer, or metastasis is a lung, liver, kidney, breast, prostate, pancreas, colon, head and neck, ovarian, and / or gastrointestinal tumor, tumor-associated cell, cancer, or metastasis. In one embodiment, the bacterial cell delivers a therapeutic peptide to the tumor, tumor-associated cell, cancer, or metastasis. In one embodiment, the peptide is NIPP1 or activated caspase 3. In one embodiment, the cell does not express endogenous flhDC. In another embodiment, expression of flhDC in the bacterial cell is under the control of an inducible promoter, the bacterial cell comprises an exogenous inducible promoter that controls expression of endogenous flhDC, or the bacterial cell comprises an exogenous inducible promoter operably linked to an exogenous flhDC gene. In one embodiment, expression of flhDC is sufficient to prevent colonization of the tumor, tumor-associated cell, cancer, or metastasis by the bacteria (e.g., 1×10 6 ~1×10 10 CFU / g tumor) and then induced.

[0013] One embodiment provides a bacterial cell comprising: a) a deletion of SseJ or reduced expression of SseJ; and b) a lysis gene or lysis cassette operably linked to a Salmonella promoter induced in the cell. In one embodiment, the bacterial cell is an intratumoral bacterial cell. In another embodiment, the bacterial cell is a Clostridium, Bifidobacterium, or Salmonella cell. In an embodiment, the bacterial cell is a Salmonella cell. In one embodiment, the lysis cassette is lysine E from phage phiX174, a lysis cassette of phage iEPS5, or a lysis cassette from lambda phage. In another embodiment, the Salmonella promoter induced in the cell is a promoter for one of the genes in the Salmonella pathogenicity island 2 type III secretion system (SPI2-T3SS) selected from the group SpiC / SsaB, SseF, SseG, SseI, SseJ, SseK1, SseK2, SifA, SifB, PipB, PipB2, SopD2, GogB, SseL, SteC, SspH1, SspH2, or SirP.

[0014] In one embodiment, the cell of any one of claims 28 to 33, wherein the cell does not contain endogenous flhDC expression. In another embodiment, the cell contains an exogenous inducible promoter operably linked to an endogenous or exogenous flhDC gene. In another embodiment, the exogenous inducible promoter is operably linked to an endogenous flhDC gene. In another embodiment, the exogenous inducible promoter is operably linked to an exogenous flhDC gene. In an embodiment, the exogenous inducible promoter comprises the arabinose-inducible promoter PBAD (L-arabinose), LacI (IPTG), nahR (acetylsalicylic acid (ASA)), or salR (acetylsalicylic acid (ASA)).

[0015] In one embodiment, the bacterial cell comprises a plasmid that expresses a peptide. In one embodiment, the peptide is a therapeutic peptide such as NIPP1 or activated caspase 3. One aspect provides a composition comprising a population of cells as described herein and a pharmaceutically acceptable carrier.

[0016] One embodiment provides a method for colonizing a tumor and / or tumor-associated cells, comprising administering a population of bacterial cells described herein to a subject in need thereof. In one embodiment, the tumor-associated cells are tumor cells, intratumoral immune cells, or intratumoral stromal cells. In one embodiment, a method for treating cancer is provided, comprising administering to a subject in need thereof an effective amount of a population of bacterial cells described herein to treat the cancer. Another embodiment provides a method for inhibiting tumor growth / proliferation or reducing tumor volume / size, comprising administering to a subject in need thereof an effective amount of a population of bacterial cells described herein to suppress tumor growth or reduce tumor volume. A further embodiment provides a method for treating metastasis, reducing the formation / number of metastases, or inhibiting the spread of metastases, comprising administering to a subject in need thereof an effective amount of a population of bacterial cells described herein to treat metastasis, reduce the formation / number of metastases, or inhibit the spread of metastases. In one aspect, the tumor, tumor-associated cell, cancer, or metastasis is a lung, liver, kidney, breast, prostate, pancreas, colon, head and neck, ovarian, and / or gastrointestinal tumor, tumor-associated cell, cancer, or metastasis. In another aspect, the bacterial cell delivers a therapeutic peptide, such as NIPP1 or activated caspase 3, to the tumor, tumor-associated cell, cancer, or metastasis. In one embodiment, endogenous expression of flhDC is under the control of an exogenous inducible promoter. In another aspect, expression of flhDC is under the control of an inducible promoter, and the bacterial cell comprises the exogenous inducible promoter operably linked to the exogenous flhDC gene. In a further aspect, expression of flhDC is induced after the tumor, tumor-associated cell, cancer, or metastasis is colonized by the bacterium.

[0017] One embodiment provides a bacterial cell comprising: a) constitutive or inducible expression of a therapeutic peptide, wherein the therapeutic peptide is activated caspase-3, and the activated caspase-3 is expressed as an activated protein without further processing; and b) a lysis gene or lysis cassette operably linked to a Salmonella promoter induced within the cell. In one embodiment, the bacterial cell is an intratumoral bacterial cell. In one embodiment, the bacterial cell is a Clostridium, Bifidobacterium, or Salmonella cell. In another embodiment, the bacterial cell is a Salmonella cell. In one embodiment, the lysis cassette is lysine E from phage phiX174, a lysis cassette of phage iEPS5, or a lysis cassette from lambda phage. In one embodiment, the Salmonella promoter induced in the cell is a promoter for one of the genes in the Salmonella pathogenicity island 2 type III secretion system (SPI2-T3SS) selected from the group SpiC / SsaB, SseF, SseG, SseI, SseJ, SseK1, SseK2, SifA, SifB, PipB, PipB2, SopD2, GogB, SseL, SteC, SspH1, SspH2, or SirP.

[0018] In another embodiment, the bacterial cell does not contain endogenous flhDC expression. In one embodiment, the bacterial cell contains an exogenous inducible promoter operably linked to an endogenous or exogenous flhDC gene. In one embodiment, the exogenous inducible promoter is operably linked to an endogenous flhDC gene. In another embodiment, the exogenous inducible promoter is operably linked to an exogenous flhDC gene. In one embodiment, the exogenous inducible promoter comprises the arabinose-inducible promoter PBAD (L-arabinose), LacI (IPTG), nahR (acetylsalicylic acid (ASA)), or salR acetylsalicylic acid (ASA).

[0019] In embodiments, the bacterial cell comprises a deletion of SseJ or has reduced expression of SseJ. One embodiment provides cells that express at least one additional exogenous therapeutic peptide, such as NIPP1.

[0020] Another aspect provides a composition comprising a population of cells described herein and a pharmaceutically acceptable carrier. One embodiment provides a method for colonizing a tumor and / or tumor-associated cells, comprising administering a population of bacterial cells described herein to a subject in need thereof. In one embodiment, the tumor-associated cells are tumor cells, intratumoral immune cells, or intratumoral stromal cells. One embodiment provides a method for treating cancer, comprising administering to a subject in need thereof an effective amount of a population of bacterial cells described herein to treat the cancer. One embodiment provides a method for inhibiting tumor growth / proliferation or reducing tumor volume / size, comprising administering to a subject in need thereof an effective amount of a population of bacterial cells described in any one of the claims described herein to suppress tumor growth or reduce tumor volume. One embodiment provides a method for treating metastasis, reducing the formation / number of metastases, or inhibiting the spread of metastases, comprising administering to a subject in need thereof an effective amount of a population of bacterial cells described herein to treat metastasis, reduce the formation / number of metastases, or inhibit the spread of metastases. In one embodiment, the tumor, tumor-associated cell, cancer, or metastasis is a lung, liver, kidney, breast, prostate, pancreas, colon, head and neck, ovarian, and / or gastrointestinal tumor, tumor-associated cell, cancer, or metastasis. In one embodiment, the bacterial cell delivers the caspase to the tumor, tumor-associated cell, cancer, or metastasis. In another embodiment, the bacterial cell delivers at least one additional exogenous therapeutic peptide, such as NIPP1. In an embodiment, endogenous expression of flhDC is under the control of an exogenous inducible promoter. In another embodiment, expression of flhDC is under the control of an inducible promoter, and the bacterial cell comprises the exogenous inducible promoter operably linked to the exogenous flhDC gene. In one embodiment, the bacterial cell does not express endogenous flhDC. In one embodiment, expression of flhDC is induced after the tumor, tumor-associated cell, cancer, or metastasis is colonized by the bacterium.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief explanation of the drawings]

[0022] [Figure 1A] The intracellular lifestyle of Salmonella is controlled by flhDC. A) The design goal was to engineer a bacterial vehicle that (1) synthesizes (makes) a protein drug (yellow / purple), (2) actively infiltrates into cancer cells, and (3) releases the drug. Over time, the drug escapes from the Salmonella vacuole (SCV, red). [Figure 1B] B) Salmonella (light blue, arrow) infiltrates cancer cells (red). [Figure 1C] C) Seventy percent of Salmonella (red, white arrow) were intracellular (colocalized red and green, black arrow) within tumors in vivo (***, P<0.001). [Figure 1D] D) In ​​tumors in mice, Salmonella infiltrated multiple cell types, including immune cells, carcinoma (epithelial) cells, and other associated (stromal) cells. [Figure 1E] E) In cancer cell monolayers, flhDC re-expression (flhDC+) increased invasion (black arrows) compared with non-expressing controls (flhDC-; ***, P<0.001). [Figure 1F] F) In three-dimensional tumor-on-a-chip, flhDCs + Salmonella carrying a green intracellular reporter invaded more than flhDCs − controls (**, P<0.01). [Figure 1G] G) After administration to tumor-bearing mice, re-expression of flhDC increased the infiltration of cancer cells and immune cells (*, P<0.05). [Figure 2A]Design of ID Salmonella to export proteins into cells. A) Salmonella carrying either the PsifA-GFP or PsseJ-GFP reporter construct expressed GFP after invasion (white arrow). Extracellular expression from PsseJ-GFP (black arrow) was less than that from PsifA-GFP (***, P<0.001). Intracellular activity of the PsseJ promoter was four-fold greater than extracellular activity (***, P<0.001). [Figure 2B] B) Induction of PBAD-LysE at 96 h (arrow) induced bacterial lysis at a rate of 0.39 h-1. [Figure 2C] C) When administered to MCF7 cancer cells, 68% of intracellular Salmonella harboring PsseJ-lysE were lysed, significantly more than the PsseJ-GFP control (***, P < 0.001). [Figure 2D] C) Salmonella carrying PsseJ-lysE and Plac-GFP delivered GFP into the cell cytoplasm. Only the released, but not the intracellular, GFP was stained. [Figure 2E] E) Intracellular ID Salmonella were lysed at a rate of 0.33 h-1 (half-life = 2.1 h). [Figure 2F] F) In liquid culture, PBAD-lysE and PsseJ-lysE Salmonella grew at a rate similar to that of untransformed controls (white bars). When intracellular, PsseJ-lysE Salmonella lysed at a rate similar to that of PBAD-lysE Salmonella induced in culture (black bars). [Figure 2G] G) Bacterial EGFP production per colony forming unit (CFU). [Figure 2H] H) After infiltration and before lysis, Salmonella (light blue, white arrow) were present within LAMP1-stained SCVs (red, yellow arrow). After lysis, GFP (green, black arrow) remained within the membrane of the SCVs. From 6 to 24 hours after infiltration, the percentage of released GFP in the cytosol, but not in the SCVs, increased from 25% to 75% (***, P<0.001). [Figure 2I]I) In phalloidin-stained cancer cells (red), released GFP (green, black arrows) translocated from the SCV near the nucleus (blue) throughout the cytoplasm. [Figure 2J] J) ID Salmonella (left) lyses and GFP diffuses through the cytosol of cancer cells (right). Temporal profile of GFP intensity centered around lysed bacteria. [Figure 3A] PsseJ and flhDC are components of ID Salmonella delivery to tumors. A) Most released GFP (green, black arrow) was derived from lysed Salmonella in LAMP1-stained SCVs (red, yellow arrow). Cytoplasmic bacteria (light blue, white arrow) did not lyse (***, P<0.001) or release GFP. Only released GFP was stained. [Figure 3B] B) The predominantly cytoplasmic ΔsifA remained intact (red, white arrows) and was less lysed than the predominantly vacuolar ΔsseJ and ID Salmonella (green, black arrows) (*** <P<0.001)。 [Figure 3C] C) GFP (green, arrow) was delivered only when Salmonella was transformed with both PBAD-flhDC and PsseJ-LysE (***, P<0.001). [Figure 3D] D) After injection of 2 × 106 bacteria / mouse into 4T1 tumor-bearing BALB / c mice, ID Salmonella delivered GFP to cancer cells (arrows). [Figure 3E] E) Delivered GFP was present in extracts from tumor (T) but not liver (L) or spleen (S). [Figure 3F] F) Administration of PBAD-flhDC-induced ID Salmonella to 4T1 tumor-bearing BALB / c mice delivered GFP (arrows) to more cells than flhDC-controls (***, P < 0.001). [Figure 3G] G) Luciferase-expressing ID Salmonella was injected intravenously into BALB / c mice bearing 4T1 tumors, and bacterial density in the tumors was measured for 14 days using bioluminescence imaging. [Figure 4A] Efficacy of ID Salmonella. A) Anti-actin nanobody (NB) and GFP (Ctr) were delivered into 4T1 cancer cells along with ID Salmonella. Beta-actin was immunoprecipitated with the delivered nanobody and was enriched 2.5-fold compared to the control. [Figure 4B] B) ID Salmonella delivery of NIPP1-CD and CT Casp-3 caused more death (red, white arrows) in Hepa 1-6 cells compared to controls (***, P<0.001, top). Cells infiltrated with control Salmonella (green, black arrows) or not infiltrated with control Salmonella (yellow arrows) did not die. [Figure 4C] C) Delivery of NIPP1-CD and CT Casp 3 caused cell death (red) in microfluidic tumor masses (*, P<0.05; **, P<0.01). Death increased over time as Salmonella infiltrated into the cells and delivered the protein (*, P<0.05). [Figure 4D] D) Delivery of CT Casp-3 reduced 4T1 mammary tumor growth compared to GFP-delivered bacterial controls (*, P<0.05, n=3). [Figure 4E] E) Nineteen days after injection, the volume of CT-Casp-3-treated Hepa 1-6 liver tumors was 12% of that of controls (***, P<0.001, n=3, left). Treatment with CT-Casp-3 reduced tumor growth rate compared with Salmonella controls (P<0.05, middle), significantly increased survival (P<0.05, right), and cured one mouse. [Figure 5A] Tumor selectivity of ΔflhD and ΔsifA Salmonella. A) Tumor colonization of ΔflhD Salmonella was unchanged compared to the parental control. However, liver colonization of ΔflhD Salmonella was 10-fold less than the control (*, P<0.05). [Figure 5B] B) Although not statistically significant, the colony formation levels of all three flhDC-overexpressing tumors were lower than those of the parental controls (P=0.34). [Figure 5C]C) Nonflagellated, flhDC-expressing ΔfliGHI Salmonella colonized the liver 8- and 12-fold more than ΔflhD and ΔfliGHI + ΔflhD strains, respectively (*, P < 0.05). [Figure 5D] D) ΔfliGHI, ΔflhD, and ΔfliGHI+ΔflhD Salmonella did not differ in tumor colonization levels. [Figure 6A] flhDC activity is required for increased bacterial dispersal within tumors. A) Mice bearing 4T1 tumors were injected with ΔflhD Salmonella. 48 hours after bacterial injection, half of the mice received arabinose at 48 and 72 hours after bacterial injection to induce flhDC expression. [Figure 6B] B) Non-flhDC-induced Salmonella were non-motile and formed clearly separated colonies in either necrotic (yellow arrows) or viable tissue (green arrows). [Figure 6C] C) 75% of distinct colonies were present in necrosis, while only 25% of colonies were located in viable tumor tissue (**, P<0.01). [Figure 6D] D) The bacterial growth rate in necrotic tissue (0.12 h-1) was slightly higher than that in viable tumor tissue (0.11 h-1) (*, P<0.05). [Figure 6E] This corresponded to a doubling time of E) 6 hours in necrotic versus 6.5 hours in viable tumor tissue (*, P<0.05). [Figure 6F] F) Dense bacterial colony size (red border) was visibly larger in flhDC-induced compared to uninduced tumors. Scale bar is in μm. [Figure 6G] G) Dense colony size was 50% greater in tumors treated with flhDC induction as opposed to uninduced tumors (*, P<0.05). [Figure 6H]H) The abundance of satellite colonies (green arrows) outside the main dense bacterial colonies was visibly greater in tumors containing flhDC induction as opposed to non-induced Salmonella. Scale bar 200 um. [Figure 6I] I) There was a two-fold greater abundance of isolated satellite colonies in tumors containing flhDC inducers as opposed to non-induced Salmonella (*, P<0.05). [Figure 7A] flhDC activity increases the dispersal of intracellular Salmonella within tumors in vitro and in vivo. A) Microfluidic tumor-on-a-chips were infected with either flhDC-induced or non-induced IR Salmonella. These bacteria selectively expressed GFP inside the cells. [Figure 7B] B) flhDC-induced Salmonella (green) were dispersed throughout the tumor mass, while non-induced bacteria were faintly detectable toward the leading edge of the tumor mass (white arrows). Scale bar 100 μm. [Figure 7C] C) Intracellular bacterial abundance was 50- to 75-fold greater in flhDC-induced tumors, as opposed to non-induced Salmonella, for x>0.5 (**, P<0.01; ***, P<0.001). [Figure 7D] D) flhDC-induced intracellular bacterial loads continued to increase over time compared with uninduced controls (*, P<0.05; **, P<0.01; ***, P<0.001). [Figure 7E] E) Mice were infected with IR Salmonella and one group was given arabinose to express flhDC. [Figure 7F] F) High density uninduced Salmonella contained significantly fewer intracellular bacteria (yellow arrows) compared to induced colonies (yellow border). [Figure 7G] G) The proportion of intracellular flhDC-induced Salmonella was three-fold greater than non-induced colonies within the tumor (*, P<0.05). [Figure 7H]H) The dispersion of intracellular bacteria within tumors was greater after flhDC induction. Euclidean distance mapping of intracellular bacteria showed that tumor coverage was (I) 50% greater when flhDC was expressed (*, P<0.05). [Figure 8A] In vivo, flhDC expression was required for intracellular protein delivery to widely distributed cells within tumors. A) When flhDCs were induced, intracellular delivery occurred in cells that were more spatially dispersed within the tumor (white arrows). Euclidean distance mapping demonstrated that tumors treated with flhDC-induced Salmonella had cells with GFP delivery that were 60% more spatially dispersed within the tumor compared with uninduced controls (B) (*, P<0.05). [Figure 8B] In vivo, flhDC expression was required for intracellular protein delivery to widely distributed cells within tumors. A) When flhDCs were induced, intracellular delivery occurred in cells that were more spatially dispersed within the tumor (white arrows). Euclidean distance mapping demonstrated that tumors treated with flhDC-induced Salmonella had cells with GFP delivery that were 60% more spatially dispersed within the tumor compared with uninduced controls (B) (*, P<0.05). [Figure 9A] Engineered Salmonella are more effective for intracellular delivery than cytoplasmic Salmonella. A) ΔsifA Salmonella colonized tumors 10-fold less than the parental control strain (*, P<0.05). [Figure 9B] B) ΔsifA Salmonella colonized the liver 15-fold less than the parental control strain (*, P < 0.05). [Figure 9C] C) Cytoplasmic ΔsifA Salmonella remained almost exclusively intact (red) within cancer cells, whereas the majority of FID Sal was lysed within cancer cells (green dot FID Sal panel). [Figure 9D] D) FID Sal lysed at least 18-fold more than ΔsifA Salmonella at any given time point (***, P < 0.001). [Figure 10A] flhDC activity reduces Salmonella activity by allowing vacuolar escape. A) 4T1 cells in monolayer were infected with either ID Sal or FID Sal. [Figure 10B] B) Overall bacterial infiltration was greater for FID Sal-treated cells (green and red dots), but bacterial lysis (green) was reduced and more FID Sal remained intact (red) after cancer cell infection compared to ID Sal. [Figure 10D] D) 60% of the control ID Sal was dissolved, whereas only 40% of the FID Sal was dissolved (**, P<0.01). [Figure 10E] E) 4T1 cells were infected with either control or flhDC-expressing Salmonella. [Figure 10F] F) Control Salmonella were primarily located within vacuoles (red circles), however, more numerous flhDC-induced Salmonella were present in the cytosol (white circles). [Figure 10G] G) 90% of control Salmonella were present within vacuoles inside cancer cells compared with 70% of flhDC-induced bacteria (**, P<0.01). [Figure 10H] H) ID Sal was more likely to lyse intracellularly as bacteria remained within the vacuole (white arrow). [Figure 10I] I) A significant proportion of FID Sal lysed inside the cell (white arrow), whereas a small but significant proportion of bacteria escaped the intracellular vacuole and therefore did not lyse (turquoise arrow). [Figure 10J] J) The presence of a significant amount of cytoplasmic undissolved FID Sal was observed in vivo (white arrow). [Figure 11A]Overexpression of flhDC in Salmonella with impaired vacuolar escape maintains high cell invasion and rescues lytic efficiency. A) 4T1 cells infected with ID Sal had lower invasion but were lysed intracellularly with high efficiency (green dots). More FID Sal invaded 4T1 cancer cells but had lower lytic efficiency (green dots). ΔsseJ FID Sal invaded 4T1 cancer cells and lysed intracellularly with high efficiency (red and green dots). B) ΔsseJ FID Sal invaded cancer cells threefold more than the ID Sal control (**, P<0.01). C) ΔsseJ FID Sal lysed cancer cells 20% more efficiently than ID Sal and (D) delivered 2.5-fold and 2-fold more protein into cells than ID Sal or FID Sal, respectively (**, P<0.01). [Figure 11B] Overexpression of flhDC in Salmonella with impaired vacuolar escape maintains high cell invasion and rescues lytic efficiency. A) 4T1 cells infected with ID Sal had lower invasion but were lysed intracellularly with high efficiency (green dots). More FID Sal invaded 4T1 cancer cells but had lower lytic efficiency (green dots). ΔsseJ FID Sal invaded 4T1 cancer cells and lysed intracellularly with high efficiency (red and green dots). B) ΔsseJ FID Sal invaded cancer cells threefold more than the ID Sal control (**, P<0.01). C) ΔsseJ FID Sal lysed cancer cells 20% more efficiently than ID Sal and (D) delivered 2.5-fold and 2-fold more protein into cells than ID Sal or FID Sal, respectively (**, P<0.01). [Figure 11C]Overexpression of flhDC in Salmonella with impaired vacuolar escape maintains high cell invasion and rescues lytic efficiency. A) 4T1 cells infected with ID Sal had lower invasion but were lysed intracellularly with high efficiency (green dots). More FID Sal invaded 4T1 cancer cells but had lower lytic efficiency (green dots). ΔsseJ FID Sal invaded 4T1 cancer cells and lysed intracellularly with high efficiency (red and green dots). B) ΔsseJ FID Sal invaded cancer cells threefold more than the ID Sal control (**, P<0.01). C) ΔsseJ FID Sal lysed cancer cells 20% more efficiently than ID Sal and (D) delivered 2.5-fold and 2-fold more protein into cells than ID Sal or FID Sal, respectively (**, P<0.01). [Figure 11D] Overexpression of flhDC in Salmonella with impaired vacuolar escape maintains high cell invasion and rescues lytic efficiency. A) 4T1 cells infected with ID Sal had lower invasion but were lysed intracellularly with high efficiency (green dots). More FID Sal invaded 4T1 cancer cells but had lower lytic efficiency (green dots). ΔsseJ FID Sal invaded 4T1 cancer cells and lysed intracellularly with high efficiency (red and green dots). B) ΔsseJ FID Sal invaded cancer cells threefold more than the ID Sal control (**, P<0.01). C) ΔsseJ FID Sal lysed cancer cells 20% more efficiently than ID Sal and (D) delivered 2.5-fold and 2-fold more protein into cells than ID Sal or FID Sal, respectively (**, P<0.01). [Figure 12] Modulating flhDC expression increased the tumor selectivity and intracellular delivery distribution of engineered Salmonella. Salmonella lacking flhDC expression selectively colonized tumors over strains without regulated flhDC expression. In tumors, flhDC expression enabled Salmonella to disperse and invade tumor cells. Expression of flhDC in the engineered ΔsseJ strain enabled vacuolar retention of Salmonella, leading to higher lytic efficiency and overall protein delivery within tumor cells. [Figure 13A] Genomic integration of inducible flhDCs leads to infiltration of not only cancer cells but also parental and plasmid-based inducible flhDC lines. A) After arabinose induction of both episomal and chromosomally integrated flhDC lines, Salmonella invaded cancer cells (red) equally well as the parental strain (green dots). [Figure 13B] B) The non-inducible knock-in strain was equally non-invasive as the non-inducible plasmid-based system. After induction, EBV-002 with the flhDC gene circuit integrated into its chromosome was more invasive than either the non-inducible plasmid-based or genomic knock-in strains (*, P<0.05). [Figure 14A] flhD expression is regulated by salicylic acid in EBV-002. A) EBV-002 was transformed with a flhD construct that was inducible with salicylic acid. The flhD gene was tagged at the C-terminus with either a low-, medium-, or high-activity degradation tag to suppress flhD activity in the uninduced state. As expected, none of the three strains invaded cancer cells without salicylic acid induction. However, after induction, only EBV-002 transformed with flhD containing the low- or medium-activity degradation tag invaded large numbers of cells (green dots). EBV-002 containing flhD with the high-activity degradation tag was only weakly invasive after induction. [Figure 14B] B) PBAD induction of flhD only increased intracellular invasion of EBV-002 by 2-fold compared to the uninduced control. EBV-002 invaded a significant number of cells without the degradation tag to suppress flhD activity in the uninduced state. However, salicylic acid-induced samples (2) and (3) invaded approximately 30-fold more cells than the uninduced control. EBV-002 (sample 4), which contains a highly active degradation tag on flhD, only invaded cancer cells 5-fold more than the uninduced control. All inductions of samples (2), (3), and (4) were statistically significant at P<0.01. [Figure 15A]Clinical EBV-002 is triggered by aspirin to swim and invade cancer cells. EBV-002 with a genomic deletion of flhD was engineered to express flhDC using a salicylic acid-responsive gene circuit. A) Without salicylic acid, bacteria remained nonmotile. After inducing bacteria with salicylic acid, all bacteria became highly motile, as indicated by the bacterial pathway (uninduced, blue; induced, red). [Figure 15B] B) Salicylic acid-induced EBV-002 was 12.7-fold more motile than uninduced bacteria (***, P<0.001). [Figure 15C] C) Aspirin induction of flhDCs strongly suppressed EBV-002-mediated cancer cell invasion. Aspirin-induced EBV-002 (green) invaded almost all cancer cells (white arrows). [Figure 15D] D) Aspirin-induced EBV-002 invaded cancer cells 30 times more than non-induced EBV-002 (***, P<0.001). [Figure 16A] Determination of the minimum amount of salicylic acid required to induce EBV-002 cell invasion. A) Concentrations of salicylic acid above 500 nM induced microscopically visible amounts of intracellular EBV-002. [Figure 16B] B) A minimum of 500 nM of salicylic acid was sufficient to induce high levels of cell infiltration (**, P<0.01). [Figure 17A] Biodistribution and protein delivery of EBV-003 and EBV-001. A) EBV-003 colonization remained unchanged in the liver and spleen compared with EBV-001, but the EBV-003 strain colonized tumors 10.7-fold more often than the first-generation strain (**, P<0.01). [Figure 17B] B) EBV-003 delivered 31-fold more protein into the tumor compared to EBV-001. Similar to EBV-001, EBV-003 did not deliver detectable amounts of protein to either the liver or spleen. [Figure 18A]Induction of flhD by salicylate increases EBV-003 penetration and intracellular invasion within viable tumor tissue. A) Uninduced (left) and induced (right) EBV-003-containing tumors. More bacteria (red Xs) were present intracellularly within or directly adjacent to actively dividing tumor cells (solid red outline) in induced compared to uninduced samples. [Figure 18B] B) Close histological examination revealed that uninduced EBV-003 present near actively dividing tumor tissue did not penetrate into the tissue, whereas induction of flhD in EBV-003 significantly increased the presence of intracellular bacteria in actively dividing tumor cells (white arrows). [Figure 18C] C) There was a three-fold enrichment of EBV-003 invasive cancer cells in flhD -induced EBV-003 bacteria compared with uninduced samples (*, P < 0.05). [Figure 19A] Intracellular protein delivery of EBV-003 within breast tumors. A) Inducible EBV-003 delivered proteins intracellularly into cells within actively dividing areas of the tumor (white arrows). [Figure 19B] B) Intracellular protein delivery was detected in only 1 of 4 mice with non-induced EBV-003. However, protein delivery was detected in 5 of 6 mice with salicylate-induced EBV-003. [Figure 20A] Colonization selectivity of EBV-003 in breast cancer liver metastases versus healthy liver tissue. A) Except for small metastatic lesions, healthy liver tissue (left) contained very limited numbers of EBV-003 colonies. On the other hand, livers with several large metastatic lesions (right) were heavily colonized by EBV-003 (indicated by solid white borders). 85% of these colonies were within or directly adjacent to actively dividing tumor cells (red arrows), indicated by the presence of dark blue nuclei. [Figure 20B]B) On closer inspection, the few colonies present in healthy liver tissue (1) were not significantly smaller than the bacteria in metastatic lesions (2). This indicates that EBV-003 colonies grow orders of magnitude more abundantly in metastatic tissue compared to healthy liver tissue, on which they preferentially colonize metastases. (The red arrow pointing to the right indicates the portion of the liver with metastatic lesions. The green arrow pointing to the left indicates the side of healthy liver tissue. The red line denotes the border between the two.) [Figure 20C] C) EBV-003 colony size within metastatic lesions was 118-fold larger than colony size within healthy liver tissue, demonstrating the ability of the bacterium to grow by orders of magnitude only within tumor tissue (***, P=2.2×10-26). [Figure 21A] Intracellular infiltration of EBV-003 within spontaneous liver metastases. A) Significant numbers of both flhDC-uninduced and -induced EBV-003 intracellularly infiltrated metastatic cancer cells within the liver (white arrows). [Figure 21B] B) 87% and 83% of uninduced and induced EBV-003, respectively, intracellularly invaded or directly adjacent to cancer cells within metastatic lesions. [Figure 22A] Intracellular protein delivery of EBV-003 within metastatic breast cancer cells in the liver. A) EBV-003 (green) delivered proteins (red) into metastatic breast cancer cells (white arrows) in the liver. [Figure 22B] B) flhDC-induced EBV-003 delivered proteins into metastatic tumor cells three-fold more frequently than non-induced EBV-003. DETAILED DESCRIPTION OF THE INVENTION

[0023] Detailed Description of the Invention The majority of proteins are intracellular. Using macromolecular therapy to specifically target intracellular pathways, especially in cancer cells, increases the potential treatment options for any patient. However, macromolecular therapy targeting intracellular pathways faces significant barriers associated with tumor targeting, distribution, internalization, and endosomal release. Engineered non-pathogenic Salmonella selectively colonizes tumors 1000 times more than any other organ, invades cytoplasmically into cancer cells, and delivers therapeutics, making bacteria an ideal delivery vehicle for cancer therapy.

[0024] However, a problem with using bacteria as anticancer agents is the toxicity of bacteria at the doses required for therapeutic efficacy, and an obstacle in cancer gene therapy is the specific targeting of the therapy directly to cancer. Another problem to be addressed is the systemic clearance of Salmonella. A further problem is the activity of cytoplasmic Salmonella (compared to SCV Salmonella). Provided herein is a novel therapeutic platform for the controlled colonization and / or invasion of engineered Salmonella in cancer cells, as well as the controlled gene and protein delivery in cancer cells, thus treating cancer.

[0025] To address these challenges, a bacterial delivery platform was developed that utilizes a mechanism unique to Salmonella to deliver protein-based drugs intracellularly. Salmonella senses the intracellular environment and accumulates inside cells when present within tumors. A genetic circuit was engineered to force entry into cancer cells and release the protein from endosomes into the cytoplasm. Intracellular lysis makes the platform self-limiting, reducing the chance of unwanted infection. The delivered nanobody and protein interactor (NIPP1) bind to their targets and cause cell death. Delivery of caspase-3 to mice reduces breast tumor growth and eliminates liver tumors. Intracellular delivery of protein-based drugs to tumors opens the entire proteome for treatment.

[0026] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some embodiments of the methods and materials are described herein. As used herein, each of the following terms has the meaning associated with it in this section.

[0027] For purposes of clarity and concise description, features may be described herein as part of the same or separate embodiments, but it will be understood that the scope of the present invention may include embodiments having all or any combination of the described features.

[0028] References herein to "one embodiment," "an embodiment," and the like indicate that the described embodiment may include a particular aspect, feature, structure, portion, or characteristic, but that not every embodiment necessarily includes that aspect, feature, structure, portion, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment as referenced elsewhere in this specification. Furthermore, when a particular aspect, feature, structure, portion, or characteristic is described in connection with an embodiment, it is within the knowledge of one of ordinary skill in the art that such aspect, feature, structure, portion, or characteristic affects or connects with other embodiments, whether or not explicitly stated.

[0029] As used herein, the indefinite articles "a," "an," and "the" are to be understood to include plural references unless the context clearly indicates otherwise.

[0030] As used herein, the term "and / or" should be understood to mean "either or both" of the elements so conjoined, e.g., elements that are conjunctively present in some instances and disjunctively present in other instances.

[0031] As used herein, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating a list of items, "and / or" or "or" should be interpreted as inclusive, e.g., including at least one of a number of items, but also including two or more, and optionally including additional unlisted items. Only terms clearly indicated to the contrary, e.g., "only one of" or "exactly one of," or, when used in the claims, "consisting of," refer to the inclusion of exactly one element of a number or list of elements. In general, as used herein, the term "or" should only be interpreted to indicate exclusive alternatives (i.e., "one or the other, but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of."

[0032] As used herein, the terms "including," "includes," "having," "has," "with," or variations thereof, are intended to be as inclusive as the term "comprising."

[0033] As used herein, the term "about" means plus or minus 10% of the indicated value. For example, about 100 means 90 to 110. Numerical ranges recited herein by endpoints include all numbers and portions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It should also be understood that all numbers and portions thereof are presumed to be modified by the term "about."

[0034] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to any subject for whom diagnosis, treatment, or therapy is desired, including mammals. Mammals include, but are not limited to, humans, livestock, sport animals, and pets. A "subject" is a vertebrate such as a mammal, including a human. Mammals include, but are not limited to, humans, livestock, sport animals, and companion animals. The term "animal" includes dogs, cats, fish, gerbils, guinea pigs, hamsters, horses, rabbits, pigs, mice, monkeys (e.g., apes, gorillas, chimpanzees, orangutans), rats, sheep, goats, cows, and birds.

[0035] The terms "treatment," "treating," and the like are used herein generally to mean obtaining a desired pharmacological and / or physiological effect, such as preventing or inhibiting, or attempting to prevent or inhibit, the onset or progression of a disorder, and / or causing or attempting to cause a reduction, suppression, regression, or remission of a disorder and / or its symptoms. The effect may be preventative, in that a disease or its symptoms are completely or partially prevented, and / or therapeutic, in that a partial or complete cure is achieved for the disease and / or adverse effects attributable to the disease. As one skilled in the art will appreciate, various clinical and scientific methodologies and assays may be used to assess the onset or progression of a disorder, and similarly, various clinical and scientific methodologies and assays may be used to assess the reduction, regression, or remission of a disorder or its symptoms. Additionally, treatment may be applied to a subject or cell culture (in vivo or in vitro).

[0036] The terms "inhibit," "inhibiting," and "inhibition" refer to slowing, stopping, or reversing the growth or progression of a disease, infection, condition, group of cells, protein, or expression thereof. Inhibition can be, for example, greater than about 20%, 40%, 60%, 80%, 90%, 95%, or 99% compared to growth or progression that occurs in the absence of treatment or contact.

[0037] "Expression" refers to the production of RNA from DNA and / or protein directed by genetic material (e.g., RNA (mRNA)). Inducible expression, as opposed to constitutive expression (expressed all the time), is expression that occurs only under certain conditions, such as in the presence of a particular molecule (e.g., arabinose) or environmental factor.

[0038] As used herein with respect to nucleic acids (or proteins) and hosts, the term "exogenous" refers to a nucleic acid, or a protein encoded by such a nucleic acid, that does not occur in (and cannot be obtained from) that particular type of cell as found in nature. Therefore, a non-naturally occurring nucleic acid is considered exogenous to the host once it is placed within the host. It is important to note that a non-naturally occurring nucleic acid can contain a nucleic acid subsequence or fragment of a nucleic acid sequence found in nature, but the nucleic acid as a whole is not naturally occurring. For example, a nucleic acid molecule containing a genomic DNA sequence within an expression vector is a non-naturally occurring nucleic acid and therefore exogenous to the host cell once introduced into the host because the nucleic acid molecule as a whole (genomic DNA and vector DNA together) is not naturally occurring. Therefore, any vector, autonomously replicating plasmid, or virus (e.g., retrovirus, adenovirus, or herpesvirus) that is not naturally occurring as a whole is considered to be a non-naturally occurring nucleic acid. Therefore, genomic DNA fragments produced by PCR or restriction endonuclease treatment, as well as cDNA, are considered to be non-naturally occurring nucleic acids because they exist as separate molecules not found in nature. Thus, exogenous sequences may be integrated into the genome of a host. Therefore, any nucleic acid containing a promoter sequence and a polypeptide-encoding sequence (e.g., cDNA or genomic DNA) in an arrangement not found in nature is also considered to be a non-naturally occurring nucleic acid. Naturally occurring nucleic acids may be exogenous to a particular host microorganism. For example, an entire chromosome isolated from a cell of yeast x is an exogenous nucleic acid to a cell of yeast y when the chromosome is introduced into the cell of yeast y.

[0039] In contrast, the term "endogenous," as used herein with respect to a nucleic acid (e.g., a gene) (or protein) and a host, refers to a nucleic acid (or protein) that occurs within (and can be obtained from) that particular host as found in nature. Moreover, a cell that "endogenously expresses" a nucleic acid (or protein) expresses that nucleic acid (or protein) as would a host of the same particular type, as would be found in nature. Furthermore, a host that "endogenously produces" or "endogenously produces" a nucleic acid, protein, or other compound produces that nucleic acid, protein, or compound as would a host of the same particular type, as would be found in nature.

[0040] Flagella are filamentous protein structures found in bacteria, archaea, and eukaryotes, but they are most commonly found in bacteria. Flagella are typically used to propel cells through liquids (i.e., bacteria and sperm). However, flagella have numerous other specialized functions. Flagella are usually found in gram-negative bacilli. Gram-positive rods (e.g., Listeria species) and cocci (some Enterococcus species, Vagococcus species) also possess flagella.

[0041] The engineered Salmonella can be any strain of Salmonella that has been designed to lyse and deliver proteins intracellularly. The term "contacting" refers to the act of touching, making contact, or bringing into close or near proximity, including at the cellular or molecular level, e.g., in a solution, in a reaction mixture, in vitro, or in vivo, to effect a physiological reaction, a chemical reaction, or a physical change.

[0042] An "effective amount" is an amount sufficient to produce beneficial or desired results, such as preclinical or clinical results. An effective amount can be administered in one or more administrations. The term "effective amount," as applied to the compounds(s), biologics, and pharmaceutical compositions described herein, refers to the amount necessary to provide the desired therapeutic result. For example, an effective amount is a level effective to treat, cure, or alleviate the symptoms of the disorder and / or disease for which the therapeutic compound, biologic, or composition is administered. The amount effective for the particular therapeutic goal sought will depend on various factors, including the disorder being treated and its severity and / or stage of onset / progression; the bioavailability and activity of the particular compound, biologic, or pharmaceutical composition used; the route or method of administration and the site of introduction into the subject; the clearance rate of the particular compound or biologic and other pharmacokinetic properties; the duration of treatment; the inoculation regimen; agents used in combination with or simultaneously with the particular compound, biologic, or composition; the age, weight, sex, diet, physiological condition, and general health of the subject being treated; and similar factors well known to those skilled in the relevant scientific fields. Some variation in dosage may occur depending on the condition of the subject being treated, and the physician or other individual administering the treatment will, in any event, determine the appropriate dose for the individual patient.

[0043] As used herein, "disorder" refers to a disorder, disease, or condition, or other deviation from healthy or normal biological activity, and the terms can be used interchangeably. The term refers to any condition that impairs normal function. The condition may be caused by a sporadic or inherited genetic abnormality. The condition may also be caused by a non-genetic abnormality. The condition may also be caused by injury to the subject from an environmental agent, such as, but not limited to, cutting, crushing, burning, piercing, stretching, shearing, injecting, or otherwise altering the subject's cell(s), tissue(s), organ(s), system(s), or the like.

[0044] As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. All of these terms also include their progeny, that is, any and all subsequent generations. It is understood that all progeny may not be identical due to intentional or unintentional mutations.

[0045] The "coding region" of a gene consists of nucleotide residues on the coding strand of the gene and those on the non-coding strand of the gene that are homologous or complementary, respectively, to the coding region of an mRNA molecule produced by transcription of the gene.

[0046] As used herein, "complementary" refers to the broad concept of subunit sequence complementarity between two nucleic acids, for example, two DNA molecules. If a nucleotide position in both molecules is occupied by a nucleotide that normally has the ability to base pair with each other, the nucleic acids are considered to be complementary to each other at this position. Thus, two nucleic acids are complementary to each other if a significant number (at least 50%) of corresponding positions in each molecule are occupied by nucleotides that normally base pair with each other (e.g., A:T and G:C nucleotide pairs). Thus, it is known that an adenine residue in a first nucleic acid region has the ability to form specific hydrogen bonds (base pairing) with a residue in a second nucleic acid region that is antiparallel to the first region when the residue is thymine or uracil. Similarly, it is known that a cytosine residue in a first nucleic acid strand has the ability to base pair with a residue in a second nucleic acid strand that is antiparallel to the first strand when the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or different nucleic acid if at least one nucleotide residue in the first region has the ability to base pair with the residues in the second region when the two regions are arranged in an antiparallel manner. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby when the first portion and the second portion are arranged in an antiparallel manner, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues in the first portion have the ability to base pair with the nucleotide residues in the second portion. More preferably, all nucleotide residues in the first portion have the ability to base pair with the nucleotide residues in the second portion.

[0047] "Encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of the gene or cDNA.

[0048] As used herein, an "essentially pure" preparation of a particular protein or peptide is one in which at least about 95%, by weight, and preferably at least about 99%, of the protein or peptide in the preparation is the particular protein or peptide.

[0049] A "fragment" or "segment" is a portion of an amino acid sequence comprising at least one amino acid or a portion of a nucleic acid sequence comprising at least one nucleotide. The terms "fragment" and "segment" are used interchangeably herein.

[0050] As used herein, a "functional" biomolecule is a biomolecule in a form that exhibits a property, which is characterized by that property. A functional enzyme is, for example, an enzyme that exhibits the characteristic catalytic activity by which the enzyme is characterized.

[0051] As used herein, "homology" refers to the similarity of subunit sequences between two polymer molecules, e.g., two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or two polypeptide molecules. If both subunit positions of two molecules are occupied by the same monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half of the positions in two compound sequences (e.g., 5 positions out of 10 subunits of the polymer) are homologous, the two sequences are 50% homologous; if 90% of the positions, e.g., 9 out of 10, are matched or homologous, the two sequences share 90% homology. For example, the DNA sequences 3'ATTGCC5' and 3'TATGGC share 50% homology.

[0052] As used herein, "homology" is used synonymously with "identity." The determination of percent identity between two nucleotide or amino acid sequences can be achieved using mathematical algorithms.For example, a useful mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA 87:2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl. Acad. Sci. USA 90:5873-5877).This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990, J. Mol. Biol. 215:403-410), and can be accessed, for example, at the National Center for Biotechnology Information (NCBI) World Wide Web site, which has a universal resource locator, using the BLAST tool at the NCBI website. BLAST nucleotide searches can be performed with the NBLAST program (designated "blastn" on the NCBI website) using the following parameters to obtain nucleotide sequences homologous to the nucleic acids described herein: gap penalty = 5; gap extension penalty = 2; mismatch penalty = 3; match reward = 1; expectation score = 10.0; and word size = 11. BLAST protein searches can be performed with the XBLAST program (designated "blastn" on the NCBI website) or the NCBI "blastp" program using the following parameters: expectation score 10.0, BLOSUM62 scoring matrix, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997, Nucleic Acids Res. 25:3389-3402). Alternatively, PSI-Blast or PHI-Blast can be used to perform an iterated search that detects distant relationships between molecules (Id.) and relationships between molecules that share common patterns.When utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blast programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used.

[0053] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, exact matches are typically counted.

[0054] As used herein, the term "hybridization" is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between nucleic acids) are affected by factors such as the degree of complementarity between the nucleic acids, the stringency of the conditions involved, the length of the hybrid formed, and the G:C ratio within the nucleic acids.

[0055] As used herein, "instructional material" includes publications, records, diagrams, or any other medium of expression that can be used to communicate the usefulness of the peptides of the invention in the kit for causing relief of the various diseases or disorders listed herein. Optionally, or alternatively, the instructional material may describe one or more methods of alleviating a disease or disorder in a mammalian cell or tissue. The instructional material of the kit of the invention may, for example, be affixed to a container containing the identified compound invention or may be shipped together with a container containing the identified compound. Alternatively, the instructional material may be shipped separately from the container, with the intention that the instructional material and the compound be used cooperatively by the recipient.

[0056] The term " nucleic acid " typically refers to large polynucleotide.By " nucleic acid ", it means any nucleic acid, whether it is composed of deoxyribonucleoside or ribonucleoside, and whether it is composed of phosphodiester bond or modified bond such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethyl ester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone bond, and the combination of these bonds.The term nucleic acid also specifically includes the nucleic acid that is composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).

[0057] As used herein, the term "nucleic acid" encompasses not only RNA but also single-stranded and double-stranded DNA and cDNA. Furthermore, the terms "nucleic acid," "DNA," "RNA," and similar terms also include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. For example, so-called "peptide nucleic acids," which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. By "nucleic acid," we mean any nucleic acid, regardless of whether it is composed of deoxyribonucleosides or ribonucleosides, and whether it is composed of modified linkages such as phosphodiester linkages or phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethyl ester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate, or sulfone linkages, and combinations of such linkages. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine, and uracil). Conventional notation is used herein to describe polynucleotide sequences, i.e., the left-hand end of a single-stranded polynucleotide sequence is the 5' end, and the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of nucleotides to a nascent RNA transcript is referred to as the transcription direction. The DNA strand having the same sequence as the mRNA is referred to as the "coding strand," the sequence on the DNA strand that is 5' to a reference point on the DNA is referred to as the "upstream sequence," and the sequence on the DNA strand that is 3' to a reference point on the DNA is referred to as the "downstream sequence."

[0058] As used herein, the term "nucleic acid construct" encompasses DNA and RNA sequences encoding the particular gene or gene fragment desired, whether obtained by genomic or synthetic methods.

[0059] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNAs may contain introns.

[0060] The term "oligonucleotide" typically refers to a short polynucleotide, generally no more than about 50 nucleotides. It will be understood that where a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence in which "T" is replaced by "U" (i.e., A, U, G, C).

[0061] A "substantially homologous nucleic acid sequence" refers to a nucleic acid sequence that corresponds to a reference nucleic acid sequence, where the corresponding sequence encodes a peptide having substantially the same structure and function as the peptide encoded by the reference nucleic acid sequence, e.g., with only amino acid changes that do not significantly affect peptide function. Preferably, a substantially identical nucleic acid sequence encodes a peptide encoded by the reference nucleic acid sequence. The percent identity between a substantially similar nucleic acid sequence and the reference nucleic acid sequence is at least about 50%, 65%, 75%, 85%, 95%, 99% or more. The substantial identity of nucleic acid sequences can be determined by comparing the sequence identity of two sequences, for example, by physical / chemical methods (i.e., hybridization) or by sequence alignment via a computer algorithm. Suitable nucleic acid hybridization conditions for determining whether a nucleotide sequence is substantially similar to a reference nucleotide sequence are 7% sodium dodecyl sulfate SDS, 0.5 M NaPO, 1 mM EDTA at 50°C with a wash in 2X standard saline citrate (SSC), 0.1% SDS at 50°C; preferably 7% (SDS), 0.5 M NaPO, 1 mM EDTA at 50°C with a wash in 1X SSC, 0.1% SDS at 50°C; preferably 7% SDS, 0.5 M NaPO, 1 mM EDTA at 50°C with a wash in 0.5X SSC, 0.1% SDS at 50°C; and more preferably 7% SDS, 0.5 M NaPO, 1 mM EDTA at 50°C with a wash in 0.1X SSC, 0.1% SDS at 65°C.Suitable computer algorithms for determining substantial similarity between two nucleic acid sequences include the GCS program package (Devereux et al., 1984 Nucl. Acids Res. 12:387), and the BLASTN or FASTA programs (Altschul et al., 1990 Proc. Natl. Acad. Sci. USA. 1990 87:14:5509-13; Altschul et al., J. Mol. Biol. 1990 215:3:403-10; Altschul et al., 1997 Nucleic Acids Res. 25:3389-3402). The default settings provided with these programs are suitable for determining substantial similarity of nucleic acid sequences for the purposes of the present invention.

[0062] Describing two polynucleotides as "operably linked" means that a single- or double-stranded nucleic acid segment comprises two polynucleotides arranged within the nucleic acid segment in such a manner that at least one of the two polynucleotides is capable of exerting a physiological effect and is thereby characterized over the other. For example, a promoter operably linked to the coding region of a gene can promote transcription of the coding region.

[0063] As used herein, the term "pharmaceutically acceptable carrier" refers to a chemical composition with which a suitable compound or derivative can be combined and which, following that combination, can be used to administer the suitable compound to a subject. "Pharmaceutically acceptable" means physiologically tolerable for human or veterinary use. As used herein, "pharmaceutical composition" includes formulations for human and veterinary use.

[0064] As used herein, the term "purified" and similar terms refer to the enrichment of a molecule or compound relative to other components normally associated with the molecule or compound in its natural environment. The term "purified" does not necessarily indicate that complete purity of a particular molecule has been achieved during the process. As used herein, a "highly purified" compound refers to a compound that is greater than 90% pure. In particular, purified sperm cell DNA refers to DNA that does not produce significant detectable levels of non-sperm cell DNA upon PCR amplification of purified sperm cell DNA and subsequent analysis of the amplified DNA. A "significant detectable level" is an amount of contaminant that would be visible in the presented data and would need to be addressed / accounted for during analysis of forensic evidence.

[0065] A "recombinant polynucleotide" refers to a polynucleotide having sequences that are not joined together in nature. The amplified or assembled recombinant polynucleotide may be contained within a suitable vector, and the vector can be used to transform a suitable host cell.

[0066] A recombinant polynucleotide may serve a non-coding function as well (eg, promoter, origin of replication, ribosome binding site, etc.). A host cell containing a recombinant polynucleotide is called a "recombinant host cell." A gene expressed in a recombinant host cell, wherein the gene contains the recombinant polynucleotide, produces a "recombinant polypeptide."

[0067] A "recombinant polypeptide" is a polypeptide produced upon expression of a recombinant polynucleotide. A "recombinant cell" is a cell that contains a transgene. Such a cell may be a eukaryotic or prokaryotic cell. Transgenic cells also include, but are not limited to, embryonic stem cells containing the transgene, cells obtained from chimeric mammals whose cells are derived from transgenic embryonic stem cells containing the transgene, cells obtained from transgenic mammals or their fetal or placental tissue, and prokaryotic cells containing the transgene.

[0068] The term "modulate" refers to either stimulating or inhibiting a function or activity of interest. " Small interfering RNA (siRNA) " refers to an isolated dsRNA molecule that is composed of both sense and antisense strands. In one embodiment, it is more than 10 nucleotides in length. siRNA also refers to a single transcript that has both sense and complementary antisense sequences from target gene, for example, hairpin. siRNA not only includes any form of dsRNA (the proteolytic cleavage product of larger dsRNA, partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA), but also includes modified RNA that differs from naturally occurring RNA by adding, deleting, substituting and / or modifying one or more nucleotides.

[0069] As used herein, the term "specifically binds" means when a compound or ligand functions under binding reaction or assay conditions that determine the presence of the compound in a sample of heterogeneous compounds, or it means that one molecule, such as a binding moiety, e.g., an oligonucleotide or an antibody, preferentially binds to another molecule, such as a target molecule, e.g., a nucleic acid or protein, in the presence of other molecules in a sample.

[0070] When used in reference to the interaction between a peptide (ligand) and a receptor (molecule), the terms "specific binding" or "specifically bind" also refer to interactions that depend on the presence of a specific structure (i.e., the amino acid sequence of the ligand or ligand-binding domain within a protein); in other words, the peptide contains a structure that allows it to recognize and bind to a specific protein structure within the binding partner rather than to molecules in general. For example, if a ligand is specific for binding pocket "A," then in a reaction containing labeled peptide ligand "A" (such as an isolated phage-displayed peptide or an isolated synthetic peptide) and unlabeled "A" in the presence of a protein containing binding pocket A, the unlabeled peptide ligand will reduce the amount of labeled peptide ligand bound to the binding partner; in other words, a competitive binding assay.

[0071] As used herein, the term "standard" refers to what is used for comparison.For example, it can be a known standard drug or compound that is administered and used to compare results when administering a test compound, or it can be a standard parameter or function that is measured to obtain a control value when measuring the effect of a drug or compound on a parameter or function.Standard can also refer to an "internal standard," such as a drug or compound that is added to a sample in a known amount and is useful for determining things such as purification or recovery when the sample is processed or subjected to a purification or extraction procedure before measuring the marker of interest.Internal standards are often purified markers of interest that are labeled, for example, with a radioisotope, and can be distinguished from endogenous markers.

[0072] The method described herein involves conventional molecular biology techniques.These techniques are generally known in the art and are described in detail in methodological treatises such as Molecular Cloning: A Laboratory Manual, 2nd ed., vol.1-3, ed.Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Current Protocols in Molecular Biology, ed.Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates).Methods for chemical synthesis of nucleic acid are discussed in, for example, Beaucage and Carruthers, Tetra.Letts.22:1859-1862,1981, and Matteucci et al., J.Am.Chem.Soc.103:3185,1981.

[0073] As used herein, the terms "including," "includes," "having," "has," "with," or variations thereof, are intended to be as inclusive as the term "comprising."

[0074] The words "comprises," "comprising," and the like may have the meaning ascribed to them in U.S. patent law and may mean "includes," "including," and the like. As used herein, "including" or "includes," or the like, means including, but is not limited to.

[0075] I. Bacteria / Flagella Bacteria useful in the present invention include, but are not limited to, Clostridium, Bifidobacterium, Escherichia coli, or Salmonella, T3SS-dependent bacteria such as Shigella, Salmonella, and Yersinia pestis. Furthermore, E. coli can be used if the T3SS system is located within E. coli.

[0076] Salmonella Examples of Salmonella strains that can be employed in the present invention include Salmonella typhi (ATCC No. 7251) and Salmonella typhimurium (ATCC No. 13311). Attenuated Salmonella strains include S. typhi-aroC-aroD (Hone et al. Vacc. 9:810 (1991)), S. typhimurium-aroA mutant (Mastroeni et al. Micro. Pathol. 13:477 (1992)), and Salmonella typhimurium 7207. Additional attenuated Salmonella strains that can be used in the present invention include (i) aro (Hoiseth et al. Nature, 291:238-239 (1981)), gua (McFarland et al. Microbiol. Path., 3:129-141 (1987)), nad (Park et al. J. Bact., 170:3725-3730 (1988)), thy (Nnalue et al. (ii) auxotrophic mutants such as cya (Curtiss et al. Infect. Immun., 55:955-962 (1987)), and asd (Curtiss, see above) mutants; (iii) cya (Curtiss et al. Infect. Immun., 55:3035-3043 (1987)), crp (Curtiss et al. (1987) see above), phoP / phoQ (Groisman et al. Proc. Natl. Acad. Sci., USA, 86:7077-7081 (1989); and Miller et al. Proc. Natl. Acad. Sci., USA, 86:5054-5058 (1989)), phop.sup.c (Miller et al. (iii) mutants that inactivate global regulatory functions, such as recA (Buchmeier et al., J. Bact., 172:2485-2490 (1990)), or ompR (Dorman et al., Infect. Immun., 57:2136-2140 (1989)) mutants; (iv) mutants that inactivate global regulatory functions, such as recA (Buchmeier et al., MoI. Micro., 7:933-936 (1993)), htrA (Johnson et al., MoI. Micro., 5:401-407 (1991)), htpR (Neidhardt et al., Biochem. Biophys. Res. Com., 100:894-900 (1981)), hsp (Neidhardt et al. Ann. Rev. Genet. 18:295-329 (1984)), and groEL (Buchmeier et al. Sci. 248:730-732 (1990)) mutants; IsyA (Libby et al. Proc. Natl. Acad. Sci. USA 91:489-493 (1994)), pag or prg (Miller et al. (1990), see above, and Miller et al. (1989), see above), iscA or virG (d'Hauteville et al. MoI. Micro. 6:833-841 (1992)), plcA (Mengaud et al. (v) mutants affecting DNA topology, such as topA (Galan et al. Infect. Immun., 58:1879-1885 (1990)); (vi) mutants that disrupt or modify the cell cycle, such as min (de Boer et al. Cell, 56:641-649 (1989)); (vii) mutants that disrupt or modify the cell cycle, such as sacB (Recorbet et al. J. Exp. Med., 173:751-754 (1991)); and act (Brundage et al. Proc. Natl. Acad. Sci., USA, 90:11890-11894 (1993)) mutants. al. App. Environ. Micro., 59:1361-1366 (1993), Quandt et al. Gene, 127:15-21 (1993)), nuc (Ahrenholtz et al. App. Environ. Micro., 60:3746-3751 (1994)), hok, gef, kil, or phlA (Molin et al. Ann. Rev. Microbiol., 47:139-166 (1993)); (viii) introduction of genes encoding suicide systems such as rFb (Raetz in Esherishia coli and Salmonella typhimurium, Neidhardt et al., Ed., ASM Press, Washington DC pp 1035-1063 (1996)), galE (Hone et al. J. Infect. Dis., 156:164-167 (1987)), and htrB (Raetz, see above), msbB (Reatz, see above, and U.S. Pat. No. 7,514,089); and (ix) mutants that alter the biogenesis of lipopolysaccharide and / or lipid A, such as P22 (Rennell et al. Virol., 143:280-289 (1985)), ramdamurein transglycosylase (Bienkowska-Szewczyk et al. Mol. Gen. Genet., 184:111-114 (1981)), or S-genes (Reader et al. and one or more other attenuating mutations, such as the introduction of a bacteriophage lytic system, such as the lysogen encoded by the bacteriophage lysogen M. von Weinberg, et al., Virol., 43:623-628 (1971)).

[0077] Attenuated mutants can be either constitutively expressed or under the control of an inducible promoter such as a temperature-sensitive heat shock family promoter (Neidhardt et al., supra) or the anaerobically induced nirB promoter (Harbome et al., Mol. Micro., 6:2805-2813 (1992)), or a repressible promoter such as uapA (Gorfinkiel et al., J. Biol. Chem., 268:23376-23381 (1993)) or gcv (Stauffer et al., J. Bact., 176:6159-6164 (1994)).

[0078] In one embodiment, the bacterial delivery system is based on a safe, non-virulent, attenuated Salmonella strain with a partial deletion of the msbB gene. This deletion reduces the TNF immune response to bacterial lipopolysaccharide and prevents septic shock. In another embodiment, it also has a partial deletion of the purI gene. This deletion renders the bacteria dependent on an external source of purines and accelerates clearance from non-cancerous tissues (13). In mice, the virulence (LD) of the therapeutic strain was significantly reduced. 50 ) is 10,000-fold less than wild-type Salmonella (72, 73). In preclinical studies, attenuated Salmonella was administered systemically to mice and dogs without toxic side effects (17, 27). Two FDA-approved phase I clinical trials have been conducted, demonstrating that this therapeutic strain can be safely administered to patients (20). In one embodiment, the bacterial strain is VNP20009, a derivative strain of Salmonella typhimurium. Deletion of two genes, msbB and purI, resulted in its complete attenuation (by preventing toxic shock in the animal host) and dependence on an external source of purines for survival. This dependence renders the organism incapable of replicating in normal tissues such as the liver or spleen, but it remains capable of growing in tumors where purines are available.

[0079] Furthermore, the insertion of a fail-safe circuit into the bacterial vector prevents unwanted infection and defines the end of therapy without the need for antibiotics to eliminate bacteria (e.g., Salmonella).

[0080] Flagellum 1) flhDC sequence In one embodiment, the flhDC sequence is the bicistronic flhDC coding region found in Salmonella typhimurium strain 14028s or a derivative thereof.

[0081] Accession number- fhD-NCBI reference sequence: NC_016856.1 flhC- NCBI reference sequence: NC_016856.1

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[0085] Other sequences can also be used to control flagellar activity, including, for example, motA, motB, flhE, cheZ, cheY cheB, cheR, cheM, cheW, cheA, fliA, fliY, fliZ, fliB, fliS, fliE, fliF, fliJ, fliL, fliM, fliN, fliO, flip, fliQ, fliR, fliG, fliH, fliI, fliT, fliD, fliC, fljB, ycrG, flgN, flgM, flgA, flgB, flgC, flgD, flgE, flgF, flgG, flgH, flgI, flgJ, flgK, and / or flgL.

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[0132] II. Vectors / Plasmids In the present compositions and / or methods, DNA, RNA (e.g., nucleic acid-based gene interference agents), or proteins may be produced by recombinant methods. The nucleic acid is inserted into a replicable vector for expression. Many such vectors are available. Vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence and a coding sequence. In some embodiments, for example, in the use of bacterial delivery agents such as Salmonella, the gene and / or promoter (sequence of interest) may be integrated into the host cell chromosome or may be presented on a plasmid / vector, for example.

[0133] Expression vectors usually contain a selection gene, also called a selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with a vector containing the selection gene will not survive in the culture medium. Typical selection genes encode (a) proteins that confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, (b) proteins that complement auxotrophic deficiencies, or (c) proteins that supply important nutrients unavailable from complex media.

[0134] Expression vectors can contain a promoter recognized by the host organism and operably linked to a nucleic acid sequence, such as a nucleic acid sequence encoding an open reading frame. Promoters are untranslated sequences located upstream (5') to the start codon of a structural gene (generally within about 100-1000 bp) that control transcription of the specific nucleic acid sequence to which they are operably linked. In bacterial cells, the region controlling global regulation can be called the operator. Promoters typically fall into two classes: inducible and constitutive. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of a nutrient or a change in temperature. Numerous promoters recognized by a variety of potential host cells are well known.

[0135] Suitable promoters for use with prokaryotic hosts include the β-lactamase and lactose promoter systems, alkaline phosphatase, tryptophan (trp) promoter systems, hybrid promoters such as the tac promoter, and starvation promoters (Matin, A. (1994) Recombinant DNA Technology II, Annals of the New York Academy of Sciences, 722:277-291). However, other known bacterial promoters are also suitable. Such nucleotide sequences have been published, allowing one of skill in the art to operably link them to DNA coding sequences. Promoters for use in bacterial systems can also contain a Shine-Dalgarno (SD) sequence operably linked to the coding sequence.

[0136] Construction of suitable vectors containing one or more of the above-listed components employs standard ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to generate the required plasmid.

[0137] In some embodiments of the invention, the expression vector is a plasmid or bacteriophage vector suitable for use in Salmonella, and the DNA, RNA, and / or protein is provided to the subject through expression by engineered Salmonella (which in one aspect is attenuated) administered to the patient. As used herein, the term "plasmid" refers to any nucleic acid encoding an expressible gene, including linear or circular nucleic acids and double-stranded or single-stranded nucleic acids. The nucleic acid can be DNA or RNA, may contain modified nucleotides or ribonucleotides, and may be chemically modified by means such as methylation or the inclusion of protecting groups or cap or tail structures.

[0138]

[0013] One embodiment provides a Salmonella strain comprising a lysis gene or cassette operably linked to an intracellularly induced Salmonella promoter. In one embodiment, the promoter is a promoter from the group SpiC / SsaB (Accession No. CBW17423.1), SseF (Accession No. CBW17434.1), SseG (Accession No. CBW17435.1), SseI (Accession No. CBW17087.1), SseJ (Accession No. CBW17656.1 or NC_016856.1), SseK1 (Accession No. CBW2 0184.1), SseK2 (accession number CBW18209.1), SifA (accession number CBW17257.1), SifB (accession number CBW17627.1), PipB (accession number CBW17123.1), PipB2 (accession number CBW18862.1), SopD2 (accession number CBW17005.1), GogB (accession number CBW18646.2 ), SseL (Accession No. CBW18358.1), SteC (Accession No. CBW17723.1), SspH1 (Accession No. STM14_1483), SspH2 (Accession No. CBW18313.1), or SirP (examples / embodiments of sequences that can be used in the present compositions / methods are provided by the accession numbers and sequences provided throughout the specification, other sequences, including sequences with greater than about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, and 100% identity, may also be used in the compositions / methods of the invention).

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[0158] In one embodiment, the Salmonella gene under the control of an inducible promoter is selected from the group consisting of ftsW (accession number CBW16230.1), ftsA (accession number CBW16235.1), ftsZ (accession number CBW16236.1), murE (accession number CBW16226.1), mukF (accession number CBW17025.1), imp (accession number CBW16196.1), secF (accession number CBW16503.1), eno (accession number CBW19030.1), hemH (accession number CBW16582.1), tmk (accession number CBW17233.1), dxs (accession number CBW16516.1), uppS (accession number CBW16324.1), cdsA (accession number CBW16325.1), a ccA (accession number CBW16335.1), pssA (accession number CBW18718.1), msbA (accession number CBW17017.1), tsf (accession number CBW16320.1), trmD (accession number CBW18749.1), cca (accession number CBW19276.1), infB (accession number CBW19355.1), rpoA (accession No. CBW19477.1), rpoB (accession no. CBW20180.1), rpoC (accession no. CBW20181.1), holA (accession no. CBW16734.1), dnaC (accession no. CBW20563.1), or eng (EngA accession no. CBW18582.1, EngB accession no. CBW20039.1).

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[0186] Other inducible promoters for use in the present invention include those that inducibly control flagella:

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[0191] These include, but are not limited to: III. Therapeutic DNA, RNA, and Peptides The present invention delivers therapeutic DNA, RNA, and / or peptides to cancer cells.

[0192] Gene silencing through RNAi (RNA interference) by the use of short interfering RNA (siRNA) can be used for therapeutic gene silencing. Short hairpin RNA (shRNA) transcribed from small DNA plasmids within target cells has also been shown to mediate stable gene silencing, achieving gene knockdown levels comparable to those obtained by transfection with chemically synthesized siRNA.

[0193] RNAi agent is the agent that regulates the expression of RNA by RNA interference mechanism.The RNAi agent that is used in one embodiment of the present invention is small ribonucleic acid molecule (also referred to herein as interfering ribonucleic acid), that is, the oligoribonucleotide that exists in duplex structure, for example, two separate oligoribonucleotides that hybridize with each other (for example, siRNA), or a single ribonucleotide that assumes small hairpin formation to produce duplex structure (for example, shRNA).

[0194] dsRNA can be prepared according to any of the many methods available in the art, including in vitro and in vivo methods as well as synthetic chemistry approaches.Single-stranded RNA can also be produced by using the combination of enzymatic synthesis and organic synthesis, or by total organic synthesis.The use of synthetic chemistry method allows the introduction of desired modified nucleotide or nucleotide analogue into dsRNA.

[0195] In certain embodiments, instead of RNAi agent being interfering ribonucleic acid, for example, siRNA or shRNA as described above, RNAi agent can be as described above, encoding interfering ribonucleic acid, for example, shRNA.In other words, RNAi agent can be the transcription template of interfering ribonucleic acid.In these embodiments, transcription template is typically the DNA that encodes interfering ribonucleic acid.DNA can be in vector, and various different vectors are known in the art, for example, plasmid vector, virus vector etc.

[0196] Alternatively, the active agent may be a ribozyme. As used herein for purposes of the specification and claims, the term "ribozyme" is interchangeable with "catalytic RNA" and refers to an RNA molecule capable of catalyzing a chemical reaction.

[0197] Exemplary target genes include, but are not limited to, EZH2 (the accession number for human EZH2 mRNA is NM_004456), NIPP1 (the accession number for human NIPP1 mRNA is NM_002713), and PP1 (the accession numbers for human PP1 mRNA are PP1α mRNA: NM_002708, PP1β mRNA: NM_206876, and PP1γ mRNA: NM_002710). EZH2, NIPP1, and PP1 interfere with cancer cell processes, eliminating and / or reducing cancer stem cells. This will stop tumor spread / growth and prevent metastasis formation.

[0198] In another embodiment, the epigenetic target is NIPP1 (accession number NM_002713), EZH2 (accession number NM_004456), PP1α (accession number NM_002708), PP1β (accession number NM_206876), PP1γ (accession number NM_002710), Suz12 (accession number NM_015355), EED (accession number NM_003797), EZH1 (accession number NM_001991), RbAp48 (accession number NM_005610), Jarid2 (accession number NM_004973), YY1 (accession number NM_003403), CBX 2 (accession number NM_005189), CBX4 (accession number NM_003655), CBX6 (accession number NM_014292), CBX7 (accession number NM_175709), PHC1 (accession number NM_004426), PHC2 (accession number NM_198040), PHC3 (accession number NM_198040), No. NM_024947), BMI1 (Accession No. NM_005180), PCGF2 (Accession No. NM_007144), ZNF134 (Accession No. NM_003435), RING1 (Accession No. NM_002931), RNF2 (Accession No. NM_0072120), PHF1 (Accession No. No. NM_024165), MTF2 (accession number NM_007358), PHF19 (accession number NM_001286840), SETD1A (accession number XM_005255723), SETD1B (accession number NM_015048), CXXC1 (accession number NM_001101654), ASH2L (accession number NM_004674), DPY30 (accession number NM_032574), RBBP5 (accession number NM_005057), WDR5 (accession number NM_017588), KMT2A (accession number NM_001197104), KMT2D (accession number XM_00 6719616), KMT2B (accession number NM_014727), KMT2C (accession number NM_170606), KAT8 (accession number NM_032188), KDM6A (accession number NM_001291415), NCOA6 (accession number NM_014071), PAGR1 (accession number NM_024516), PAXIP1 (accession number NM_007349), ASH1L (accession number NM_018489), SMARCA2 (accession number NM_003070), SMARCA4 (accession number NM_001128844), BPTF (accession number NM_182641),or SMARCA1 (accession number NM_001282874) (e.g., mRNA).

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[0334] In some embodiments, the therapeutic peptide expressed by the bacterial cell is a caspase, such as caspase 3 (eg, expressed in its active form), or NIPP1. IV. Cancer Treatment Bacteria such as Salmonella, Clostridium, and Bifidobacterium have a natural tropism for cancers, such as solid tumors. Types of cancer that can be treated using the methods of the present invention include solid tumors, such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial tumor, lymphangiosarcoma, lymphangioendothelial tumor, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and papillary adenocarcinoma). , cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma).

[0335] In some aspects, the subject is treated with radiation and chemotherapy before, after, or during administration of the bacterial cells described herein. V. Administration The present invention includes the administration of the attenuated Salmonella strains described herein, as well as methods for preparing pharmaceutical compositions and methods for administering the same, which methods involve formulating a pharmaceutically acceptable carrier with one or more of the attenuated Salmonella strains described herein.

[0336] The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous administration can contain the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate; and an agent for adjusting tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0337] Suitable carriers for intravenous administration include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ, USA), or phosphate-buffered saline (PBS). It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of other (undesirable) microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols (mannitol, sorbitol, sodium chloride, etc.), in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0338] Injectable solution can be prepared by incorporating active compound in appropriate solvent with one of the above-mentioned components or the combination of above-mentioned components in the required amount.Generally, dispersion is prepared by incorporating active compound into the vehicle that contains basic dispersion medium and various other components as discussed above.For the powder that is used to prepare injectable solution, the preferred preparation method is vacuum drying and freeze-drying, which produces the powder of active ingredient and any additional desired components from before.

[0339] Oral compositions generally contain inert diluents or edible carriers.For example, they can be encapsulated in gelatin capsules.For the purpose of oral therapeutic administration, active compound can be incorporated with excipients and used in the form of tablets, troches or capsules.

[0340] Pharmaceutically compatible binders, and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches, and the like can contain any of the following ingredients, or compounds of a similar nature: binders such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch or lactose; disintegrating agents such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring.

[0341] For administration by inhalation, the bacteria are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0342] Systemic administration can also be carried out by transmucosal or transdermal means.For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation.Such penetrants are generally known in the art, and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration.Transmucosal administration can be achieved through the use of nasal sprays or suppositories.For transdermal administration, the bacteria is formulated into ointments, salves, gels, or creams that are generally known in the art.

[0343] For ease of administration and uniformity of dosage, it is particularly advantageous to prepare compositions in dosage unit form.As used herein, dosage unit form refers to a physically separate unit that is suitable as a unit dose for the subject to be treated, and each unit contains a predetermined amount of active compound that is calculated to produce desired therapeutic effect, in association with required pharmaceutical carrier.The specification of dosage unit form of the present invention is indicated by and directly depends on the specific characteristics and specific therapeutic effect of active compound that should be achieved and the inherent limitation of the technology that formulates these active compounds for individual treatment.

[0344] When administered to a patient, the attenuated Salmonella can be used alone or in combination with any physiological carrier. Generally, the dose is from about 1.0 cfu / kg to about 1 x 10 12 cfu / kg, optionally from about 1.0 cfu / kg to about 1 x 10 10 cfu / kg, optionally from about 1.0 cfu / kg to about 1 x 10 8 cfu / kg, optionally about 1 x 10 2 cfu / kg ~ approx. 1 x 10 8 cfu / kg, optionally about 1 x 10 4 cfu / kg ~ approx. 1 x 10 8 cfu / kg, optionally about 1 x 10 5 cfu / kg ~ approx. 1 x 10 12 cfu / kg, optionally about 1 x 105 cfu / kg ~ approx. 1 x 10 10 cfu / kg, optionally about 1 x 10 5 cfu / kg ~ approx. 1 x 10 8 cfu / kg range. [Example]

[0345] The following examples are provided to demonstrate and further illustrate certain embodiments and aspects of the present invention and should not be construed as limiting its scope. Example I introduction Delivering protein drugs to the cytoplasm of cancer cells will increase the number of treatable cancer targets. Over 60% of pathways that control cell function are intracellular (1), and nearly all are difficult to access. Intracellular pathways control most of the hallmarks of cancer (2) and are the focus of a significant portion of cancer research. Due to their specificity, protein biologics are excellent candidates for interfering with these pathways. However, transporting functional proteins across the cell membrane is technically challenging. Effective intracellular delivery, coupled with specific protein drugs, has the potential to provide new treatments for previously untreatable cancers.

[0346] material and method bacterial culture All bacterial cultures (both Salmonella and DH5α) were grown in LB (10 g / L sodium chloride, 10 g / L tryptone, and 5 g / L yeast extract). Resistant strains of bacteria were grown in the presence of carbenicillin (100 μg / ml), chloramphenicol (33 μg / ml), kanamycin (50 μg / ml), and / or 100 μg / ml DAP.

[0347] Bacterial strains and plasmid construction Fifteen strains of Salmonella Enterica serovar Typhimurium were used throughout the experiments (Table S1). All plasmids contained a ColE1 origin and either chloramphenicol or ampicillin resistance (Table S2). All assembled DNA constructs were transformed into chemically competent DH5α E. coli (New England Biolabs, Ipswich, MA, USA) and then electroporated into Salmonella. All cloning reagents, buffer reagents, and primers were from New England Biolabs, Fisher Scientific (Hampton, NH, USA), and Invitrogen (Carlsbad, CA, USA), respectively, unless otherwise noted.

[0348] For electroporation, Salmonella cultures were grown to an optical density of 0.6–0.8, washed twice with 25 ml of ice-cold water, and resuspended in 400 μL of ice-cold water. DNA (200 ng for plasmids or 1–2 μg for linear DNA) was mixed with 50 μL of the bacterial suspension and electroporated in a 1 mm electroporation cuvette at 1800 V and 25 μF with a 5 ms time constant.

[0349] The parental control strain (Par) was based on an attenuated therapeutic strain of Salmonella (VNP20009), which contains three deletions—ΔmsbB, ΔpurI, and Δxyl—that eliminate most virulence in vivo. A strain (VNP200010) lacking the asd gene was used to allow for balanced lethal plasmid maintenance (1). A second strain (ΔflhD Par) was the basis for many of the strains in the study (Table S1). This strain was generated by first deleting flhD and then deleting asd.

[0350] Gene deletions were created using a modified Lambda Red recombination protocol (2). Salmonella were transformed with pkd46 (Yale CGSC E. coli stock center) and grown from single colonies in 50 ml of LB. At an optical density of 0.1, arabinose was added to the bacterial culture to a final concentration of 20 mM. When the optical density reached 0.6–0.8, the bacteria were centrifuged at 3,000 × g and washed twice with 25 ml of ice-cold ultrapure water (Millipore). The pelleted Salmonella were resuspended in 400 μL of ice-cold water. A linear DNA segment was designed to insert an in-frame deletion into a gene (here, flhD). It was generated by PCR amplification of FRT-KAN-FRT from plasmid pkd4 using primers vr121 and vr309 (Table S3). The PCR product contained kanamycin resistance flanked by FRT recombination sites and a 50-base pair region homologous to flhD. After electroporation, Salmonella were recovered in LB at 37°C for 2 hours and left at room temperature overnight. The recovered solution was plated on kanamycin (50 μg / ml) agar plates and incubated at 37°C until colonies formed. Colonies were screened for knockout by colony PCR. Successful transformants were plated on kanamycin plates and grown overnight at 43°C to remove pkd46 from the bacteria.

[0351] A similar process was used to delete asd. Transformants with successful flhD deletion were transformed with pkd46. A PCR product was generated to insert an in-frame deletion into asd by PCR, amplifying FRT-CHLOR-FRT from plasmid pkd3 using primers vr266 and vr268 (Table S3). This PCR product contained chloramphenicol resistance flanked by FRT recombination sites and a 50-base pair region of homology to asd. During recovery, electroporated bacteria were plated on agar containing 33 μg / ml chloramphenicol and 100 μg / ml diaminopimelic acid (DAP). Successful transformants were grown in the presence of chloramphenicol, kanamycin, and DAP.

[0352] To generate an intracellular reporting strain of Salmonella, the parent Salmonella strain (Par) was transformed with a plasmid containing PsseJ-GFP (plasmid P1; Table S2). Construction of this plasmid began by first creating a promoterless GFP plasmid from pLacGFP and pQS-GFP [1]. The pQS-GFP plasmid contains chloramphenicol resistance, a ColE1 replication origin, and the asd gene. Expression of ASD is required in the Δasd strain, creating a balanced lethal system that maintains gene expression in vivo. The Plac-GFP gene circuit was amplified from the plasmid pLacGFP using primers nd1 and nd2 (Table S4). The PCR product and plasmid were digested with Aat2 and Pci1 and ligated with T4 DNA ligase (NEB, catalog no. M0202S). The PsseJ promoter was amplified from the genome of SL1344 Salmonella using primers nd3 and nd4 (Table S4). This PCR product and the backbone plasmid were ligated after digestion with XbaI and PciI.

[0353] A strain re-expressing flhDC (flhDC Sal, Table S1) was generated by transforming ΔflhD Salmonella with plasmid P2 (Table S2). Plasmid P2 was generated from transient plasmid P3. Plasmid P3 was generated by amplifying flhDC from Salmonella genomic DNA using primers vr46 and vr47 (Table S4) and ligating it into plasmid PBAD-his-mycA (Invitrogen; catalog no. V430-01). The PCR product was digested with NcoI, XhoI, and DpnI (NEB, catalog nos. R0193S, R0146S, and R0176L). The PBAD-his-mycA plasmid was digested with NcoI and XhoI and treated with calf intestinal phosphatase (NEB, catalog no. M0290) for 3 hours. The PCR product was ligated into the plasmid backbone using T4 DNA ligase (NEB, Cat. No. M0202S).

[0354] The Plac-GFP-myc circuit was inserted into P3 by Gibson assembly. (1) The insert (Plac-GFP-myc) was amplified from the plasmid pLacGFP(1) using primers vr394 and vr395 (Table S4), which added a homology region to the backbone and a myc tag. (2) The backbone plasmid (P3) was amplified using primers vr385 and vr386, which added homology to the insert. (3) Both PCR products were digested with DpnI for 3 hours and (4) ligated by Gibson assembly (HiFi Master Mix, NEB, catalog no. E2621L). The gene for aspartate semialdehyde dehydrogenase (asd) was inserted by Gibson assembly by amplifying asd from genomic Salmonella DNA using primers vr424 and vr425 and amplifying the plasmid backbone with primers vr426 and vr427.

[0355] A strain re-expressing flhDC and producing GFP after invasion (flhDC reporting, Table S1) was created by transforming ΔflhD Salmonella with plasmid P4 (Table S2). The PsseJ-GFP-myc gene circuit was amplified from P1 using primers vr269 and vr270, and the backbone of plasmid P3 was amplified using primers vr271 and vr272. The two PCR products were ligated by Gibson assembly.

[0356] To generate the PsifA intracellular promoter-reporter strain, the PsifA promoter was cloned from Salmonella genomic DNA using primers nd5 and nd6 and inserted into P1 using XbaI and the PciI-generated plasmid P5. The PsifA reporter strain was created by transforming plasmid P5 into background Salmonella by electroporation. The generation of the PsseJ reporter strain has been described above. To investigate lysis in Salmonella, the lysis gene E (LysE) was placed under the control of PBAD. LysE was cloned using primers nd7 and nd8 and inserted into pBAD / Myc-His A (Invitrogen) using NcoI and KpnI to form plasmid P6.

[0357] Intracellular delivery (ID) Salmonella was created by cloning the Lysin E gene behind the PsseJ promoter. LysE was amplified using primers nd9 and nd10 and cloned into P1 using XbaI and Aat2. The Plac-GFP circuit was added to this plasmid by cloning it from plasmid pLacGFP using primers nd11 and nd12 and inserting it using SacI to create plasmid P7. This plasmid constitutively expresses myc-tagged GFP for bacterial identification in both live and fixed cell assays.

[0358] Genomic knockouts ΔsifA and ΔsseJ were created using the modified Lambda Red recombination protocol described above for the generation of ΔflhD Salmonella. Salmonella was transformed with pkd46. Linear DNA with homologous flanking regions was generated by PCR of the pkd4 plasmid using primers vr432 and vr433 for ΔsseJ and vr434 and vr435 for ΔsifA. After electroporation and recovery, colonies were screened for knockouts by colony PCR of the junction site of the inserted PCR amplicon. Successful transformants were plated on kanamycin plates (50 μg / ml) and grown overnight at 43°C to remove pkd46.

[0359] ID Salmonella re-expressing flhDC (flhDC-ID Sal) was generated by transforming ΔflhD with plasmid P8. Plasmid P8 was generated by amplifying the Pssej-LysE gene circuit from P7 using primers vr398 and vr399 and ligating it into plasmid P2 using Gibson assembly. The P2 backbone plasmid was amplified using primers vr396 and vr397.

[0360] A strain of ID Salmonella constitutively expressing luciferase (ID Sal-luc; Table S1) was created by cloning Plac-luc from pMA3160 (Addgene) using primers ch1 and ch2. The P7 plasmid backbone was amplified with primers ch3 and ch4, and the fragments were ligated by Gibson assembly to form plasmid P9 (Table S2).

[0361] To generate ID Salmonella expressing an anti-b-actin nanobody (NB), a PBAD-inducible nanobody was cloned in place of flhDC in plasmid P8. The actin nanobody (Chromotek, catalog number acr) was amplified using primers vr466 and vr467. The delivery plasmid backbone was amplified using primers vr448 and vr449. The two PCR products were ligated by Gibson assembly to generate plasmid P10.

[0362] To generate ID Salmonella expressing the central domain of NIPP1 (NIPP1-CD), NIPP1-CD was cloned into the pLacGFP plasmid. NIPP1-CD and the backbone plasmid were amplified using primers nd13 to nd16 linked by Gibson assembly. The pLac-NIPP1-CD circuit was cloned using primers nd11 and nd17 (Table S4) and inserted into P7 using SacI to generate plasmid P11.

[0363] To generate ID Salmonella that deliver CT caspase-3 (CT Casp-3) intracellularly, we transformed parental Salmonella with plasmid P12. This plasmid was generated by PCR amplification of template DNA encoding CT caspase-3 from the constitutively double-stranded (CT) caspase-3-encoding plasmid pC3D175CT using primers vr450 and vr451. The pC3D175CT plasmid (Hardy Lab DNA Archive Box 7, line 62) was constructed similarly to the caspase-6 CT expression construct [3] using Quikchange mutagenesis on a construct encoding full-length human caspase-3 in the pET23 expression vector (Addgene). Plasmid pC3D175CT encodes human caspase-3 residues 1 to 175, followed by a TAA stop codon, a ribosome binding sequence, and coding sequence for an initiating methionine and an inserted serine, followed by coding sequence for residues 176 to 286, adding a six-histidine tag. The backbone of plasmid P8 was PCR amplified using primers vr448 and vr449, and the PCR products were ligated as previously described.

[0364] [Table 1-1]

[0365] [Table 1-2]

[0366] [Table 2]

[0367] [Table 3]

[0368] [Table 4-1]

[0369] [Table 4-2]

[0370] [Table 4-3]

[0371] cell culture Four cancer cell lines were used: 4T1 mouse breast cancer cells, Hepa1-6 mouse hepatocellular carcinoma cells, MCF7 human breast cancer cells, and LS174T human colorectal cancer cells (ATCC, Manassas, VA, USA). All cancer cells were grown and maintained in Dulbecco's minimal Eagle's medium (DMEM) containing 3.7 g / L sodium bicarbonate and 10% fetal bovine serum. For microscopy studies, cells were incubated in DMEM with 20 mM HEPES buffer and 10% FBS. To generate tumor spheroids, a single-cell suspension of LS174T cells was transferred to a PMMA-coated cell culture flask (2 g / L PMMA in 100% ethanol, dried before use).

[0372] Salmonella invasion into cancer cells in vitro To observe invasion into cancer cells, Salmonella was administered to murine 4T1 breast cancer cells grown on coverslips using an invasion assay. Cells and bacteria were stained with phalloidin and anti-Salmonella antibodies and imaged under a 100x oil immersion microscope. General procedures for the invasion assay, immunocytochemistry, and microscopy are detailed in the following sections.

[0373] Invasion assay For the invasion assay, cancer cells were grown on cover slips for fixed-cell imaging or on well plates for live-cell imaging. For fixed-cell imaging, glass cover slips were placed in 12-well plates and sterilized with UV light for 20 minutes in a biosafety hood. Mouse 4T1 or human MCF7 cells were seeded onto the cover slips at 40% confluency and incubated overnight in DMEM. Simultaneously, Salmonella was grown to an optical density (OD at 600 nm) of 0.8. After incubation, Salmonella was added to the 4T1 cultures at a multiplicity of infection (MOI) of 10 and allowed to infect the cells for 2 hours. After this invasion period, the cultures were washed five times with 1 ml of phosphate-buffered saline (PBS) and resuspended in 2 ml of DMEM supplemented with 20 mM HEPES, 10% FBS, and 50 μg / ml gentamicin. The added gentamicin removes extracellular bacteria. After 6 hours of incubation, the medium was removed and the coverslips were fixed with 10% formalin in PBS for 10 minutes.

[0374] A similar procedure was used for live-cell imaging. Cells were grown directly on well plates in DMEM (3.7 g / L sodium bicarbonate, 10% FBS) to 30-50% confluency. After growth to an OD of 0.8, Salmonella was added to the cell culture at an MOI of 25 for 2 hours. After invasion, cancer cells were washed five times with PBS, and 2 ml of DMEM with 50 μg / ml gentamicin was added to each well. Cells and bacteria were directly imaged under a microscope.

[0375] immunocytochemistry Using immunocytochemistry, detailed images of Salmonella invading cancer cells grown on coverslips were obtained. Coverslips were fixed with formalin and then blocked for 30 minutes with staining buffer (PBS with 0.1% Tween 20, 1 mM EDTA, and 2% bovine serum albumin [BSA]). The Tween 20 in this buffer selectively permeabilizes mammalian cell membranes while leaving bacterial membranes intact.

[0376] After permeabilization, coverslips were stained with (1) rabbit anti-Salmonella polyclonal antibody (Abcam, ab35156) or FITC-conjugated rabbit anti-Salmonella polyclonal antibody (Abcam, ab69253), (2) rat anti-myc monoclonal antibody (Chromotek, catalog no. 9e1-100), (3) rabbit anti-LAMP1 polyclonal antibody (Abcam, catalog no. ab24170), and (4) Alexaflor-568-conjugated phalloidin (ThermoFisher, catalog no. A12380) to identify Salmonella, released GFP, vacuolar membranes, and / or intracellular f-actin. Three different staining combinations were used: (1) Salmonella alone, (2) Salmonella, released GFP, and actin, and (3) Salmonella, released GFP, and vacuoles.

[0377] For staining of Salmonella alone (combination 1), coverslips were stained with FITC-conjugated anti-Salmonella antibody for 1 hour at 30° C. and washed three times with staining buffer. For Salmonella, released GFP, and actin staining (combination 2), coverslips were stained with anti-Salmonella and anti-myc primary antibodies for 1 h at 30°C and washed twice with staining buffer. To identify Salmonella, GFP, and intracellular f-actin, coverslips were incubated with secondary antibodies at a 1:200 dilution for 1 h at 30°C: Alexaflor-647 chicken anti-rabbit (ThermoFisher, catalog no. A21443), Alexaflor-488 donkey anti-rat (ThermoFisher, catalog no. A21208), and Alexaflor-568-conjugated phalloidin, respectively.

[0378] For Salmonella, released GFP, and vacuole staining (combination 3), coverslips were sequentially stained with anti-LAMP1 primary antibody for 1 hour at 30°C and washed three times with staining buffer. Coverslips were incubated with Alexaflor-647 chicken anti-rabbit secondary antibody (ThermoFisher, catalog no. A21443) at a 1:200 dilution for 1 hour at 30°C and washed four times with staining buffer. Coverslips were then stained with FITC-conjugated anti-Salmonella antibody and anti-myc primary antibody and washed three times with staining buffer. Coverslips were incubated with Alexaflor-568 goat anti-rat secondary antibody (ThermoFisher, A11077) at a 1:200 dilution for 1 hour at 30°C to identify GFP.

[0379] After all staining, coverslips were washed three times with staining buffer and mounted on glass slides using 20 μL of mounting medium with DAPI (ProLong Gold Antifade Mountant, ThermoFisher, catalog no. P36962). The mounted coverslips were allowed to harden overnight at room temperature.

[0380] Microscopy Samples were imaged on a Zeiss Axio Observer Z.1 microscope. Fixed cells on coverslips were imaged using a 100x oil-immersion objective (1.4 NA). Tumor sections were imaged using 10x and 20x objectives (0.3 and 0.4 NA, respectively). Time-lapse fluorescence microscopy of live cells in well plates and tumor chip devices was housed in a humidified 37°C environment and imaged using 5x, 10x, 63x, or 100x objectives (0.2, 0.3, 1.4, and 1.4 NA, respectively). Fluorescence images were acquired using either 480 / 525 or 525 / 590 excitation / emission filters. All images were background subtracted and uniformly contrast-enhanced. Some image analysis was automated using computer code (MATLAB, Mathworks).

[0381] Intracellular Salmonella in tumors To determine the proportion of intracellular tumor-colonizing Salmonella, BALB / c mice bearing 4T1 tumors were inoculated with 2 × 10 6 CFU of intracellularly reporting Salmonella (harboring PsseJ-GFP; Table S1) was injected. 96 hours after bacterial injection, mice were sacrificed, and tumors were excised, sectioned, and stained as described in the immunohistochemistry section below. Tumor sections were stained to identify Salmonella and GFP produced by intracellular Salmonella. The percentage of intracellular Salmonella was determined by identifying Salmonella (n = 1,258) within eight images and determining the number that colocalized with GFP.

[0382] immunohistochemistry Resected tumor sections were fixed in 10% formalin for 3 days. Fixed tumor samples were then stored in 70% ethanol for 1 week. Tumor samples were embedded in paraffin and sectioned into 5 μm sections. Deparaffinization was performed by washing the sectioned tissue three times in 100% xylene, twice in 100% ethanol, once in 95% ethanol, once in 70% ethanol, once in 50% ethanol, and once in DI water. Each washing step was performed for 5 minutes. Antigen retrieval was performed by incubating the tissue sections in 20 mM sodium citrate (pH 7.6) buffer at 95°C for 20 minutes. The samples were left in the sodium citrate buffer until the temperature decreased to 40°C. The samples were then rehydrated with two quick (<1 minute) rinses in DI water, followed by one 5-minute wash in TBS-T.

[0383] Before staining, tissue sections were blocked for 1 hour with Dako blocking buffer (Dako, catalog no. X0909). To identify Salmonella and GFP, tissue sections were stained with a 1:100 dilution of either (1) a FITC-conjugated rabbit anti-Salmonella polyclonal antibody (Abcam, catalog no. ab69253) and (2) a rat anti-myc monoclonal antibody (Chromotek, catalog no. 9e1-100) or a rat anti-GFP monoclonal antibody (Chromotek, catalog no. 3h9-100) in Tris-buffered saline with 0.1% Tween 20 (TBS-T) containing 2% BSA (FisherScientific, catalog no. BP9704-100). Sections were washed three times in TBS-T with 2% BSA and incubated with Alexaflor-568 goat anti-rat secondary antibody (ThermoFisher, Cat. No. A11077). After washing the sections three times with TBS-T, 40 μL of mounting medium with DAPI (ThermoFisher, Cat. No. P36962) and a coverslip were added to each slide. Slides were incubated at room temperature for 24 hours until the mounting medium solidified.

[0384] Flow cytometry analysis of bacterial infiltration within tumors Flow cytometry was used to identify cells in tumors infiltrated by Salmonella and the effect of flhDC induction on infiltration. The types of cells infiltrated by Salmonella were determined by isolating infiltrated Salmonella-containing cells using EPCAM and anti-CD45 antibodies and classifying them into carcinoma, immune, and other tumor-associated cells. The effect of flhDC induction on cell infiltration was determined by comparing mice receiving non-flhDC-induced and flhDC-induced bacteria and counting the percentage of cells of the three cell types.

[0385] Two groups of mice were given 2 × 10 6CFU of flhDC Salmonella (Table S1) were injected via the tail vein. To induce production from the PBAD-flhDC gene construct, the flhDC-induced group (n = 9) received 100 μg of arabinose in 400 μL PBS via intraperitoneal (IP) injection 48 and 72 hours after bacterial injection. The control, non-flhDC-induced group (n = 8) received IP injections at the same time. 96 hours after bacterial injection, the mice were sacrificed, and tumors were excised and cut in half. Tumors were processed into single-cell suspensions, stained, and analyzed by flow cytometry.

[0386] To create single-cell suspensions from excised tumors, they were minced with a sterile razor blade in 5 ml of RPMI containing 20 mM HEPES, 10% FBS, 1 mg / ml collagenase D (Roche, catalog number 11088866001), 200 units / ml DNAse I (Roche, catalog number 04716728001), and 50 μg / ml gentamicin (ThermoFisher, catalog number BP918-1) to prevent bacterial overgrowth / infiltration. Once tumor pieces were less than 5 mm in length, the tumor slurry was added to a 7 ml douncer and dounced 10 times. The slurry was placed in a single well of a 6-well plate and incubated at 37°C for 2 hours. To separate the cells, the suspension was filtered through a 40 μm cell strainer (ThermoFisher, catalog number 22-363-547) and centrifuged at 300 × g for 5 minutes. Red blood cells (RBCs) were lysed by incubating the single-cell suspension with RBC lysis buffer (150 mM ammonium chloride, 12 mM sodium bicarbonate, and 0.1 mM EDTA) for 10 minutes. The cell suspension was added to 10 ml of D-PBS (Hyclone, catalog number SH30256001) and spun at 300 × g for 5 minutes.

[0387] Single-cell suspensions were fixed for 10 minutes at room temperature in PBS containing 1 mM EDTA and 5% formaldehyde. Fixed cells were spun at 600 × g for 5 minutes and resuspended in blocking buffer for 1 hour. The blocking buffer was TBS-T with 2% BSA and 1 mM EDTA. 0.1% Tween 20 permeabilizes cancer cells but not bacteria, as described in the immunocytochemistry section above. Cell suspensions were sequentially stained with FITC-conjugated anti-Salmonella antibody (Abcam, catalog no. ab69253), PE dazzle 594 anti-CD326 (EpCAM, BioLegend, catalog no. 118236), and APC anti-CD45 (Biolegend, catalog no. 103112) at concentrations of 1:2000, 1:2000, and 1:1000, respectively. First, anti-Salmonella antibodies were added to the cells for 45 minutes, followed by four washes and six washes with staining buffer (2% BSA, 1 mM EDTA, and 0.1% Tween in PBS). EpCAM and anti-CD45 were then added for 45 minutes, followed by two washes. Fluorescence minus one (FMO) of each sample was used as a gating control for each fluorophore. Samples were analyzed on a custom-built flow cytometer (dual LSR Fortessa 5-laser, BD). All fluorophores were compensated with compensation beads (BD, catalog no. 552845) and did not leak more than 2% into any other channel. Cells were first identified if they contained intracellular Salmonella. Non-immune cells (cancer and other related cells) were identified by staining samples with all antibodies except CD45 (i.e., the FMO gating control). Non-cancer cells (immune cells and other related cells) were identified by samples stained with all antibodies except anti-EpCAM (CD326).

[0388] Effect of flhDC induction on bacterial invasion into cells in culture To determine the effect of expressing flhDC on bacterial invasion, 4T1 cells were grown on glass coverslips as described in the infection assay section above. Inducible flhDC Salmonella (Table S1) were grown in LB with 20 mM arabinose to induce flhDC expression. Control (flhDC-) bacteria were grown without arabinose. Cancer cells were infected with both induced flhDC+ and flhDC- Salmonella at an MOI of 10 (n=4 for each condition). For the induced flhDC+ condition, 20 mM arabinose was added to the mammalian cultures to maintain expression. 18 hours after invasion, cancer cells were stained to identify intracellular Salmonella (Salmonella alone, combination 1) as described in the immunocytochemistry section above. Three images were acquired at 20x for each coverslip, resulting in a total of 12 images per condition. Invasion was quantified by randomly identifying 20 cancer cells from the DAPI channel of each image. Each cell was defined as invaded if Salmonella staining colocalized with the nucleus or was within 10 μm of the nucleus. The invasion percentage was defined as the number of invaded cells relative to the total number of cells.

[0389] In vitro effect of flhDCs on infiltration into tumor masses To quantify invasion into tumor mass, engineered Salmonella were administered to a tumor-on-a-chip device developed in our laboratory (6, 7). The microfluidic tumor-on-a-chip device was fabricated using negative-tone photoresist and PDMS-based soft lithography. A master chip was constructed by spin-coating a layer of SU-8 2050 onto a silicon wafer at 1250 RPM for 1 min. This speed corresponded to a thickness of 150 μm of SU-8 2050. The silicon wafer was baked at 65 °C for 5 min, followed by baking at 95 °C for 30 min. The microfluidic design printed on a high-resolution transparency was placed above the silicon wafer in a mask aligner. The silicon wafer with the overlaid mask was then illuminated with UV light (22 J / cm). 2) for 22 seconds. The silicon wafer was baked at 65°C for 5 minutes, followed by baking at 95°C for 12 minutes. The wafer was then developed in PGMEA developer solution for 10 minutes and / or until the microfluidic features were microscopically distinct with sharp and defined edges.

[0390] Soft lithography was used to create a multilayer tumor-on-a-chip device with 12 tumor chambers (two conditions with six chambers each). PDMS (Sylgard 184) was used in a ratio of 9:1 and 15:1 for the channel and valve layers, respectively. To achieve a PDMS thickness of 200 μm, the channel layer was placed on a spin coater at 220 rpm for 1 minute. The silicon wafer was degassed for 45 minutes to remove air bubbles within the PDMS. The silicon wafer was baked at 65°C for approximately 1 hour, or until both PDMS layers were partially cured. The top valve layer of PDMS was cut, removed from the silicon wafer, and aligned on top of the channel layer using a stereomicroscope. The combined layers were baked at 95°C for 1 hour to covalently bond the two layers. The multilayer PDMS device and glass slide were plasma-treated for 2.5 minutes in a plasma cleaner (Harrick). A vacuum pump was used to pneumatically actuate the valve and place the PDMS on the plasma-treated glass slide. The valve was left actuated until the device was ready for use.

[0391] The tumor-on-a-chip was sterilized with 10% bleach, followed by 70% ethanol for 1 hour each. The microfluidic chip was equilibrated with culture medium (DMEM with 20 mM HEPES, pH 7.4) for 1 hour. The tumor spheroid was positioned within the tumor chamber using valve actuation. The rear valve of the chamber was opened while the outflow channel was closed. After the tumor mass was positioned, the valve was reset so that the rear valve was closed and the inflow and outflow channels were open.

[0392] Prior to administration to the device, flhDC-reporting Salmonella (Table S1) were grown in LB with 20 mM arabinose to induce flhDC expression. These Salmonella have an inducible flhDC (PBAD-flhDC) and produce GFP when intracellular (PsseJ-GFP). Control (flhDC-) Salmonella of the same strain were grown without arabinose. Bacteria were centrifuged and collected at 2 × 10 7 The cells were resuspended in culture medium (DMEM with 20 mM HEPES) at a density of 100 CFU / ml. For the induced flhDC+ condition, 20 mM arabinose was added to the medium. Bacteria-containing medium (flhDC+ and flhDC-, n = 6 chambers each) was added to each device at 2 × 10 6 For total CFU delivery, perfusion was performed through the tumor-on-chip device at 3 μm / min for 1 h. Bacterial administration was followed by bacteria-free medium (with 20 mM HEPES) for 48 h.

[0393] The device was imaged at 30-minute intervals. Infiltration was quantified at 31 hours by measuring GFP expression by invaded bacteria within the tumor mass. Regions of interest were defined around the border of the tumor mass. The extent of infiltration was determined as the mean GFP fluorescence intensity of each tumor mass. Intensity was normalized by the intensity of the average tumor mass administered with control (flhDC-) Salmonella.

[0394] Intracellular activity of the PsifA and PsseJ promoters Salmonella carrying GFP reporting constructs for the PsifA and PsseJ promoters were grown in LB. These intracellular reporting and PsifA strains contain the constructs PsseJ-GFP and PsifA-GFP, respectively (Table S1). Both bacterial strains were administered to MCF7 cancer cells in 6-well plates at an MOI of 25, as described in the invasion assay section above. Live cells were imaged 3 hours after invasion at 20x magnification. Images of extracellular bacteria were captured in LB cultures in 6-well plates at 20x magnification. Extracellular promoter activity was determined as the mean fluorescence intensity of bacteria from each triplicate well and normalized to the mean intensity of PsseJ bacteria. The increase in promoter activity following cell invasion was determined by averaging the fluorescence intensity of intracellular bacteria in triplicate wells and comparing it to the mean intensity of extracellular bacteria.

[0395] Bacterial death caused by inducing expression of lysine E Salmonella strain PBAD-LysE (Table S1) was grown in LB in 3 ml culture tubes to an average OD of 0.25. OD was measured every 30 minutes for 3 hours. After 90 minutes of growth, three cultures were induced with 10 mM arabinose. Arabinose was not added to three control cultures. Growth and mortality rates were determined by fitting exponential functions to the bacterial density starting at time zero (for growth) and 90 minutes (for bacterial death).

[0396] Intracellular lysis and GFP delivery To visualize and quantify triggered intracellular lysis and GFP delivery, ID Salmonella were administered to cancer cells on coverslips and in well plates as described in the invasion assay section above. ID Salmonella constitutively express GFP (Plac-GFP) and express lysine E after activation of PsseJ (PsseJ-LysE).

[0397] To quantify the extent and rate of lysis, ID Salmonella was administered to MCF7 cancer cells at an MOI of 25. Parental Salmonella constitutively expressing GFP (transformed with the plasmid pLacGFP) was used as a control. Transmitted light images of the cancer cells and fluorescent images of the bacteria were acquired at 20x magnification every 30 minutes for 10 hours. 200 cancer cells from triplicate wells were randomly selected from the first image transmitted for each condition. Cells were scored if any bacteria invaded over the experimental time period and when these intracellular bacteria were lysed. Percent lysis was defined as the number of cells with lysed bacteria over the total number of cells observed. The rate of intracellular lysis was determined by binning the number of cells with lysed bacteria per hour and fitting an exponential function to the cumulative percentage of cells with lysed bacteria.

[0398] Comparisons of growth and mortality were made between (1) the growth rate of parent Salmonella in LB, (2) the growth rate of PBAD-LysE Salmonella in LB, (3) the mortality rate of PBAD-LysE Salmonella after induction with arabinose, (4) the growth rate of PsseJ-LysE Salmonella in LB, and (5) the lysis (mortality) rate of PsseJ-LysE Salmonella after invasion into cancer cells.

[0399] To generate images of bacterial lysis and GFP delivery, ID Salmonella was administered to 4T1 cancer cells grown on coverslips at an MOI of 10. After 6 hours, coverslips were fixed and stained for Salmonella and released GFP (antibody combination #2) as described in the immunocytochemistry section above. Images were acquired using oil immersion at 100x magnification.

[0400] Bacterial protein content To quantify the amount of GFP produced, ID Salmonella (Table S1) was grown in LB. Bacteria were centrifuged, washed, and diluted to 10 per 40 μL of Laemmli buffer. 6 , 10 7 , 10 8 , and 10 9Bacteria were resuspended at four densities, which allowed bacterial lysis. GFP standards were loaded at three concentrations: 1, 10, and 100 ng per 40 μL of Laemmli buffer. Samples were boiled and loaded onto NuPAGE 4-12% protein gels (Invitrogen, catalog no. NPO0321BOX) in MOPS buffer. The resolved gels were transferred to PVDF blotting paper. The membranes were blocked for 1 hour with 2% bovine serum albumin in Tris-buffered saline with 5% skim milk powder and 0.1% Tween 20 (TBST+milk). Blots were incubated overnight with rat anti-GFP monoclonal antibody (Chromotek, catalog no. 3h9-100) in TBST+milk. The blot was washed three times with (TBST) and incubated with HRP-conjugated goat anti-rat secondary antibody (Dako, Cat. No. X0909) in TBST-milk for 1 hour at room temperature.

[0401] Lysis and GFP release in cells and SCVs ID Salmonella was administered to 4T1 cancer cells to assess GFP release from vacuoles. Special staining techniques were used to identify SCVs and isolate released GFP from non-released bacterial GFP. 4T1 cells were grown on glass coverslips and infected with ID Salmonella (Table S1) at an MOI of 10 using the method described in the invasion assay section.

[0402] At two time points, 6 and 24 hours, four coverslips were fixed and permeabilized as described in the immunocytochemistry section above. The blocking buffer used to permeabilize the cells contained Tween 20, which selectively permeabilizes mammalian but not bacterial cell membranes. This allowed the primary antibody to bind to GFP in the mammalian cytoplasm but not inside unlysed bacteria. After permeabilization, cells were stained for Salmonella, released GFP, and vacuoles (combined 3 in the immunocytochemistry section) using anti-Salmonella, anti-myc, and anti-LAMP1 antibodies.

[0403] After mounting, coverslips were imaged under oil immersion at 100x magnification. Images were background subtracted, and a border was drawn around the cells (n = 24 at 6 h and n = 7 at 24 h). The emitted GFP was divided into two groups: vacuolar and cytoplasmic. Vacuolar GFP was surrounded by the LAMP1-stained region. Cytoplasmic GFP was all other GFP inside the cell. For each cell, the percentage of vacuolar and cytoplasmic GFP was determined as the sum of pixel intensities within the region divided by the sum of intensities within both regions (i.e., the total within the cell).

[0404] To visualize the localization of released GFP within cells over time, ID Salmonella was administered to 4T1 cancer cells. Cancer cells were grown on glass coverslips and infected with ID Salmonella (Table S1) at an MOI of 10. At two time points, 6 and 24 hours, four coverslips were fixed and permeabilized as described above. Cells were stained for Salmonella, released GFP, and β-actin (combination 2) using anti-Salmonella and anti-myc antibodies and phalloidin. Actin staining allows visualization of structure and boundaries. Images were acquired using oil immersion at 100x magnification.

[0405] Dynamic measurements of GFP release and diffusion To measure the rate of GFP diffusion through cells after lysis, MCF7 cancer cells were grown on glass-bottom 96-well plates (ThermoFisher, catalog no. 160376) with imaging coverslips. ID Salmonella was administered at an MOI of 25 using the method for live-cell imaging described in the invasion assay section. After washing away extracellular bacteria and adding gentamicin, a single cell with an intracellular bacterium was identified, and transmission and fluorescence images were acquired at 63x magnification every minute for 14 hours. This process was repeated 10 times. Fluorescence images were selected to begin with intact bacteria and end after GFP diffusion. These images were converted to stacks in Zen (Zeiss), and intensity was measured along a line passing through the center of the bacteria at time zero (before lysis) until diffusion was complete. The GFP time and spatial intensity profile was fit to a radial diffusion equation.

[0406]

number

[0407] In this equation, C is the GFP concentration and D is the effective diffusion coefficient of GFP in the cytosol. If there is an instantaneous release of material from r=0 at t=0 (i.e., dissolution), then equation (1) has an analytical solution.

[0408]

number

[0409] The cytoplasmic diffusion coefficient D of released GFP was determined using a least-squares fit to fit the GFP intensity profile to equation (2). Location of GFP emission To quantify the location of intracellular GFP release, 4T1 cancer cells were inoculated with ID Salmonella at an MOI of 10 onto glass coverslips using the method described in the invasion assay section. At 6 h, three coverslips were fixed, permeabilized, and stained using anti-Salmonella, anti-myc, and anti-LAMP1 antibodies to identify Salmonella, released GFP, and vacuoles (combined 3 immunocytochemistry sections). After mounting, coverslips were imaged under oil immersion at 100x magnification. Images were background-subtracted, and Salmonella were identified in seven 86.7 x 66.0 μm regions across the three coverslips. Any bacteria within the region were classified as non-lysed or lysed if they colocalized with released GFP. The location of each lysed Salmonella was determined based on colocalization with LAMP1 staining as either inside or outside the SCV. The ratio of released GFP in the vacuole was the number of lysed Salmonella in the SCV relative to the total lysed Salmonella.

[0410] Dependence of protein release on its presence within the SCV To determine the dependence of protein release on the presence within the SCV, cancer cells were administered ID Salmonella carrying two gene knockouts. 4T1 cancer cells were grown on coverslips and infected with ΔsifA, ΔsseJ, or ID Salmonella (n = 3 for each condition). All three strains contained the PsseJ-lysE and Plac-GFP-myc gene circuits (Table S1). The ΔsifA strain accumulated primarily within the cell cytoplasm, while the ΔsseJ strain accumulated primarily within the SCV and did not escape into the cytoplasm. Bacteria were administered at an MOI of 10 as described in the invasion assay section. Six hours after invasion, cancer cells were fixed, permeabilized, and stained for Salmonella and GFP release as described in the immunocytochemistry section. Nine images from three coverslips were acquired at 20x magnification for each condition. Images were background subtracted. Percent lysis was calculated using pixel-by-pixel image analysis in MATLAB. Lysis was identified as pixels that stained positively for GFP-myc. The permeabilization technique prevented GFP staining inside unlysed Salmonella. Unlysed Salmonella were identified as pixels that stained for Salmonella but not for GFP-myc. The total bacterial pixels were the sum of these values. Percent lysis was the number of lysed pixels relative to the total bacterial pixels.

[0411] Dependence of protein delivery on invasion and intracellular lysis Four strains of Salmonella were administered to cancer cells to determine the requirement for two engineered gene circuits, PsseJ-LysE and PBAD-flhDC, for protein delivery. Two strains were used: flhDC Sal and flhDC-ID Sal (Table S1). Both strains lack flhD and express only flhDC after induction with arabinose. The flhDC-ID Sal strain also contains the PsseJ-LysE circuit, which induces lysis after cell invasion. Prior to invasion, two cultures of flhDC Sal and flhDC-ID Sal bacteria were grown in LB with 20 mM arabinose to induce flhDC expression. Two cultures were grown without arabinose. For microscopic analysis, 4T1 cancer cells were grown on coverslips and infected with one of four strains: PsseJ-LysE-, flhDC-; PsseJ-LysE-, flhDC+; PsseJ-LysE+, flhDC-; or PsseJ-LysE+, flhDC+ at an MOI of 10. For flow cytometry, 4T1 cells were grown on 6-well plates and infected with the same four strains at an MOI of 10. To maintain expression, 20 mM arabinose was added to the two induced flhDC+ conditions on both coverslips and well plates.

[0412] For microscopy, coverslips were fixed, permeabilized, and stained for released GFP as described in the immunocytochemistry section. Nine images for each condition were acquired at 20x magnification and background subtracted. Protein (GFP) delivery was determined using pixel-by-pixel image analysis in MATLAB. A pixel was positive for delivery if it stained for GFP-myc. Total delivery was calculated as the sum of the intensities of all delivery-positive pixels. Values ​​were normalized by the PsseJ-LysE- and flhDC- conditions.

[0413] For flow cytometry, cells were washed with PBS, added with 0.05% trypsin (ThermoFisher, catalog no. 25300-054), and then processed into a single-cell suspension by gentle pipetting. Cells were fixed with 5% formaldehyde in PBS with 1 mM EDTA and incubated in blocking buffer for 30 minutes. Cells were intracellularly stained with a 1:2000 dilution of FITC-conjugated anti-Salmonella antibody (Abcam, catalog no. ab69253) and a 1:200 dilution of rat anti-myc monoclonal antibody (Chromotek, catalog no. 9e1-100) for 30 minutes. Cells were washed three times with blocking buffer. Cells were incubated with DyLight 750 anti-rat secondary antibody (ThermoFisher, catalog no. SA5-10031) at a 1:200 dilution for 1 hour at room temperature. Samples were analyzed on a custom-built flow cytometer (dual LSR Fortessa 5-laser, BD). All fluorophores were compensated with compensation beads (BD, catalog no. 552845) and did not leak into any other channel by more than 2%. Control cells not infected with Salmonella were used as a gating control to identify uninfected cells in the samples based on Salmonella staining. Cells administered with non-lysed bacteria (i.e., PsseJ-LysE-) were stained with anti-Salmonella antibody, anti-rat secondary antibody, but not anti-myc primary antibody to identify cells without GFP delivery.

[0414] Intracellular delivery of GFP to cells in tumors by ID Salmonella To identify and quantify GFP delivery to tumor cells, five BALB / c mice bearing 4T1 tumors were treated with 2 × 10 6CFU of ID Salmonella were injected into the mice (Table S1). Ninety-six hours after bacterial injection, mice were sacrificed, and tumors, livers, and spleens were excised. Tumors were cut in half. One half was fixed and stained for imaging, and the other half was cryopreserved for protein quantification. The liver and spleen were also cryopreserved. Fixed tumors were embedded, sectioned, and deparaffinized as described in the immunohistochemistry section. Tumor sections were stained overnight with a 1:50 dilution of goat anti-GFP (Abcam, ab6556) to identify GFP, followed by incubation with a 1:50 dilution of Alexa Fluor 488-conjugated donkey anti-goat antibody (ThermoFisher, catalog no. A21208) for 1 hour at room temperature. After counterstaining with DAPI and mounting, sections were imaged at 20x magnification.

[0415] To quantify the amount of delivered protein, half the tumor, as well as the liver and spleen, were snap-frozen in liquid nitrogen and stored at -80°C. Lysates were prepared in a buffer containing 50 mM Tris-HCl, 0.3% Triton-X 100, 0.1% NP-40, and 0.3 M NaCl at pH 7.4. The buffer was supplemented with 25 mM NaF, 5 μM leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM benzamidine, and 1 mM dithiothreitol. Similar to cancer cells in culture, this buffer lyses mammalian cells but not bacterial membranes, separating the delivered protein from intact bacterial proteins. Samples were homogenized on ice using a blender (Polytron) and a homogenizer (Potter-Elvehjem). Samples were incubated on ice for 20 minutes, centrifuged at 664 × g and 4 °C for 10 minutes, and the supernatants were collected. Immunoblotting was performed after 10% SDS-PAGE using anti-GPF (Abcam, catalog no. ab6673) and anti-β-actin (GeneTex, catalog no. GTX26276, clone AC-15). Immunoblots were visualized using eCL reagent (PerkinElmer) on an ImageQuant LAS4000 imaging system (GE Healthcare).

[0416] Effect of flhDCs on protein delivery in mice To determine the effect of flhDCs on protein delivery, nine BALB / c mice bearing 4T1 tumors were inoculated with 2 × 10 cells via the tail vein. 6 CFU of flhDC-ID Salmonella (Table S1) were injected into the flhDC+ mice. Prior to injection, flhDC-ID Sal cultures were grown in LB with 20 mM arabinose to induce flhDC expression. A second culture was grown without arabinose. At 48 and 72 hours after bacterial injection, flhDC+ mice were intraperitoneally injected with 100 μg of arabinose in 400 μL of PBS to maintain expression. flhDC− mice received simultaneous intraperitoneal injections of PBS. Ninety-six hours after bacterial injection, mice were sacrificed, and tumors (n=4 for flhDC− and n=5 for flhDC+) were excised and sectioned as described in the immunohistochemistry section. Tumor sections were stained to identify GFP using a rat anti-GFP monoclonal antibody (Chromotek, catalog no. 3h9-100) and an Alexaflor-568 goat anti-rat secondary antibody (ThermoFisher, catalog no. A11077). After counterstaining with DAPI, sections were imaged at 10x magnification. Images were background subtracted and analyzed using a computational code in MATLAB. Delivery was quantified at 20 random points within the transition zone of each tumor. Points were scored as positive if cells within 20 μm contained delivered GFP. Cells were considered to have delivered protein if GFP filled the entire cytoplasm. The percentage delivery was calculated by dividing the number of positive points by the total number of random points.

[0417] Transient colonization of ID Salmonella in tumors To determine tumor density over time, 2 x 10 luciferase-expressing cells were cultured in 100 wells. 7CFU of ID Salmonella (ID Sal-luc, Table S1) were intravenously injected into five BALB / c mice bearing orthotopic 4T1 tumors in the mammary fat pad. Bacterial colonization was tracked in real time by bioluminescence imaging. 24, 48, 72, 168, and 336 hours after bacterial injection, mice were intraperitoneally injected with 100 μL of 30 mg / ml luciferin in sterile PBS, anesthetized with isoflurane, and imaged with an IVIS animal imager (PerkinElmer, SpectrumCT). Bacterial density within the tumor was determined as proton flux from the tumor. After final imaging (on day 14), tumors were excised and minced in an equal volume of sterile PBS. Homogenized tumors were plated on agar plates. Colonies were counted after overnight growth at 37°C.

[0418] Biodistribution and virulence of ID Salmonella To determine the biodistribution of Salmonella, 1 × 10 cells were injected into five tumor-free BALB / c mice. 7 1 × 10 ID Salmonella were injected into four tumor-free BALB / c mice. 14 days later, six organs were excised and weighed: spleen, liver, lungs, kidneys, heart, and brain. Organs were minced in an equal volume of sterile PBS, diluted 10-fold and 100-fold, and plated on agar plates. Colonies were counted after overnight growth at 37°C. To measure the virulence of ID Salmonella, 1 × 10 ID Salmonella were injected into four tumor-free BALB / c mice. 7 Four control mice were injected with 100 mg of ID Salmonella. Four control mice were injected with sterile saline. After 14 days, whole blood was isolated from anesthetized mice by percutaneous cardiac puncture. The collected blood was divided between clot-activated serum tubes and EDTA anticoagulant tubes for chemistry and CBC analysis, respectively. Chemical profiling and comprehensive hematology were performed on serum and whole blood samples by Idexx Laboratories (Grafton, MA, USA).

[0419] Delivery of nanobodies by ID Salmonella To measure nanobody delivery, ID Salmonella was administered to cancer cells, and the extent of binding to the protein target was determined by immunoprecipitation. 4T1 cancer cells were grown to 80% confluency in T75 flasks and infected with either NB or ID Salmonella (as a control, Table S1) at an MOI of 10, as described in the invasion assay section. The β-actin nanobody expressed by NB Salmonella is tagged with a FLAG sequence at the C-terminus. Prior to administration, NB Salmonella was grown in LB with 20 mM arabinose to induce nanobody expression, and 20 mM arabinose was added to the NB culture to maintain expression. 24 hours after invasion, cancer cells were harvested using a cell lifter and centrifuged at 600 × g for 10 minutes. The cell pellet was resuspended in 10 ml of lysis buffer (20 mM HEPES, 1 mM EDTA, 10% glycerol w / v, 300 mM sodium chloride, and 0.1% Tween), which lysed only cancer cells but not intact bacteria. The cell suspension was homogenized in a douncer using a tight-fitting plunger. The cell lysate was clarified by centrifugation at 20,000 × g for 20 minutes at 4 ° C. The lysate was incubated with 50 μL of anti-FLAG purification resin (Biolegend, catalog number 651502) overnight at 4 ° C. The FLAG resin was washed three times with lysis buffer. Fifty microliters of Laemmli buffer was added directly to the bead solution and boiled at 95 ° C for 5 minutes. The boiled beads were loaded onto an SDS-PAGE gel (15% polyacrylamide, cast in-house) in MOPS buffer for Western blotting, as described in the Bacterial Protein Content section. The gel was transferred to nitrocellulose blot paper. The blot was incubated with a mouse anti-actin monoclonal antibody (Cell Signaling Technology, Cat. No. 8H10D10) and an HRP-conjugated goat anti-mouse secondary antibody (ThermoFisher, Cat. No. 31450) to identify β-actin.

[0420] Cytotoxicity of CT-Casp-3 and NIPP1-CD delivery to cells in culture To measure the cytotoxicity of protein drug delivery, ID Salmonella was administered to cancer cells in culture. Hepa 1-6 liver cancer cells were grown to 80% confluency in 6-well plates. NIPP1-CD, CT-Casp-3 Salmonella, and control ID Salmonella were administered at an MOI of 10 as described in the invasion assay section. Prior to invasion, CT-Casp-3 Salmonella cultures were grown in LB with 20 mM arabinose for 1 hour to induce CT-Casp-3 expression. 20 mM arabinose was added to all wells to maintain expression. Ethidium homodimer (500 ng / ml) was added to each well to stain dead cells with permeable membranes. Three images per well were taken every 30 minutes for 24 hours (for nine images per condition) at 20x magnification. For each run, one transmission image and two fluorescence images were acquired: bacterially produced GFP (480 / 525 excitation / emission) and ethidium homodimer (525 / 590 excitation / emission). Images were background subtracted. Cancer cells invaded by Salmonella were identified from the fluorescence time-lapse images. Cell death was calculated as the percentage of dead Salmonella-invaded cells (colocalized with ethidium homodimer staining) relative to the total number of Salmonella-invaded cells.

[0421] Delivery of CT-Casp-3 and NIPP1-CD to the tumor mass To measure cell death in tumor masses after delivery of CT-Casp-3 or NIPP1-CD, ID Salmonella were administered to the tumor-on-a-chip device. The microfluidic device was fabricated as described in the section on the in vitro effect of flhDCs on tumor mass invasion. Two independent device experiments were performed: (1) NIPP1-CD vs. ID control Salmonella, each with six chambers, and (2) CT-Casp-3 vs. ID control Salmonella, each with four and three chambers. Prior to administration to the device, CT-Casp-3 Salmonella were grown in LB with 20 mM arabinose to induce CT-Casp-3 expression. NIPP1-CD and ID Salmonella were grown in LB without arabinose. All bacteria were centrifuged and 2 × 10 7 The bacteria were resuspended in culture medium (DMEM with 20 mM HEPES) at a density of 100 CFU / ml. For CT-Casp-3 Salmonella, 20 mM arabinose was added to the medium. Bacteria-containing medium containing 500 ng / ml ethidium homodimer was added to each device. 2 × 10 6 For total CFU delivery, perfusion was performed through the tumor-on-a-chip device at 3 μm / min for 1 h. Bacteria-free medium with 20 mM HEPES and ethidium homodimer followed bacterial administration. Transmission and fluorescence images were acquired at 5x magnification every 30 min for 24 h. Killing was calculated by first defining the boundaries of the tumor mass. Fluorescence images were segmented to identify areas of dead cells stained with ethidium homodimer. The extent of killing was the percentage of the tumor mass that was killed. The final percentage of killing was determined at 24 h.

[0422] Tumor response to delivery of CT-Casp-3 in mice The effects of delivering CT-Casp-3 were measured using two mouse models: 4T1 mouse breast cancer cells in BALB / c mice and Hepa1-6 mouse liver cancer cells in C57L / J mice. For both models, three conditions were tested by injecting saline, ID Salmonella, or CT-Casp-3 Salmonella. The saline control established the baseline growth rate of the tumor. The ID Salmonella control established the effect of colonizing bacteria and intracellular lysis on tumor growth rate. For both mouse models, 1 x 10 cells were injected into three groups of six mice. 5 Tumor cells were injected subcutaneously. 3 They were then placed in three conditions: saline, or 4 x 10 7 Mice were injected with either 100 mg of CFU or CT-Casp-3 Salmonella. 48 and 72 hours after injection, mice were intraperitoneally injected with 100 mg of arabinose in 400 μL of PBS. Bacteria or saline were injected into the tumor every 5 days. Tumors were measured twice a week, and the volume was calculated using the formula (length)*(width). 2 ) / 2. 3 Mice were sacrificed when tumor growth reached 1. Tumor growth rate was determined by fitting an exponential function to tumor volume as a function of time.

[0423] statistics For pairwise comparisons, Student's t-test was used. Statistical significance was established when P<0.05. ANOVA with Bonferroni correction was used when comparing multiple data points.

[0424] [Table 5]

[0425] Results and Discussion Here, we describe the creation of an intracellular protein delivery system based on the natural properties of Salmonella (Figure 1A). In the intestine, Salmonella has a partially intracellular lifestyle. To avoid clearance, Salmonella invades epithelial cells using proteins expressed by Salmonella pathogenicity island 1 (SPI1) (3, 4). After invasion, Salmonella resides within early and late endosomes, which are then remodeled into Salmonella-containing vacuoles (SCVs) by expressing the pathogenicity island 2 (SPI2) gene (5-7). SCVs enable intracellular survival (5, 8) and protect Salmonella from intracellular defense mechanisms (9, 10). A key step in SPI2 gene activation is sensing the endosomal environment. These sensing mechanisms, unique to Salmonella, are required for protein delivery into cells.

[0426] Although it is well established that Salmonella invade enterocytes (4, 11), their location within tumors is less clear, despite extensive documentation of tumor colonization (12-16). Preferential accumulation and exponential growth in tumors are essential characteristics of therapeutic Salmonella (17, 18). When administered in culture, Salmonella readily invade carcinoma cells (Figure 1B). To determine their location within tumors, Salmonella carrying a fluorescent intracellular reporter were injected into tumor-bearing BALB / c mice. In these tumors, >70% of Salmonella were intracellular (P < 0.001, n = 5, Figure 1C), demonstrating their suitability as a delivery vehicle. In cells dissociated from tumors using collagenase, bacteria were present within carcinoma, immune, and other tumor-associated cells (Figure 1D).

[0427] The development of a therapeutic Salmonella into an intracellular protein delivery system involved three steps (Figure 1A). The design goal was to engineer Salmonella to (1) make the drug, (2) invade into cells, and (3) release the drug intracellularly. The use of bacteria changes what is traditionally meant by "delivery." Unlike typical delivery vehicles, bacteria manufacture protein drugs at the disease site (19), delivering exponentially more molecules than were originally present in the injected bacteria. Sequentially, the first step was to generate a platform strain with controlled invasion and release. The final step was to genetically transform this platform strain to synthesize different protein drugs. In the final engineered strain of intracellular delivery (ID) Salmonella, each of these three processes (production, invasion, and release) was controlled by specialized genetic circuits.

[0428] In this system, the invasion of ID Salmonella into cells is controlled by the regulatory factor flhDC (Figure 1E-G). Expression of flhDC is required for Salmonella to invade cancer cells (Figure 1E). When flhDC is not expressed, Salmonella invaded less than 2% of cells, which was 54-fold less than that of Salmonella with re-expressed flhDC (84%; P<0.001; Figure 1E). Invasion is dependent on flhDC because it regulates flagella production and the type III secretion system (20). In vitro, in microfluidic tumor masses (21), re-expression of flhDC increased cell invasion and colony formation by 53-fold (P<0.01, Figure 1F). In tumors, re-expression of flhDC increased the infiltration of both carcinoma and immune cells (P<0.05, Figure 1G).

[0429] The second component of ID Salmonella, release, required the development of a system to trigger autonomous lysis after cell invasion (Figure 2). This goal was achieved by identifying a Salmonella promoter that is triggered intracellularly, but not extracellularly. After invasion into cells, the SPI2 gene is activated, leading to the formation of Salmonella-containing vacuoles (SCVs) (8). When linked to a GFP reporter, the promoters of two SPI2-related genes, PsseJ and PsifA, both become activated after invasion into cancer cells (Figure 2A, left). However, extracellular expression of PsseJ was 5.8-fold less than that of PsifA (P < 0.001, Figure 2A), indicating that PsseJ is more sensitive to cell invasion.

[0430] To release the synthesized protein cargo, bacteria must lyse after invasion. Triggered expression of the lysine gene E (LysE) from bacteriophage ΦX1174 results in rapid bacterial death (Figure 2B). Salmonella carrying the PsseJ-LysE construct and constitutively expressing GFP (as a model protein drug) lysed after invasion into cancer cells (Figure 2C) and excreted GFP into the cytoplasm (Figure 2D). Bacterial lysis occurred over a 10-hour period after invasion (Figure 2E). Basal expression of LysE by the PsseJ-LysE cycle did not affect bacterial health, and intracellular induction activated the system at a rate close to its maximal rate (Figure 2F). Each bacterium was able to deliver an average of 163,000 GFP molecules (Figure 2G).

[0431] After bacterial lysis, the delivered protein escapes the SCV and fills the cell cytoplasm (Figure 2H-I). This escape is significant because immediately after invasion, most Salmonella reside within the SCV (Figure 2H, left). When ID Salmonella lyse, clusters of released GFP protein are contained within the SCV (Figure 2H, center). Over time, the protein escapes the SCV and fills the entire cytoplasm (Figure 2I), a translocation that occurs for most cells (P<0.001, Figure 2H, right). GFP diffuses through the cytoplasm with an effective diffusion coefficient of 0.15 μm² / min (Figure 2J).

[0432] As designed, bacterial lysis depended on presence within the SCV (Fig. 3A-B). After invasion, some ID Salmonella escaped into the cytoplasm and were not surrounded by the SCV membrane (Fig. 3A, left). More than 95% of the GFP released from the Salmonella originated from inside the SCV (P < 0.001, Fig. 3A, right). After invasion into cancer cells, ID Salmonella with the ΔsifA deletion, which are primarily cytoplasmic (23), did not lyse despite containing the PsseJ-LysE construct. In comparison, ID Salmonella with ΔsseJ, which are primarily vacuolar (24), almost completely lysed (P < 0.001, Fig. 3B). Most ID Salmonella without these deletions localized to the SCV, lysed, and delivered proteins (P < 0.001, Fig. 3B). This dependency indicates that the Pssej promoter is activated only after SCV localization and not when in the cytoplasm. This specific sensing of the SCV environment is an exclusive feature of Salmonella.

[0433] Protein delivery depended on two engineered systems: PBAD-flhDC for invasion and PsseJ-LysE for release (Fig. 3C). Salmonella without flhDC expression did not invade cells, and Salmonella without PsseJ-LysE did not release GFP cargo (Figs. 3C and S2). Compared to controls, the presence of both systems increased protein delivery by 548-fold (P < 0.001, Fig. 3C).

[0434] When administered systemically to tumor-bearing mice, ID Salmonella specifically delivered proteins to tumor cells, and this delivery was dependent on flhDCs (Figure 3D-F). ID Salmonella infiltrated the cells and delivered GFP, which filled the cytoplasm of the cells (Figure 3D). This system achieved a tumor density of 1.5 × 10 per gram of tumor. 8The bacteria delivered 60 ± 12 μg GFP / g tumor (Figure 3E). No GFP was detected in the liver or spleen of any mice (Figure 3E). When tumor-bearing mice were challenged with ID Salmonella that did not express flhDC, very little GFP was delivered (Figure 3F). Re-expression of flhDC increased the percentage of cells that received GFP six or more times (P < 0.001).

[0435] Protein delivery using ID Salmonella is safe and self-limiting (Figure 3G). After intravenous administration, tumor density of ID Salmonella peaked at 72 hours and then decreased by 97% over 11 days (Figure 3E). The decreased density, caused by intracellular lysis, limits exposure to the treatment and improves safety compared to non-lytic Salmonella. After administration to healthy, tumor-free mice, ID Salmonella did not accumulate in the lungs, heart, kidneys, or brain, did not affect liver function, or elicit adverse immune responses.

[0436] To demonstrate its broad capabilities, we engineered ID Salmonella to express three different proteins that affect intracellular physiology (Figure 4): a nanobody (anti-actin), a protein inhibitor (NIPP1-CD), and an endogenous protein (CT casp-3). The central domain of nuclear inhibitor of protein phosphatase 1 (NIPP1-CD) removes PP1 from its holoenzyme, inducing cell death (25). Constitutively active two-chain caspase-3 (CT Casp-3) is an engineered active form of caspase-3 and is the dominant executioner caspase that leads to apoptotic cell death (26, 27).

[0437] In one embodiment, the bicistronic mRNA encodes a caspase, eg, a large subunit, followed by a ribosome binding site and a small subunit, eg, on a PBAD-inducible promoter.

[0438] [ka]

[0439] [ka]

[0440] [ka]

[0441] [ka]

[0442] [ka]

[0443] After bacterial delivery via invasion and lysis, the anti-actin nanobody bound to cellular actin (Figure 4A), demonstrating specific targeting of intracellular proteins. Delivery of both NIPP1-CD and CT Casp-3, potential therapeutic proteins, caused more cell death than control (P<0.001, Figure 4B, left). Induced cell death depended on invasion and protein delivery (Figure 4B, right). When administered to the microfluidic tumor device, ID Salmonella delivering NIPP1-CD (P<0.05) and CT Casp-3 (P<0.01) caused cell death that increased over time as the bacteria infiltrated the tumor mass (Figure 4C).

[0444] In mice, delivery of CT Casp-3 was effective against both liver cancer and triple-negative breast cancer (Figure 4D-E). After 14 days of treatment, delivery to BALB / c mice reduced 4T1 mammary tumor volume by twofold compared with controls (P<0.05, Figure 4D). Administration of ID Salmonella bearing CT Casp-3 significantly reduced liver Hepa 1-6 tumor volume in C57L / J mice (P<0.001, Figure 4E, left) and tumor growth rate by 28-fold (P<0.05, Figure 4E, center), equivalent to a doubling time increase from 5 to 148 days. Tumor volume decreased for more than 50 days in two mice, and survival was significantly increased compared with bacterial controls (P<0.05, Figure 4E, right). Treatment with CT Casp-3 completely eliminated the tumor from one mouse, which remained disease-free for more than 124 days.

[0445] conclusion Described herein is an autonomous, intracellular Salmonella vehicle that efficiently delivers properly folded and active proteins into cells. This bacterial strain is safe, eliminates tumors, and increases survival. An engineered genetic circuit produces the protein drug, causes hyperinvasion (flhDC), and triggers bacterial lysis after cell invasion. Because the system is autonomous, it requires no intervention and is self-timed. Protein delivery is triggered at the most favorable time for each individual bacterium, ensuring that the protein is placed inside the cell rather than in the extracellular environment. The accumulation of ID Salmonella in different cell types in tumors (Figures 1D and 1G) suggests that this system can be used to deliver proteins to non-cancerous tumor-associated cells, such as macrophages or endothelial cells.

[0446] Taken together, two essential qualities of ID Salmonella enable the use of protein drugs that are not currently feasible. Intracellular Salmonella delivery (1) transports intact, functional proteins across the cell membrane, and preferential tumor accumulation (2) maintains safety for protein drugs that would otherwise act broadly against healthy cells. Both NIPP1-CD and CT Casp-3 have exclusively intracellular targets and would be toxic if delivered systemically. The specific accumulation of ID Salmonella eliminates these issues by concentrating the therapy specifically in the intracellular environment of tumors (Figures 1C and 3E).

[0447] The use of ID Salmonella to deliver CT Casp-3 could address the need for effective treatments for unresectable hepatocellular carcinoma (HCC). For the 840,000 patients diagnosed with HCC annually, there is currently no curative treatment (28, 29). Current therapies have toxic side effects and only modestly increase survival (29-31). Treatment with CT Casp-3 ID Salmonella is potentially curative (Figure 4E) and safer. Inclusion of the PsseJ-LysE cycle renders ID Salmonella self-limiting. The delivered bacteria lyse after cell invasion (Figure 3F), reducing the likelihood of unwanted infection.

[0448] Delivery using ID Salmonella will enable the targeting of inaccessible cancer pathways and accelerate the generation of new cancer therapies. These therapies can be created by encoding genes for specific protein drugs into Salmonella expression cassettes. Nanobodies (Figure 4A) can be designed to specifically inhibit pathways necessary for cancer survival and progression. Using bacteria to deliver proteins into cells expands the number of accessible pathways, unlocking numerous targets across the soluble proteome for therapy, and increasing the efficacy and safety of cancer treatments.

[0449] [Table 6-1]

[0450] [Table 6-2]

[0451] [Table 6-3]

[0452] Example II introduction Intracellularly targeted macromolecular therapies present an opportunity for cancer treatment. The mammalian proteome consists of 60% intracellular proteins, while only 30% are surface-associated and extracellularly exposed (1). However, macromolecules face barriers to tumor specificity, distribution, cellular internalization, and endosomal release (2). Improved drug delivery systems are needed to circumvent these delivery limitations and enhance the therapeutic efficacy of intracellularly active therapies. Salmonella is ideally suited for tumor-selective intracellular protein delivery. Salmonella colonizes and infiltrates tumors with high specificity, selectively delivering protein therapeutics inside tumor cells. Herein, we report the discovery that flhDC expression is crucial for protein delivery into tumor cells using Salmonella. To this end, we sought to determine the mechanism by which flhDC expression enables intracellular therapeutic delivery in vivo. The specific mechanism by which engineered flhDC-expressing Salmonella exhibited resistance to intracellular therapeutic delivery was also evaluated. Understanding these mechanisms will help create improved tumor-targeted intracellular delivery strains of Salmonella.

[0453] Salmonella typhi strains that systemically infect humans carefully regulate flagellum expression in vivo. Typhoid bacteria that spread systemic infection in humans implement a genetic program to downregulate the expression of the flagellum synthesis regulator, flhDC, in the blood (3-5) (6, 7). One reason for this is that flagellin is a TLR5 / NLRC4 agonist that potently activates antimicrobial immune responses (8, 9). However, in tumor tissue, activation of the Salmonella transcription factor, flhDC, is required for intracellular invasion and delivery into cancer cells (10). Therefore, developing methods to control flhDC activity in engineered Salmonella is necessary to enable high levels of therapeutic delivery within tumors.

[0454] Modulation of flhDC activity in Salmonella has significant implications for determining tumor selectivity and reducing systemic virulence. Unlike tumors, clearance organs such as the liver and spleen possess high concentrations of functional immune cells that mount robust responses following pathogenic insults. The liver is a key clearance organ and a specific and essential site for immune-mediated Salmonella clearance (11). The motility regulator, flhDC, regulates flagella expression but also acts as a broad regulator of Salmonella lifestyle and virulence (10, 12, 13). Flagella expression in Salmonella in macrophages or epithelial cells triggers excessive NLRC4 inflammasome-dependent pyroptosis. Salmonella hijacks this inflammatory pathway, resulting in exaggerated antimicrobial responses in both macrophages and the intestine, leading to immune dysfunction (14, 15). Because the liver contains a large number of Kupffer cells, flagellated Salmonella can induce significant pyroptosis in these liver-specific macrophages. Although pyroptosis is required in limited amounts to eliminate pathogens, flagellated Salmonella induce high levels of pyroptosis, which cripples the antimicrobial immune response (14, 16). Macrophages are more effective at eliminating Salmonella that express low levels of flhDC due to reduced flagellum expression, and limited pyroptosis results in less immune dysfunction (16). Because tumors do not share the same level of immune function, low flagellin expression does not affect tumor colonization (17).

[0455] Following cell invasion, there are two known mechanisms by which Salmonella delivers therapeutics into the cytosol: (1) the bacteria invade, escape the intracellular vacuole, rupture, and deliver the therapeutic into the cytosol (18-20); or (2) the bacteria are genetically engineered to lyse and deliver the therapeutic from the Salmonella-containing vacuole into the cytosol. Several variants of cytoplasmic bacteria (ΔsifA Salmonella, listeriolysin O-expressing bacteria) have been used for therapeutic delivery into tumor cells (18-20). In scenario (1), therapeutic delivery requires the bacteria to reside in the cytoplasm of cancer cells and spontaneously lyse without any control. This mechanism relies on ubiquitin-dependent degradation of the bacteria (21) and subsequent cytoplasmic release of the therapeutic. Additionally, cytoplasmic pathogens are known to strongly activate NF-kB signaling and initiate innate immune responses to eliminate the bacteria (9, 21, 22). Therefore, a high presence of cytoplasmic Salmonella is detrimental to immune evasion.

[0456] Salmonella has evolved to reside within intracellular vacuoles, which provide protection for bacteria inside the cell (23, 24). Bacteria modify vacuoles to provide protection against degradation and clearance (25, 26). In addition, the presence of vacuoles appears to be particularly important for bacteria in the systemic circulation, as demonstrated by Salmonella typhi. The spi-2 protein, SseJ, is required for Salmonella to escape the SCV (27). SseJ-expressing Salmonella typhi localizes to the human gastrointestinal tract (28). S. typhi lacking SseJ (29) is efficient at escaping from the gastrointestinal tract into the systemic circulation (30). Furthermore, S. typhi expresses only typhoid toxin intracellularly within the SCV (31, 32). These taxonomic boundaries between S. typhi and S. typhi suggest that the presence of vacuoles is essential for increasing bacterial fitness in vivo.

[0457] Understanding the dynamics between the vacuole and cytoplasmic Salmonella expressing flhDC will aid in engineering intracellular delivery strains of Salmonella. Herein, we show that intracellular lysis of engineered Salmonella occurs within the vacuole. However, flagellated intracellular Salmonella have a significant cytoplasmic presence (12). Intracellular invasion is driven by flhDC and T3SS1 activity (10, 12, 33-35). Upon cell invasion, Salmonella significantly modify the vacuole in which they reside (24, 36). During this process, some bacteria rupture the vacuole and escape (37, 38). Typically, vacuolar bacteria also downregulate flagella expression through repression of flhDC by ssrB (39). The ssrB protein is thought to be a master regulator of SPI-2 expression (33). However, flagellated cytoplasmic Salmonella have abrogated T3SS2 activity due to vacuolar escape (12). As shown here, T3SS2 activity is required to enable intracellular lysis and protein delivery using therapeutic Salmonella.

[0458] We demonstrate here how regulated expression of flhDC can improve tumor colonization and therapeutic delivery in vivo compared to existing delivery strategies. Furthermore, we elucidate the mechanisms underlying flhDC-induced resistance to therapeutic delivery and genetic engineering strategies for rescuing therapeutic delivery in Salmonella strains. We hypothesize that selective intratumoral flhDC expression is important for increasing tumor specificity, colonization, and protein delivery to a spatially distributed set of tumor cells. We further hypothesize that engineered Salmonella that inducibly express flhDC can deliver more protein intracellularly compared to exclusively cytosolic Salmonella. We also hypothesize that flhDC activity enabled lytic resistance in engineered Salmonella that could be rescued. To test these hypotheses, cell-based assays, tumor-on-a-chip models, and in vivo experiments were employed to quantitatively understand the mechanisms underlying intracellular therapeutic delivery using engineered Salmonella. Discovering the key mechanisms governing therapeutic delivery using Salmonella will address the limitations of current delivery methods and provide the basis for potentially improving the delivery efficiency of engineered bacteria against a wide variety of cancers.

[0459] material and method bacterial culture All bacterial cultures (both Salmonella and DH5α) were grown in LB (10 g / L sodium chloride, 10 g / L tryptone, and 5 g / L yeast extract). Resistant strains of bacteria were grown in the presence of carbenicillin (100 μg / ml), chloramphenicol (33 μg / ml), kanamycin (50 μg / ml), and / or 100 μg / ml DAP.

[0460] Cloning One of the three plasmids was used in every experiment. The first plasmid, P1, was created by cloning the flhDC gene into the PBAD his-myc plasmid (Invitrogen, catalog number V430-01). The flhDC gene was PCR-transcribed from VNP20009 genomic DNA using primers vr46 and vr47. The PCR product was digested with NcoI, XhoI, and DpnI (NEB, catalog numbers R0193S, R0146S, and R0176L). The PBAD-his-myc backbone was digested with NcoI, XhoI, and calf intestinal phosphatase (NEB, catalog number M0290). Both products were cleaned up using PCR cleanup columns (Zymo Research). 50 ng of the digested vector backbone and 500 ng of the digested PCR product were ligated together using T4 DNA ligase (NEB). The ligated product was transformed into DH5a E. coli. Positive transformants were confirmed by sequencing (plasmid P1a). To add the plac-GFP-myc gene circuit to the plasmid, plasmid P1a was PCR amplified using primers vr385 and vr386. The plac-GFP-myc gene circuit was PCR amplified from a previously generated plasmid (40) using primers vr394 and vr395. Both PCR products were digested with DpnI. 50 ng of the P1a PCR product and 500 ng of the P1b PCR product were ligated together using 2x Hifi DNA Assembly Master Mix (NEB). The resulting product was transformed into DH5a E. coli, and the complete P1b plasmid was purified from a positive colony. To generate the complete plasmid P1, the P1b backbone was amplified using PCR with primers vr426 and vr427. The ASD gene was amplified from the previously generated plasmid, PCS2 (40), using primers vr424 and vr425. 50 ng of the P1b PCR product and 500 ng of the ASD PCR product were ligated together using 2x Hifi DNA Assembly Master Mix. The resulting ligation was transformed into chemically competent DH5a E. coli.The complete P1 plasmid was purified from colonies that screened positive for GFP, ASD, and PBAD-flhDC.

[0461] To create plasmid P2, plasmid P1 was PCR amplified using primers vr396 and vr397. The psseJ-lysinE gene circuit was amplified from synthetic DNA (Genscript) using primers vr398 and vr399. The two PCR products were DpnI digested and purified using PCR cleanup columns (Zymo Research). 50 ng of backbone PCR and 500 ng of psseJ-lysinE PCR product were used in a ligation reaction with 2x Hifi Assembly Master Mix (NEB) to create plasmid P2a. Plasmids were purified from colonies screened positive for plasmid assembly for downstream use. To create complete P2, plasmid P2a was PCR amplified using primers vr426 and vr427. The ASD gene was amplified as previously described using primers vr424 and vr425. Both PCR products were DpnI digested and purified using PCR cleanup columns as previously described. 50 ng of the P2a PCR product was ligated together with 500 ng of the ASD PCR product using 2x Hifi DNA Assembly Master Mix. The resulting ligation was transformed into DH5a E. coli, and the complete P2 plasmid was purified from colonies that screened positive for GFP, ASD, PBAD-flhDC, and sseJ-lysinE.

[0462] To create plasmid P3 (sseJ-GFP-myc + PBAD-flhDC), plasmid P1a was PCR amplified using primers vr271 and vr272. The sseJ-GFP-myc gene circuit was PCR amplified from a previously generated plasmid (10) using primers vr269 and vr270. The resulting PCR product was DpnI digested and purified using PCR cleanup columns. 50 ng of the P1a backbone and 500 ng of the psseJ-GFP-myc PCR product were ligated together using 2x Hifi DNA Assembly Master Mix. The resulting ligation was transformed into DH5a E. coli. For downstream applications, the complete P3 plasmid was purified from colonies that screened positive for psseJ-GFP-myc and PBAD-flhDC.

[0463] [Table 7]

[0464] [Table 8]

[0465] [Table 9]

[0466] KK All engineered strains were based on VNP20009 and strain details can be found in the table below.

[0467] [Table 10]

[0468] Gene knockouts were generated using a modified Lambda Red recombination procedure. (41, 42) Master gene-edited strains were generated by transforming a plasmid containing the required phage lambda gene pkd46 into VNP20009 using electroporation.

[0469] Six genome knockout strains of Salmonella were created. Three of the knockouts were generated by growing Salmonella containing pkd46 to an optical density of 0.1, at which point the bacteria were supplemented with 20 mM arabinose to induce expression of the lambda gene. When the bacteria reached an optical density of 0.8, 1 microgram of DpnI-digested PCR product amplified from pkd4 (vr121 / vr309 for ΔflhD, vr318 / vr319 for ΔfliGHI, vr432 / vr433 for ΔsseJ, and vr434 / vr435 for ΔsifA) was transformed into Salmonella via electroporation. Bacteria were allowed to recover in LB for 2 hours at 37°C and plated onto agar plates containing 50 micrograms / mL kanamycin. The resulting transformants were screened for insertions using antibiotic selection and junction PCR to confirm the correct location of the genomic deletion, and successful knockouts were then grown at 43°C to cure the knockout strain of the pkd46 plasmid.

[0470] To create the ΔflhD + ΔfliGHl knockout, the above ΔflhD strain was retransformed with pkd46 via electroporation, grown to an OD of 0.1, and induced with 20 mM arabinose until the bacteria reached an OD of 0.8. The fliGHl knockout PCR product was amplified from pkd3 using primers vr266 and vr268. The PCR product was digested with DpnI, and 1 microgram was transformed into the lambda-inducible ΔflhD strain using electroporation. Bacteria were allowed to recover in LB at 100 micrograms / ml for 2 hours at 37°C and plated on agar plates containing 33 micrograms / ml chloramphenicol. Successful transformants were screened as described above, and the pkd46 bacteria were cured by overnight growth at 43°C on LB containing 33 micrograms / ml chloramphenicol.

[0471] The generated plasmids were transformed into the relevant strains using electroporation, which are listed in Table 3. Mouse model Six-week-old Balb / C mice (Jackson Laboratories) received 1 × 10 5 4T1 tumor cells were injected subcutaneously into the posterior flank. 3 Once the tumor reached 100%, mice were injected intravenously with either saline or bacteria. At either 24 or 96 hours after bacterial administration, the mice were sacrificed, and the tumor, liver, and spleen were excised for downstream analysis.

[0472] Salmonella colonization of tumors and liver in vivo To quantify tumor and liver colonization, subcutaneous 4T1 tumors (approximately 500 mm) were cultured. 3Five groups of five Balb / C mice containing ΔflhD, ΔfliGHl, or ΔflhD + ΔfliGHl Salmonella were injected intravenously via the tail vein with either parental, ΔflhD, ΔfliGHl, or ΔflhD + ΔfliGHl Salmonella. Ninety-six hours after bacterial challenge, tumors and livers were excised and homogenized in two volumes (w / v) of sterile PBS. The organ slurries were serially diluted 10-fold: four times for livers and eight times for tumors. 200 μl of each dilution was plated onto agar containing the appropriate antibiotic. After drying, plates were incubated overnight at 37°C. Plates containing 10–100 colonies were counted to determine the level of bacterial colonization in either tumors or livers.

[0473] immunohistochemistry Resected tumor sections were fixed in 10% formalin for 3 days. Fixed tumor samples were then stored in 70% ethanol for 1 week. Tumor samples were embedded in paraffin and sectioned into 5 μm sections. Deparaffinization was performed by washing the sectioned tissue three times in 100% xylene, twice in 100% ethanol, once in 95% ethanol, once in 70% ethanol, once in 50% ethanol, and once in DI water. Each washing step was performed for 5 minutes. Antigen retrieval was performed by incubating the tissue sections in 20 mM sodium citrate (pH 7.6) buffer at 95°C for 20 minutes. The samples were left in the sodium citrate buffer until the temperature decreased to 40°C. The samples were then rehydrated with two quick (<1 minute) rinses in DI water, followed by one 5-minute wash in TBS-T.

[0474] Prior to staining, tissue sections were blocked for 1 hour with Dako blocking buffer (Dako). Tissue sections were stained to identify Salmonella and GFP with a 1:100 dilution of (1) a FITC-conjugated rabbit anti-Salmonella polyclonal antibody (Abcam), and (2) either a rat anti-myc monoclonal antibody (Chromotek) or a rat anti-GFP monoclonal antibody (Chromotek) in Tris-buffered saline with 0.1% Tween 20 (TBS-T) with 2% BSA (Fisher Scientific). Sections were washed three times in TBS-T with 2% BSA and incubated with Alexaflor-568 goat anti-rat secondary antibody (ThermoFisher). After washing the sections three times with TBS-T, 40 μL of Prolong Gold mounting medium with DAPI (ThermoFisher) and a coverslip were added to each slide. The slides were incubated at room temperature for 24 hours until the mounting medium solidified.

[0475] Histological detection of intracellular delivery of GFP to cells in tumors bearing FID Salmonella To identify and quantify GFP delivery to tumor cells, two groups of 10 BALB / c mice bearing 4T1 tumors were treated with 2 × 10 6 CFU of FID Salmonella were injected into one group of mice. One group was injected intraperitoneally twice with arabinose to induce flhDC expression, while the other group was injected with saline as a control. 96 hours after bacterial injection, the mice were sacrificed, and the tumor, liver, and spleen were excised. The tumors were cut in half. One half was fixed and stained for imaging as described in the immunohistochemistry section.

[0476] cell culture Two cancer cell lines were used: 4T1 mouse breast cancer cells and LS174T human colorectal cancer cells (ATCC, Manassas, VA, USA). All cancer cells were grown and maintained in Dulbecco's minimal Eagle's medium (DMEM) containing 3.7 g / L sodium bicarbonate and 10% fetal bovine serum. For microscopy studies, cells were incubated in DMEM with 20 mM HEPES buffer and 10% FBS. To generate tumor spheroids, a single-cell suspension of LS174T cells was transferred to a PMMA-coated cell culture flask (2 g / L PMMA in 100% ethanol, dried before use).

[0477] A microfluidic system for quantifying the intracellular invasive distribution of flhDC-induced Salmonella To quantify invasion into the tumor mass, engineered Salmonella were administered to a tumor-on-a-chip device developed in our laboratory (43, 44). The microfluidic tumor-on-a-chip device was fabricated using negative-tone photoresist and PDMS-based soft lithography. A master chip was constructed by spin-coating a layer of SU-8 2050 onto a silicon wafer at 1250 RPM for 1 min. This speed corresponded to a thickness of 150 μm of SU-8 2050. The silicon wafer was baked at 65 °C for 5 min, followed by baking at 95 °C for 30 min. The microfluidic design printed on a high-resolution transparency was placed above the silicon wafer in a mask aligner. The silicon wafer with the overlaid mask was then illuminated with UV light (22 J / cm). 2 ) for 22 seconds. The silicon wafer was baked at 65°C for 5 minutes, followed by baking at 95°C for 12 minutes. The wafer was then developed in PGMEA developer solution for 10 minutes and / or until the microfluidic features were microscopically distinct with sharp and defined edges.

[0478] Soft lithography was used to create a multilayer tumor-on-a-chip device with 12 tumor chambers (two conditions with six chambers each). PDMS (Sylgard 184) was used in a ratio of 9:1 and 15:1 for the channel and valve layers, respectively. To achieve a PDMS thickness of 200 μm, the channel layer was placed on a spin coater at 220 rpm for 1 minute. The silicon wafer was degassed for 45 minutes to remove air bubbles within the PDMS. The silicon wafer was baked at 65°C for approximately 1 hour, or until both PDMS layers were partially cured. The top valve layer of PDMS was cut, removed from the silicon wafer, and aligned on top of the channel layer using a stereomicroscope. The combined layers were baked at 95°C for 1 hour to covalently bond the two layers. The multilayer PDMS device and glass slide were plasma-treated for 2.5 minutes in a plasma cleaner (Harrick). A vacuum pump was used to pneumatically actuate the valve and place the PDMS on the plasma-treated glass slide. The valve was left actuated until the device was ready for use.

[0479] The tumor-on-a-chip was sterilized with 10% bleach, followed by 70% ethanol for 1 hour each. The microfluidic chip was equilibrated with culture medium (DMEM with 20 mM HEPES, pH 7.4) for 1 hour. The tumor spheroid was positioned within the tumor chamber using valve actuation. The rear valve of the chamber was opened while the outflow channel was closed. After the tumor mass was positioned, the valve was reset so that the rear valve was closed, and the inflow and outflow channels were opened.

[0480] Prior to administration to the device, flhDC-reporting Salmonella were grown in LB with 20 mM arabinose to induce flhDC expression. These Salmonella have an inducible flhDC (PBAD-flhDC) and produce GFP when intracellular (PsseJ-GFP). Control (flhDC-) Salmonella of the same strain were grown without arabinose. Bacteria were centrifuged and collected at 2 × 10 7The cells were resuspended in culture medium (DMEM with 20 mM HEPES) at a density of 100 CFU / ml. For the induced flhDC+ condition, 20 mM arabinose was added to the medium. Bacteria-containing medium (flhDC+ and flhDC-, n = 6 chambers each) was added to each device at 2 × 10 6 For total CFU delivery, perfusion was performed through the tumor-on-chip device at 3 μm / min for 1 h. Bacterial administration was followed by bacteria-free medium (with 20 mM HEPES) for 48 h.

[0481] The device was imaged at 30-minute intervals. Infiltration was quantified at 31 hours by measuring GFP expression by invaded bacteria within the tumor mass. Regions of interest were defined around the border of the tumor mass. The extent of infiltration was determined as the mean GFP fluorescence intensity of each tumor mass. Intensity was normalized by the intensity of the average tumor mass administered with control (flhDC-) Salmonella.

[0482] Microscopy and image analysis Samples were imaged on a Zeiss Axio Observer Z.1 microscope. Fixed cells on coverslips were imaged using a 100x oil-immersion objective (1.4 NA). Tumor sections were imaged using 10x and 20x objectives (0.3 and 0.4 NA, respectively). Time-lapse fluorescence microscopy of live cells in well plates and tumor chip devices was housed in a humidified 37°C environment and imaged using 5x, 10x, 63x, or 100x objectives (0.2, 0.3, 1.4, and 1.4 NA, respectively). Fluorescence images were acquired using either 480 / 525 or 525 / 590 excitation / emission filters. All images were background subtracted and uniformly contrast enhanced. All immunocytochemical image analysis was automated using computer code (MATLAB, Mathworks). Immunohistochemical imaging of bacterial distribution within tumors was automated using MATLAB. Intracellular protein delivery within mouse tumors was visually quantified.

[0483] Infection assay For infection assays, cancer cells were grown on coverslips for fixed-cell imaging. For fixed-cell imaging, glass coverslips were placed in 12-well plates and sterilized with UV light for 20 minutes in a biosafety hood. Mouse 4T1 cells were seeded onto the coverslips at 40% confluency and incubated overnight in DMEM. Simultaneously, Salmonella was grown to an optical density (OD at 600 nm) of 0.8. After incubation, Salmonella was added to the 4T1 cultures at a multiplicity of infection (MOI) of 10 and allowed to infect the cells for 2 hours. After this infiltration period, the cultures were washed five times with 1 ml of phosphate-buffered saline (PBS) and resuspended in 2 ml of DMEM supplemented with 20 mM HEPES, 10% FBS, and 50 μg / ml gentamicin. The added gentamicin removes extracellular bacteria. After 6 hours of incubation, the medium was removed and the coverslips were fixed with 10% formalin in PBS for 10 minutes.

[0484] immunocytochemistry Using immunocytochemistry, detailed images of Salmonella invading cancer cells grown on coverslips were obtained. Coverslips were fixed with formalin and then blocked for 30 minutes with staining buffer (PBS with 0.1% Tween 20, 1 mM EDTA, and 2% bovine serum albumin (BSA)). The Tween 20 in this buffer selectively permeabilizes mammalian cell membranes while leaving bacterial membranes intact.

[0485] After permeabilization, coverslips were stained with (1) rabbit anti-Salmonella polyclonal antibody (Abcam) or FITC-conjugated rabbit anti-Salmonella polyclonal antibody (Abcam), (2) rat anti-myc monoclonal antibody (Chromotek), and (3) rabbit anti-LAMP1 polyclonal antibody (Abcam) to identify Salmonella, released GFP, and vacuoles. Three different staining combinations were used: (1) Salmonella alone, (2) Salmonella and released GFP, and (3) Salmonella, released GFP, and vacuoles.

[0486] For staining of Salmonella alone (combination 1), coverslips were stained with FITC-conjugated anti-Salmonella antibody for 1 hour at 30° C. and washed three times with staining buffer. For Salmonella, released GFP, and vacuole staining (combination 2), coverslips were sequentially stained with anti-LAMP1 primary antibody for 1 hour at 30°C and washed three times with staining buffer. Coverslips were incubated with Alexaflor-647 chicken anti-rabbit secondary antibody (ThermoFisher) at a 1:200 dilution for 1 hour at 30°C and washed four times with staining buffer. Coverslips were then stained with FITC-conjugated anti-Salmonella antibody and anti-myc primary antibody and washed three times with staining buffer. Coverslips were incubated with Alexaflor-568 goat anti-rat secondary antibody (ThermoFisher) at a 1:200 dilution for 1 hour at 30°C to identify GFP.

[0487] After all staining, coverslips were washed three times with staining buffer and mounted on glass slides using 20 μL of mounting medium with DAPI (ProLong Gold Antifade Mountant, ThermoFisher). The mounted coverslips were allowed to harden overnight at room temperature.

[0488] Quantification of vacuolar percentage, degree of invasion, and lysis of engineered Salmonella To quantify what proportion of intracellular flhDC-expressing Salmonella was located within vacuoles, coverslips were infected with either a parental control strain of Salmonella or FID Salmonella, as described in the infection assay section. Coverslips were then stained for LAMP1, Salmonella, and nuclei, as described in the immunocytochemistry section. Coverslips were imaged at 100x magnification, as described in the microscopy and image analysis section. Ten to twenty cells from either the control or FID-treated groups were analyzed. Salmonella that were either colocalized or very closely bordered by LAMP1 were defined as inside the vacuole. Salmonella that closely bordered the bacteria but did not localize with LAMP1 were defined as cytoplasmic.

[0489] result Controlling flhD expression improves tumor targeting of Salmonella Suppressing Salmonella flhD expression in the systemic circulation improved bacterial tumor colonization. Tumor colonization levels were 10 8 Although the CFU / gram of tumor was significantly higher in the ΔflhD Salmonella group, liver colonization of ΔflhD Salmonella was reduced 10-fold compared to the control (Figure 5A, *, P<0.05). However, when flhDC was overexpressed before injection, tumor colonization was impaired compared to the bacterial control (Figure 5B). These results indicated that expression of flhDC before injection increased the clearance rate of Salmonella from the blood. However, suppression of flhDC before injection increased tumor colonization and specificity of Salmonella.

[0490] The lack of flhDC activity, but not simply the lack of flagella expression, reduced liver colonization while maintaining similar levels of Salmonella tumor colonization. Mice were infected with three different Salmonella strains: ΔflhD, ΔfliGHI, and ΔflhD + ΔfliGHI. The ΔfliGHI strain lacks flagella but retains flhDC activity. Both ΔflhD and ΔflhD + ΔfliGHI strains, lacking flhDC activity, colonized the liver 8.5- and 20-fold less, respectively, than the flagella-deficient ΔfliGHI strain (Figure 5C, *, P < 0.05). However, the tumor colonization levels of all three strains were not different (Figure 5D). These results indicate reduced flhDC activity and increased tumor specificity of Salmonella, not simply the lack of flagella.

[0491] flhDC expression increases intratumoral dispersal of Salmonella Suppressing flhDC expression caused Salmonella to colonize and grow primarily within tumor necrosis. Uninduced flhDC was systemically administered to mice, and flhDC expression was induced in half of the mice by arabinose administration (Fig. 6A). Salmonella that did not express flhDC were not motile and, as a result, formed spatially separated, dense colonies primarily within tumor necrosis (yellow arrows, Fig. 6B). However, a small proportion of these colonies was located within viable tumor tissue (green arrows, Fig. 6B). The proportion of these dense colonies present in necrosis was 75% compared with 25% in viable tumor tissue (Fig. 6C). Assuming that each spatially separated, dense colony originated from a single bacterium, the colony growth rate in necrosis was 0.12 h-1 compared with a slightly reduced rate of 0.11 h-1 in viable tissue (*, P < 0.05, Fig. 6D). These bacterial growth rates corresponded to doubling times of 6 h within necrosis and 6.5 h within viable tumor tissue (*, P < 0.05, Figure 6E), consistent with previous estimates (45). These results indicate that Salmonella strongly favors colonization and growth within tumor necrosis compared with viable tumor tissue.

[0492] Re-expression of flhDC in intratumoral Salmonella increased bacterial dispersal and tumor coverage. ΔflhD Salmonella carrying the PBAD-flhDC gene circuit were intravenously injected into 4T1 tumor-bearing mice, which then received two doses of arabinose intraperitoneally to induce flhDC expression in Salmonella (Figure 6A). Induction of flhDC in intratumoral Salmonella increased both bacterial colony size and intratumoral bacterial coverage (Figure 6F). Colony size of flhDC-reexpressing Salmonella increased 1.5-fold compared to uninduced controls (*, P<0.05, Figure 6G). While non-flhDC-induced Salmonella formed dense, tightly packed colonies (upper panel, Figure 6H), a greater number of flhDC-induced bacteria were located outside these dense colonies (lower panel, green arrow, Figure 6H). The number of Salmonella outside the dense central colonies (termed satellite colonies) increased twofold when flhDCs were induced in intratumoral Salmonella (*, P < 0.05, Figure 6I). These results indicate that intratumoral induction of flhDCs in Salmonella allows bacteria to move away from dense colonies within tumor necrosis and toward surviving cancer cells.

[0493] In situ expression of flhDC is required to increase intracellular invasion of Salmonella into spatially distant cells. Re-expression of flhDC increased the spatial distribution of intracellular Salmonella within tumors. Using a tumor-on-a-chip device, we quantified the spatial distribution of intracellular Salmonella (Figure 7A). These Salmonella expressed GFP in flhDC after intracellular infiltration with arabinose supplementation (intracellular reporting Salmonella, IR Sal). Arabinose induction of flhDC enabled widespread distribution of intracellularly expressed GFP Salmonella within the in vitro tumor mass (+flhDC, Figure 7B). However, non-induced ΔflhD Salmonella D (-flhDC) was detected at very low concentrations throughout the tumor mass (white arrow, Figure 7B). The presence of intracellular Salmonella gradually increased deeper into the tumor tissue, being 140-fold enriched in +flhDC compared to -flhDC Salmonella for x > 0.5 (**, P < 0.01; ***, P < 0.001; Figure 7C). The overall amount of flhDCs expressing IR Salmonella increased exponentially over time compared with -flhDC controls (*, P < 0.05; **, P < 0.01; ***, P < 0.001; Figure 7D ), indicating that flhDC induction increased intracellular Salmonella coverage in the tumor mass.

[0494] When mice were administered +flhDC IR Sal (Fig. 7E) and arabinose-induced to express flhDC (Fig. 7F), a greater proportion of bacteria were located intracellularly (inducible, white squares, Fig. 7F). Intracellular invasion of flhDC re-expressing Salmonella was increased 2.3-fold over the -flhDC IR Sal control (*, P < 0.05, Fig. 7G).

[0495] We quantified the distribution of intracellular bacteria using Euclidean distance mapping of histological sections, which quantifies the proximity of every location within the tumor to the nearest bacterium. The spatial coverage of intracellular bacteria was greater after flhDC induction, as shown by Euclidean distance modeling of histological sections (Figure 7H). Salmonella was 1.6-fold more distributed after flhDC induction (*, P < 0.05, Figure 7I). These results indicated that flhDC expression increases intracellular invasion by positioning more bacteria near a greater number of viable cancer cells. In addition, flhDC expression increases intracellular invasion in a flagella- and T3SS-1-driven manner (10).

[0496] Controlling flhDC expression improves intratumoral GFP delivery distribution Induction of flhDCs with engineered Salmonella in tumors increased protein delivery across a larger cellular area. Induced Salmonella delivered protein to a broad, spatially distributed set of cells within the tumor (Figure 8A). Euclidean distance mapping analysis of intratumoral delivery demonstrated that flhDC-induced (flhDC-intracellularly delivered Salmonella; FID Sal) Salmonella increased spatial delivery coverage by 1.6-fold compared with non-flhDC-induced (non-inducible intracellularly delivered Salmonella; UID Sal) Salmonella (***, P < 0.001; Figure 8B). These results demonstrate that flhDC-induced Salmonella increased intratumoral spatial coverage (Figures 7H, I), enabling bacteria to intracellularly deliver protein to a broad, distributed set of cells within the tumor.

[0497] Engineered Salmonella are superior in tumor colonization and protein delivery compared to exclusively cytoplasmic Salmonella The engineered Salmonella colonized tumors and delivered significantly more protein inside cancer cells than conventionally used cytosolic Salmonella. As demonstrated, ΔflhD Salmonella did not colonize tumors less than the control (Figure 5A). However, ΔsifA Salmonella colonized tumors 10-fold less than the control (*, P<0.05, Figure 9A). Liver colonization was also reduced 10-fold between ΔsifA and control Salmonella (*, P<0.05; Figure 9B), indicating that the ΔsifA strain exhibited overall poorer fitness in vivo. Using selective staining techniques to detect bacterial lysis and protein delivery as previously described, the engineered Salmonella visibly lysed more inside cells than ΔsifA Salmonella at all time points (Figure 9C). FID Sal lysed 18-fold more than ΔsifA Salmonella (Figure 9D, **, P<0.01). Cytoplasmic localization is important for protein therapeutics to have biological and anticancer activity. However, these results demonstrate that primarily cytoplasmic Salmonella are less suitable for therapeutic delivery. This is the result of a combination of poor tumor colonization, poor systemic infectivity in vivo, and poor lysis efficiency of ΔsifA compared to FID Sal. ΔsifA strains of Salmonella are unable to effectively colonize tumors and are therefore not advantageous for intracellular protein delivery.

[0498] flhDC expression reduces lysis efficiency in intracellular Salmonella Expression of flhDC in Salmonella affects intracellular lysis and protein delivery after invasion. To understand this dynamics, cancer cells were infected with lysing Salmonella (ID Sal) or control lysing Salmonella re-expressing flhDC (FID-Sal) (Figure 10A). As expected, FID Sal invaded cancer cells three times more than ID Salmonella (Figure 10B, C; **, P<0.01). However, FID Sal lysed 33% less than control ID Sal (Figure 10D, **, P<0.01). To understand why, we quantified the vacuolar / cytoplasmic distribution of control and flhDC-expressing Salmonella after cancer cell infection (Figure 10E). While most control Salmonella were contained within vacuoles (colocalized green and red), a greater proportion of flhDC re-expressing Salmonella was cytoplasmic (green only, Figure 10F). At the population level, 90% of controls were intravacuolar, compared with 70% for flhDCs re-expressing Salmonella (Figure 10G). Consequently, ID Salmonella were more likely to remain intravacuolar and lyse (white arrow, Figure 10H), while a small proportion of FID Sal was more likely to escape the vacuole and remain intact (light blue arrow, Figure 10I). In vivo, FID Sal qualitatively demonstrated a similar phenomenon (Figure 10J). Unlysed and intracellular FID Sal was distributed throughout several cells (white arrow), likely indicating that bacteria were overreplicating in the tumor cell cytoplasm. These results indicate that flhDC induction increases invasion but decreases the lytic efficiency of engineered Salmonella, likely due to vacuolar escape.

[0499] Vacuolar retention of Salmonella -overexpressing flhDC rescues lysis and protein delivery efficiency Overexpressing flhDC impaired vacuolar escape in engineered Salmonella strains and rescued lysis efficiency and overall intracellular protein delivery. We previously demonstrated that engineered ΔsseJ Salmonella lysed intracellularly with high efficiency. Therefore, we hypothesized that overexpressing flhDC in lysing ΔsseJ Salmonella (ΔsseJ FID Sal) would rescue lysis efficiency while maintaining high levels of invasion. Cells infected with ΔsseJ FID Sal exhibited an increase in invaded, lysed bacteria (white arrows, Figure 11A). ΔsseJ FID Sal invaded cancer cells 1.5-fold more than FID Sal and 3-fold more than ID Sal (Figure 11B, **, P<0.01). Intracellular ΔsseJ FID Sal also lysed 25% more efficiently than FID Sal alone (Figure 11C, **, P<0.01). The combination of these two phenomena (increased invasion and improved lysis) in the engineered strain increased overall protein delivery by 2.5-fold over FID Sal (Figure 6D, **, P<0.01). This data demonstrated that the reduced lysis efficiency resulting from flhDC activity can be rescued by overexpressing a Salmonella transcription factor engineered to remain in the vacuole.

[0500] conclusion Modulating flhDC expression in engineered Salmonella had broad implications for intracellular therapeutic delivery within tumors (Figure 12). Salmonella lacking flhDC expression colonized tumors more selectively. However, overexpression of transcription factors within systemic Salmonella reduced bacterial tumor colonization. Controlled expression of flhDC in tumors increased the spatial distribution of extracellular and intracellular Salmonella. While flhDC expression reduced the intracellular lytic efficiency of engineered Salmonella, overexpressing the vacuolar-resident ΔsseJ transcription factor rescued lytic efficiency and improved overall protein delivery in tumor cells. Collectively, the results demonstrate that modulating flhDC expression in therapeutic Salmonella improves several driving features of protein delivery in tumors (Figure 12).

[0501] Consideration Herein, we demonstrate that controlling flhDC expression in engineered bacteria maintains high colonization levels, improves tumor specificity, and increases intratumoral protein delivery distribution. Expression of flhDC also reduced intracellular lysis efficiency, which was rescued by overexpressing a transcription factor in a vacuolar-localizing strain (ΔsseJ) of Salmonella. The combination of the two genetic engineering strategies increased overall intracellular protein delivery.

[0502] The colonization pattern of flhDC-inducible Salmonella suggests that only a few hundred single bacteria infiltrate tumors and grow in situ out of 2 million injected. These ratios are supported by previous studies demonstrating that 1 in 10,000 bacteria adhere to tumor vasculature (46). In histological samples, flhDC-inducible Salmonella form spatially separated colonies that are predominantly localized to tumor necrosis (Figure 6B,C). Each of these colonies could be derived from the clonal expansion of a single bacterium that has managed to colonize the tumor. In this case, this suggests that bacterial influx into tumors, a rare event, is strongly supported by extensive necrosis and is the rate-limiting step in tumor colonization. Such rare bacterial infiltration events could explain why tumor colonization is highly variable within mouse or human populations, as previously described (47). These results could explain why extensive tumor colonization has primarily been detected in the presence of tumor necrosis in humans (47). Thus, combining tumor vascular disrupting agents with Salmonella may reduce patient-to-patient treatment variability and allow for effective colonization of small necrotic defect primary and metastatic tumors.

[0503] Two strategies can be used to potently initiate bacterial colonization within tumors: (1) coadministration of bacteria with a mild TNF-alpha inducer, as previously described (48), or (2) genetic modification of Salmonella to evade systemic innate immune recognition (e.g., flhDC modulation). In scenario (1), as previously demonstrated, coadministration with lipid A (a known TNF-alpha inducer) did not induce septic shock but increased vascular permeability, thus increasing the probability of bacterial invasion into tumors across a large number of mice. In scenario (2), flhDC suppression of injected Salmonella could help the bacteria evade innate immune detection of flagella in the systemic circulation. This could allow the bacteria to persist systemically for longer without inducing septic shock. Longer systemic persistence, in turn, could increase the probability of bacterial invasion into tumors.

[0504] Wild-type Salmonella is likely not optimized for intracellular delivery of therapy within tumors. One reason for this may be that necrotic tumor tissue favors the colonization of ceciles and nonmotile Salmonella. Because flagellated bacteria minimally colonize tumors, likely due to innate immune-mediated clearance (Figure 5B), the data suggest that tumors select for bacteria that are likely nonmotile and nonflagellated (8, 9). NonflhDC-induced bacteria did not exhibit impaired colonization levels compared with control strains (Figure 5D). Furthermore, nonflhDC-induced bacteria clustered in densely packed colonies, mostly located within tumor necrosis (Figure 6B). This suggests that Salmonella has a higher affinity for colonizing necrotic rather than viable tissue and that external control is required to enable Salmonella to invade viable tumor cells in a flhDC-dependent manner. Controllable activation of flhDC expression in intratumoral Salmonella demonstrated that a significant proportion of these bacteria invaded and delivered proteins into a spatially distributed set of cells.

[0505] The presence of a vacuole may also help prevent premature clearance of engineered Salmonella before tumor accumulation, in addition to enabling lysis of the engineered Salmonella. The current paradigm for intracellular cytoplasmic therapeutic delivery is to allow Salmonella to escape the vacuole and invade directly into the cytosol through deletion of the sifA gene (20). Similarly, bacterial variants expressing listeriolysin O have also been used to enable vacuolar escape of therapeutic Salmonella (49-51). However, it was determined that the unnatural cytoplasmic escape of Salmonella (ΔsifA) reduced tumor colonization by 100-fold compared to the parent strain (Figure 9A). This is likely because cytoplasmic pathogens elicit strong antimicrobial and NF-kB-dependent immune responses that are detrimental to bacterial fitness in vivo (21, 52-55). ΔsifA bacteria also lysed 18-fold less than FID Salmonella. These results indicate that the engineered strains have significantly improved delivery potential Salmonella compared to existing cytoplasmic delivery methods.

[0506] The engineered bacterial system described herein shares similarities with Salmonella typhi strains that have evolved to systemically infect human hosts. Humans serve as the natural host for Salmonella typhi, and upon ingestion, the bacteria surreptitiously transition from the intestine to the systemic circulation without eliciting a significant initial immune response (30). The bacteria can circulate systemically for extended periods without causing septic shock (30). Typhoid strains achieve this by encoding the capsule-like regulatory protein TviA. The transcription factor encodes the Vi capsule, which masks bacterial LPS (56). In addition, TviA suppresses flagellar and T3SS-I activity in systemically circulating bacteria through the repression of flhDC and HilA expression, respectively. 57 Masking of LPS and downregulation of flagellar and T3SS-I activity lead to evasion of innate immune recognition (57). Salmonella immediate-delivery strains also have a modified LPS through deletion of msbB, which prevents sepsis. Additionally, expression of the flagellum-activating flhDC and, to a lesser extent, T3SS-1 synthesis (10) are suppressed following systemic administration of engineered Salmonella. Engineered strains of Salmonella and Salmonella typhi also share the similarity that both types of bacteria reside primarily within intracellular vacuoles. Their presence within intracellular vacuoles prevents bacterial detection by cytoplasmic, innate immune sensors, such as Nod-like receptors, ubiquitin, and NF-kB components. These genetic modifications likely act to mask common pathogen-associated molecular patterns associated with Salmonella, increasing systemic persistence without eliciting any adverse immune response.

[0507] [Table 11-1]

[0508] [Table 11-2]

[0509] [Table 11-3]

[0510] [Table 11-4]

[0511] [Table 11-5]

[0512] Example III Chromosomal integration of flhD into EBV-002. The cell invasion ability of EBV-002 containing a single chromosomal copy of PBAD-flhDC was evaluated. Chromosomal integration of an inducible version of flhDC can create a master delivery vehicle that can be used to deliver any therapy into tumors. Creating a single master delivery vehicle can streamline the manufacturing process for any EBV-based therapy. To this end, a single copy of PBAD-flhDC was integrated into VNP20009 Salmonella in place of the endogenous flhDC gene. This chromosomally integrated strain was grown with arabinose to activate flhDC expression and used to infect cancer cells. Chromosomally integrated VNP20009 invaded cancer cells to levels similar to bacteria containing an episomal copy of flhDC (Figure 13A). Chromosomal knock-in of flhDC was also similarly inducible compared to Salmonella with episomal PBAD-flhDC (Figure 13B). This result demonstrated that it was possible to genomically integrate the flhDC-inducible gene circuit to create a master EBV-002 delivery vehicle.

[0513] Development of a clinical strain of EBV-002. A clinically compatible strain of EBV-002 was created by controlling the activation of flhDC using salicylic acid, the active ingredient in aspirin (Figure 14). Because flhDC is the master transcription factor controlling flagellar synthesis, chemotaxis, and motility, bacteria are highly sensitive to even low expression levels of the protein. As a result, we hypothesized that expression levels in the uninduced state must be tightly repressed to completely suppress uninduced cell invasion. To test this hypothesis, we generated four different flhDC-inducible EBV-002 strains: 1) salicylic acid-inducible flhD, 2) flhD containing a weakly active ssrA degradation sequence, 3) flhD containing a moderately active ssrA degradation sequence, and 4) flhD containing a highly active degradation tag (Figure 14). The purpose of these degradation tags was to eliminate uninduced flhD activity, which would have resulted from leaky expression from the pSal promoter. The intracellular invasion rate of each of these four strains was compared with that of a PBAD-inducible version of flhDC. As expected, sample (1) was highly motile and invasive with or without salicylic acid induction (Figure 14B), indicating that the salicylic acid promoter was leaky. Samples (2), (3), and (4) only invaded cells after salicylic acid induction and were completely noninvasive otherwise. However, samples (2) and (3) were the most intracellularly invasive after aspirin induction (Figure 14B). Most importantly, strains (2) and (3) were more invasive than the PBAD-inducible version of EBV-002 (Figure 14B). These results demonstrate that the salicylic acid induction circuit was optimized to express flhD and regulate the intracellular invasion of EBV-002 into cancer cells.

[0514] Sample (2) was characterized because this strain of EBV-002 had the highest range of activation between the uninduced and induced samples. The induced bacteria swam significantly longer distances compared to the uninduced EBV-002, which remained stationary (Figure 15A). Salicylate-induced EBV-002 swam 12.7-fold farther than the uninduced control (***, P<0.001, Figure 15B). This demonstrated that the salicylate-inducible gene circuit was able to strongly control flhDC activity in clinical EBV-002 strains. As expected, the salicylate-inducible clinical strain of EBV-002 invaded cancer cells 30-fold more than the uninduced control (***, P<0.001, Figures 15C and D). These results indicate that expressing flhD with a weakly activated degradation tag using salicylate enabled maximum control of EBV-002 intracellular invasion.

[0515] After determining which version of pSal-flhD was most effective at invading cancer cells with salicylic acid induction, we next determined the lowest amount of salicylic acid required to enable intracellular invasion. EBV-002 was induced with either 10 nanomolar (nM), 100 nM, 500 nM, 1 micromolar (uM), or 10 uM salicylic acid, and cancer cells were infected with each of these strains. It was determined that a concentration of 500 nM salicylic acid was required to enable intracellular invasion of EBV-002 (Figure 16). This result is significant because it indicates that the induction threshold for EBV-002 is well within the range of salicylic acid concentrations (10–50 uM) found in the bloodstream after a person takes aspirin orally. Together, these results indicate that EBV-002 is ripe for use as an intracellular delivery vehicle within human tumors.

[0516] Incorporation of the ΔsseJ mutation into EBV-002 to generate EBV-003. The ΔsseJ mutation was previously demonstrated to significantly increase the lytic efficiency of EBV strains. To this end, the EBV-002 strain, which contains not only the same salicylic acid-inducible flhDC gene but also an intracellular lysis cassette, was additionally engineered with the ΔsseJ mutation to create EBV-003.

[0517] In vivo efficacy of EBV-003. Biodistribution and tumor-selective protein delivery were evaluated in mice bearing subcutaneous 4T1 tumors. 3 Balb / C mice bearing subcutaneous tumors of 1 × 10 7 CFU of EBV-003 were injected intravenously. 72 hours after injection, mice were intraperitoneally injected with 5 mg of salicylic acid to induce flhDC expression in the tumor bacilli. 24 hours later, mice were sacrificed, and tumors, livers, and spleens were removed for analysis. EBV-003 colonization and protein delivery were compared with EBV-001 to evaluate any improvements. After colonization, EBV-003 colonized tumors 10.7-fold more than EBV-001, while spleen and liver colonization remained unchanged (Figure 17A, **, P<0.01). On average, EBV-003 delivered 31-fold more protein into tumor cells compared with EBV-001 (Figure 17B). However, protein delivery was not detected in the spleen or liver with either strain. These results demonstrate that EBV-003 is significantly more effective at colonizing and delivering proteins selectively into tumors while sparing healthy tissue.

[0518] To determine whether EBV-003 intracellularly infiltrated cancer cells after salicylic acid induction in vivo, female BALB / c mice were injected subcutaneously with 4T1 tumors. 3 When it becomes, 1 × 10 6CFU were injected via the tail vein. 72 hours after bacterial administration, seven mice were intraperitoneally injected with 5 mg of sodium salicylate, while four mice received saline injections as a control. 24 hours after salicylic acid administration, the mice were sacrificed, and tumors were excised, fixed, and stained for Salmonella. Histological examination revealed that salicylic acid induction increased the intracellular infiltration of viable cancer cells within quiescent tumor tissue. More bacteria (red X, Figure 18A) were dispersed throughout the quiescent tumor tissue after induction with salicylic acid (Figure 18B). Salicylic acid induction resulted in a two-fold increase in cancer cells harboring intracellular EBV-003 compared to the uninduced control (*, P<0.05, Figure 18C). These results demonstrated that EBV-003 can be induced to infiltrate cells using therapeutic doses of salicylic acid.

[0519] Intracellular protein delivery using EBV-003 was also evaluated with and without salicylate induction. After salicylate induction, protein delivery was detected in 5 of 6 tumors within the transition zone, where tumor cells were rapidly dividing (white arrows, Figure 19A). In contrast, delivery was detected only within the transition zone in 1 of 4 uninduced control mice (Figure 19B). These results demonstrated that salicylate induction of EBV-003 enabled intracellular protein delivery in vivo.

[0520] In vivo colonization, invasion, and protein delivery of EBV-003 in spontaneous breast cancer metastases in the liver. EBV-003 selectively colonized, invaded, and delivered proteins to metastatic breast cancer lesions in the liver (Figure 20). All dense bacterial colonies were found exclusively within metastatic breast cancer lesions in the liver (colonies outlined in white, Figure 20A). Furthermore, 85% of these colonies were located within directly adjacent or actively dividing tumor lesions (red arrows, Figure 20A), where therapeutic delivery is most effective. In contrast, colonies found in healthy tissue were observed much less frequently and were much smaller (Figure 20A). Bacterial colonies were rare and very small in healthy tissue (1, white arrow, Figure 20B). However, in metastatic lesions, colonies were significantly larger in area (2, white arrow, Figure 20B). Within the liver, 87.7% of colonies were found within metastatic lesions, while the other 12.3% were found within healthy liver tissue. Furthermore, the size of colonies within metastatic lesions was over 118-fold larger than that within healthy tissue (***, p=2.2×10-26, Figure 20C). This equates to an 850-fold enrichment of EBV-003 in metastatic breast cancer lesions within the liver relative to immediately adjacent healthy tissue. While we have demonstrated the ability of therapeutic Salmonella to colonize primary tumors over 1,000-fold more than any other organ, this is the first demonstration that Salmonella preferentially colonizes metastatic tumor lesions to a similarly high extent compared to immediately adjacent healthy tissue. This illustrates the superior selectivity of EBV-003 for colonizing tumor tissue, regardless of whether the tumor is a primary or metastatic lesion.

[0521] EBV-003 strains also intracellularly invaded cancer cells within liver metastases (white arrows, Figure 21A). However, there was no difference in the level of invasion between salicylate-induced and uninduced EBV-003 (Figure 21B). One reason for this may be that most metastatic lesions contained a higher proportion of viable tumor tissue and a lower amount of necrosis. As a result, EBV-003 bacteria were more likely to be in close proximity to viable tumor cells, increasing the likelihood that bacteria could invade cells intracellularly, regardless of induction status. This is in contrast to primary tumor tissue, where salicylate induction of flhDCs increased the intracellular presence of EBV-003 within quiescent tumor tissue (Figure 18A). This may be because bacteria preferentially colonize necrotic tissue, swim toward actively dividing cancer cells, and require flhDC-dependent motility for intracellular invasion. This therefore indicates that flhDC induction is necessary for intracellular infiltration within the primary tumor mass, but less so within small, non-necrotic metastatic or primary lesions.

[0522] Although EBV-003 appeared to infiltrate metastatic cancer cells in the presence or absence of flhDC activity, salicylate induction in vivo resulted in higher protein delivery. Cytoplasmic delivery into cells within metastatic tumors was histologically detected (white arrows, Figure 22A). The frequency of protein delivery to cells within metastases was more than threefold higher with induced EBV-003 than with uninduced EBV-003 (**, P<0.01, Figure 22B). Together, these results indicate that induction of flhDC improves protein delivery in both primary and metastatic breast tumors.

[0523] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event that the definitions of terms incorporated by reference conflict with those defined herein, the present specification shall control.

Claims

1. A bacterial cell of the genus Salmonella, a) SseJ deletion or reduced expression of SseJ; b) a lysis gene or lysis cassette operably linked to a Salmonella promoter induced in the cell; A bacterial cell, wherein the Salmonella promoter induced in said cell is the promoter for SseJ.

2. The cell of claim 1, wherein the lysis cassette is lysine E from phage phiX174, the lysis cassette of phage iEPS5, or the lysis cassette from lambda phage.

3. The cell of claim 1 , wherein the cell does not contain endogenous flhDC expression.

4. 2. The cell of claim 1, wherein the cell does not contain endogenous flhDC, motA, motB, flhE, cheZ, cheY cheB, cheR, cheM, cheW, cheA, fliA, fliY, fliZ, fliB, fliS, fliE, fliF, fliJ, fliL, fliM, fliN, fliO, flip, fliQ, fliR, fliG, fliH, fliI, fliT, fliD, fliC, fljB, ycrG, flgN, flgM, flgA, flgB, flgC, flgD, flgE, flgF, flgG, flgH, flgI, flgJ, flgK, and / or flgL expression.

5. 2. The cell of claim 1, wherein the cell comprises an exogenous inducible promoter operably linked to an endogenous or exogenous flhDC, motA, motB, flhE, cheZ, cheY cheB, cheR, cheM, cheW, cheA, fliA, fliY, fliZ, fliB, fliS, fliE, fliF, fliJ, fliL, fliM, fliN, fliO, flip, fliQ, fliR, fliG, fliH, fliI, fliT, fliD, fliC, fljB, ycrG, flgN, flgM, flgA, flgB, flgC, flgD, flgE, flgF, flgG, flgH, flgI, flgJ, flgK, and / or flgL gene.

6. 6. The cell of claim 5, wherein the exogenous inducible promoter is operably linked to the endogenous flhDC, motA, motB, flhE, cheZ, cheY cheB, cheR, cheM, cheW, cheA, fliA, fliY, fliZ, fliB, fliS, fliE, fliF, fliJ, fliL, fliM, fliN, fliO, flip, fliQ, fliR, fliG, fliH, fliI, fliT, fliD, fliC, fljB, ycrG, flgN, flgM, flgA, flgB, flgC, flgD, flgE, flgF, flgG, flgH, flgI, flgJ, flgK, and / or flgL gene.

7. 6. The cell of claim 5, wherein the exogenous inducible promoter is operably linked to the exogenous flhDC, motA, motB, flhE, cheZ, cheY cheB, cheR, cheM, cheW, cheA, fliA, fliY, fliZ, fliB, fliS, fliE, fliF, fliJ, fliL, fliM, fliN, fliO, flip, fliQ, fliR, fliG, fliH, fliI, fliT, fliD, fliC, fljB, ycrG, flgN, flgM, flgA, flgB, flgC, flgD, flgE, flgF, flgG, flgH, flgI, flgJ, flgK, and / or flgL gene.

8. 6. The cell of claim 5, wherein the exogenous inducible promoter comprises an arabinose-inducible promoter PBAD (L-arabinose), LacI (IPTG), salR, or nahR (acetylsalicylic acid (ASA)).

9. The cell of claim 1 , wherein the cell contains a plasmid that expresses the peptide.

10. The cell of claim 9 , wherein the peptide is a therapeutic peptide.

11. The cell of claim 9, wherein the peptide is NIPP1 or activated caspase 3.

12. A composition comprising the population of cells of claim 1 and a pharmaceutically acceptable carrier.

13. 10. A pharmaceutical composition comprising the population of cells described in claim 1 for use in a method for colonizing tumors and / or tumor-associated cells, said method comprising administering said pharmaceutical composition to a subject in need thereof.

14. The pharmaceutical composition of claim 13 , wherein the tumor-associated cells are intratumoral immune cells or intratumoral stromal cells.

15. 10. A pharmaceutical composition comprising the population of cells described in claim 1 for use in a method for treating cancer, the method comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition to treat the cancer.

16. 10. A pharmaceutical composition comprising the population of cells described in claim 1 for use in a method for inhibiting tumor growth / proliferation or reducing tumor volume / size, the method comprising administering an effective amount of the pharmaceutical composition to a subject in need thereof so as to suppress tumor growth or reduce the volume of the tumor.

17. A pharmaceutical composition comprising the population of cells described in claim 1 for use in a method for treating metastasis, reducing the formation / number of metastases, or inhibiting the spread of metastases, the method comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition to treat metastasis, reduce the formation / number of metastases, or inhibit the spread of metastases.

18. 14. The pharmaceutical composition of claim 13, wherein the tumor and / or tumor-associated cells are lung, liver, kidney, breast, prostate, pancreas, skin, colon, head and neck, ovarian, and / or gastrointestinal tumor and / or tumor-associated cells.

19. The pharmaceutical composition of claim 13 , wherein the bacterial cells deliver a therapeutic peptide to the tumor and / or tumor-associated cells.

20. 14. The pharmaceutical composition of claim 13, wherein endogenous expression of flhDC, motA, motB, flhE, cheZ, cheY cheB, cheR, cheM, cheW, cheA, fliA, fliY, fliZ, fliB, fliS, fliE, fliF, fliJ, fliL, fliM, fliN, fliO, flip, fliQ, fliR, fliG, fliH, fliI, fliT, fliD, fliC, fljB, ycrG, flgN, flgM, flgA, flgB, flgC, flgD, flgE, flgF, flgG, flgH, flgI, flgJ, flgK, and / or flgL is under the control of an exogenous inducible promoter.

21. wherein expression of flhDC, motA, motB, flhE, cheZ, cheY, cheB, cheR, cheM, cheW, cheA, fliA, fliY, fliZ, fliB, fliS, fliE, fliF, fliJ, fliL, fliM, fliN, fliO, flip, fliQ, fliR, fliG, fliH, fliI, fliT, fliD, fliC, fljB, ycrG, flgN, flgM, flgA, flgB, flgC, flgD, flgE, flgF, flgG, flgH, flgI, flgJ, flgK, and / or flgL is under the control of an inducible promoter, and the bacterial cell is expressing exogenous flhDC, motA, motB, flhE, cheZ, cheY, 14. The pharmaceutical composition of claim 13, comprising an exogenous inducible promoter operably linked to a cheB, cheR, cheM, cheW, cheA, fliA, fliY, fliZ, fliB, fliS, fliE, fliF, fliJ, fliL, fliM, fliN, fliO, flip, fliQ, fliR, fliG, fliH, fliI, fliT, fliD, fliC, fljB, ycrG, flgN, flgM, flgA, flgB, flgC, flgD, flgE, flgF, flgG, flgH, flgI, flgJ, flgK, and / or flgL gene.

22. 21. The pharmaceutical composition of claim 20, wherein the expression of flhDC, motA, motB, flhE, cheZ, cheY cheB, cheR, cheM, cheW, cheA, fliA, fliY, fliZ, fliB, fliS, fliE, fliF, fliJ, fliL, fliM, fliN, fliO, flip, fliQ, fliR, fliG, fliH, fliI, fliT, fliD, fliC, fljB, ycrG, flgN, flgM, flgA, flgB, flgC, flgD, flgE, flgF, flgG, flgH, flgI, flgJ, flgK, and / or flgL is induced after colonization of the tumor and / or tumor-associated cells with the bacterial cells.

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