Engineered microorganisms

Abstract

The present invention relates to a live attenuated Gram-negative bacterium comprising an engineered hlyCABD operon wherein the engineered hlyCABD operon is divided into a first segment and a second segment, each segment being operably linked to an independently controlled promoter wherein the first segment comprises a heterologous polynucleotide encoding a cargo molecule upstream of an hlyAs translocation sequence and the second segment comprises a heterologous polynucleotide encoding a cargo molecule upstream of an hlyAs translocation sequence. Wherein a heterologous polynucleotide encoding a cargo molecule substitutes the hlyA gene, and wherein the second segment comprises an hly gene involved in secretion. Thus, the present invention relates to bacterial delivery systems and various uses and methods thereof.

Description

Technical Field

[0001] The present invention relates to attenuated live Gram - negative bacteria that have been engineered to be capable of delivering biomolecules. Background Art

[0002] Secretion of recombinant proteins from bacterial chassis has emerged as a strategy to explore the delivery of relevant cargo from within the cell envelope [Freudl et al., 2018]. This can facilitate downstream purification and processing of biotechnologically relevant protein envelopes [Freudl, R., et al., 2018] or the active delivery of pharmacological molecules to appropriate tissues in vivo, such as tumors in immuno - oncology [Carrier, M. J., et al., 1992; Yang, E. Y. and Shah, K., 2020; Ruano - Gallego, D., et al., 2019].

[0003] Bacteria such as Salmonella enterica Typhi can naturally colonize and develop within the tumor microenvironment (TME) [Hoffman, R. M., 2011]. This can enable targeted delivery of relevant proteins and peptides in vivo to enhance the initial immune response caused by exposure to Salmonella pathogen - associated molecular patterns [Chen, J., et al., 2021]. Salmonella can generally secrete proteins through specific pathways that rely on needle - like structures to inject protein effectors into mammalian cells [Lhocine, N., et al., 2015; Park, D., et al., 2018]. After assembling the needle and piercing the mammalian host, a series of proteins (effectors) containing specific signal peptides are translocated into the host's cytosol, where they can, for example, induce phagocytosis of the bacteria [Park, D., et al., 2018]. However, fusion of a recombinant protein with a recognizable signal peptide only allows the protein to be secreted within the cell, but not into the interstitial space that may be more relevant for therapy.

[0004] Uropathogenic strains of Escherichia coli (E. coli) can export a pore-forming toxin (hemolysin) involved in erythrocyte lysis into the interspace through a specialized secretion pathway, the type 1 secretion system (T1SS) [Thomas, S., et al., 2014]. This pathway depends on two transporters, HlyB and HlyD, which bind to the inner membrane and the periplasm of the bacterium, respectively [Gentschev, I., et al., 2002]. When the toxin HlyA is expressed and the signal peptide (HlyAs), which is approximately 60 base pairs long at its C-terminus, is recognized, the TolB1D2 complex interacts with TolC, thereby opening the pore through which HlyA translocates from the cytoplasm, although the protein remains unfolded at this time. The last protein in this pathway, HlyC, activates the toxicity of HlyA by transferring an acyl group to two internal lysines (lysins) (Lys564 and Lys690), although it remains in the bacterial cytosol (i.e., before export).

[0005] In the chromosome, the hly genomic island consists of four genes, hlyCABD, and a regulatory activator, hlyR [Gentschev, I., et al., 2002; Nagamatsu, K., et al., 2015; Nieto, J.M., et al., 2000; Pourhassan, N.Z., et al., 2022; Khosa, S., et al., 2018; Madrid, C., et al., 2002]. In addition to the control exerted by the hlyR product, the expression of the toxin is also controlled by multiple genetic elements, such as: an operon polarization sequence (ops) and an RfaH-binding sequence downstream of the poorly characterized promoter (Phly) [Gentschev, I., et al., 2002; Nagamatsu, K., et al., 2015; Wang, B., M. et al., 2022], which are involved in the trans-inhibition of a Rho-independent terminator that splits the operon between hlyA and hlyB [Gentschev, I., et al., 2002]; the stress-related sensor CpxR; physicochemical sensors (such as pH or osmotic pressure) HhA or H-NS [Gentschev, I., et al., 2002; Nieto, J.M., et al., 2000; Madrid, C., et al., 2002]; and enhancer elements, such as the AU-rich region between hlyC and hlyA, which can recruit RpsA (also known as S1) [Pourhassan, N.Z., et al., 2022; Khosa, S., et al., 2018].

[0006] When designing synthetic organisms for delivering relevant proteins into the interstitial space between eukaryotic cells, such as within the tumor microenvironment (TME), the complex regulation of the hly operon may limit the yield and robustness of recombinant protein production.

[0007] Therefore, new cargo molecule delivery systems are needed to deliver cargo molecules into the interstitial space between eukaryotic cells. Summary of the Invention

[0008] The inventors of the present invention unexpectedly found that the hlyCABD operon (as Figure 1 shown) can be modified in such a way that it enables the delivery of cargo molecules into the interstitial space between eukaryotic cells ( Figure 2 ).

[0009] In a first aspect, the present invention provides an attenuated live Gram-negative bacterium comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is divided into a first segment and a second segment, each segment being operably linked to an independently controlled promoter, wherein the first segment contains a heterologous polynucleotide encoding a cargo molecule upstream of the hlyA translocation sequence, wherein the heterologous polynucleotide encoding the cargo molecule replaces the hlyA gene, and wherein the second segment contains hly genes involved in secretion.

[0010] In a second aspect, the present invention provides a vaccine composition comprising an attenuated live Gram-negative bacterium comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is divided into a first segment and a second segment, each segment being operably linked to an independently controlled promoter, wherein the first segment contains a heterologous polynucleotide encoding a cargo molecule upstream of the hlyA translocation sequence, wherein the heterologous polynucleotide encoding the cargo molecule replaces the hlyA gene, and wherein the second segment contains hly genes involved in secretion.

[0011] In a third aspect, the present invention provides a method of treating, preventing, inhibiting, preventing recurrence or controlling a disease in a subject, wherein the method comprises administering to the subject an attenuated live Gram-negative bacterium comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is divided into a first segment and a second segment, each segment being operably linked to an independently controlled promoter, wherein the first segment contains a heterologous polynucleotide encoding a cargo molecule upstream of the hlyA translocation sequence, wherein the heterologous polynucleotide encoding the cargo molecule replaces the hlyA gene, and wherein the second segment contains hly genes involved in secretion.

[0012] In a fourth aspect, the present invention provides a method of delivering a therapeutic molecule to the interstitial space between eukaryotic cells in the tumor microenvironment of a subject having a tumor, the method comprising the steps of: i) engineering an attenuated live Gram-negative bacterium, the attenuated live Gram-negative bacterium comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is divided into a first segment and a second segment, each segment operably linked to an independently controlled promoter, wherein the first segment comprises a heterologous polynucleotide encoding a cargo molecule upstream of the hlyA translocation sequence, wherein the heterologous polynucleotide encoding the cargo molecule replaces the hlyA gene, and wherein the second segment comprises hly genes involved in secretion, and ii) administering the engineered Gram-negative bacterium to a subject in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 A schematic diagram of the hly operon encoding the type 1 secretion system (T1SS) is shown. The hly operon is tightly regulated by multiple proteins and signals.

[0014] Figure 2 A schematic diagram of a reconstituted T1SS derived from Escherichia coli is shown. The cargo region and the structural region are shown as being divided into two transcriptional units, independently controlled by promoter P1 and promoter P2, respectively. In addition, two variants of the cargo region were constructed, one variant in which the hlyC gene is upstream of the secretable cargo and one variant lacking the hlyC gene, to evaluate the effect of the hlyC gene on the cargo secretion yield.

[0015] Figure 3 shows the secretion of the reporter protein mScarlet under different configurations of the T1SS. Figure 3A Demonstrates the effect of manipulating the ratio of cargo to structural protein transcription on output by varying the strength of promoter P1 and promoter P2. Analysis of the protein content in the supernatant showed that an increase in the strength of the cargo promoter led to up to a 10-fold increase in the protein detected in the supernatant. Interestingly, an increase in the strength of the promoter controlling the expression of the structural gene led to up to a 64-fold increase in protein production. Figure 3B Demonstrates that when using the same circuit and adding hlyC upstream of the cargo, the overall pattern previously observed is retained, although the protein output level is lower (as low as 1 / 4). This may be due to H-NS binding by Hha downstream of hlyC or to the cellular burden resulting from the addition of hlyC expression.

[0016] Figure 4 shows the secretion of the recombinant protein LLO (also known as listeriolysin O) under different configurations of the T1SS. Figure 4AShows the exchange of the reporter gene mScarlet with hly of Listeria monocytogenes and evaluates its output into the culture supernatant under all configurations. Although the pattern of increasing secretion with increasing promoter P2 strength was retained, the overall protein level of the output was lower than that of the reporter protein mScarlet. Figure 4B Indicates that, contrary to previous observations shown in Figure 3, the addition of hlyC does not result in a decrease in output, and the observed pattern (where enhanced hlyBD expression is associated with higher secretion) is also retained. This shows that the decrease previously observed in Figure 3 may be due to the protein expression load on the cells.

[0017] Figure 5 shows a two-plasmid system for allowing the regulation of cargo output yield. Dividing the two circuits into separate plasmids enables the regulation of cargo output yield through a combination of promoter strength and copy number. This allows for the screening of optimal experimental conditions that allow for the optimization of yield without overloading the bacterial vector. The experiment evaluated the output of mScarlet in the T1SS two-plasmid system, where the mscarlet-hlyA fusion was controlled by promoters of increasing strength (2 (SEQ NO ID: 22), 4 (SEQ NO ID: 23), 6 (SEQ NO ID: 24)), and hlyBD was also controlled by the same increasing promoters. It was found that although the output yield was the highest in the strongest promoter combination (cargo-hlyA: 6, hlyBD: 6), it caused a cell load (see Figure 5B ). The output was evaluated in relative light units (RLU) of NanoLuc, and due to the HiBit tag at the N-terminus of the cargo, the luminescence of NanoLuc was proportional to the cargo in the supernatant; growth was recorded as the absorbance at 600 nm (see Figure 5C ). Detailed Description

[0018] To make the present invention more easily understood, some terms are first defined. Additional definitions are set forth throughout the detailed description.

[0019] As used herein, the term "non-native bacterium" refers to a bacterial (prokaryotic) cell that has been genetically modified or "engineered" such that it is altered relative to a naturally occurring cell. Such genetic modification can be, for example, the integration of additional genetic information into the cell, the modification of existing genetic information, or the deletion of existing genetic information. For example, this can be achieved by transfecting a recombinant plasmid into the cell or directly modifying the bacterial genome. In addition, bacterial cells can be genetically modified by chemical mutagenesis, for example, to achieve attenuation, which is well known to those skilled in the art. Thus, the term "non-native bacterium" can refer to both recombinantly modified bacterial strains and non-recombinantly modified bacterial strains.

[0020] As used herein, "heterologous polynucleotide" refers to a polynucleotide that has been introduced into an attenuated live Gram-negative bacterium, i.e., a polynucleotide that was not previously present. Thus, the attenuated live Gram-negative bacteria disclosed herein are recombinant bacterial strains. The heterologous polynucleotide in the context of the present invention can be a DNA molecule or an RNA molecule for delivery to a eukaryotic cell. The heterologous polynucleotide in the context of the present invention can encode a protein or peptide for delivery to a eukaryotic cell. The heterologous polynucleotide in the context of the present invention can encode an RNA molecule for delivery to a eukaryotic cell. The resulting RNA molecule or protein is also referred to herein as a "cargo" or "cargo molecule". In a particularly preferred embodiment, the DNA or RNA molecule to be encoded is a mammalian DNA or RNA molecule.

[0021] As used herein, the term "prophylactic treatment" refers to a medical procedure aimed at preventing rather than treating or curing an infection or disease. In the context of the present invention, this particularly applies to vaccine compositions. The term "prevention" as used herein is not intended to be absolute and can also include partial prevention of an infection or disease and / or one or more symptoms of said infection or disease. In contrast, the term "therapeutic treatment" refers to a medical procedure well known in the art aimed at treating or curing an infection or disease or its related symptoms.

[0022] The terms "tumor", "cancer", "malignancy" and "neoplasm" are used interchangeably and refer to a cell or population of cells whose growth, proliferation or survival (e.g., disorder of cell proliferation or differentiation) is higher than that of normal corresponding cells. Usually, the growth is uncontrolled. The term "malignancy" refers to invasion of nearby tissues. The term "metastasis" refers to the spread or dissemination of a tumor, cancer or neoplasm to other parts, locations or regions within the body of an object, where the site, location or region is different from the primary tumor or cancer. In one embodiment, the cancer is malignant. In an alternative embodiment, the cancer is non-malignant.

[0023] The term "effective amount" or "pharmaceutically effective amount" refers to an amount of an agent sufficient to provide the desired biological or therapeutic result. The result can be a reduction, improvement, alleviation, mitigation, delay and / or slowing of one or more signs, symptoms or causes of a disease, or any other desired change in a biological system. For cancer, an effective amount can include an amount sufficient to shrink a tumor and / or reduce the rate of tumor growth (e.g., inhibit tumor growth) or prevent or delay other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay the development of cancer or a tumor or extend survival or induce its stabilization.

[0024] In some embodiments, a therapeutically effective amount is an amount sufficient to prevent or delay recurrence. A therapeutically effective amount can be administered in one or more administrations. A therapeutically effective amount of an agent or combination can produce one or more of the following effects: (i) reducing the number of cancer cells; (ii) shrinking the tumor size; (iii) inhibiting, retarding, slowing, preferably preventing, to some extent, the invasion of cancer cells into peripheral organs; (iv) inhibiting (i.e., slowing, preferably preventing, to some extent) tumor metastasis; (v) inhibiting tumor growth; (vi) preventing or delaying tumorigenesis and / or recurrence; and / or (vii) alleviating, to some extent, one or more symptoms associated with cancer.

[0025] For example, for the treatment of a tumor, a "therapeutically effective amount" can induce the tumor to shrink by at least about 5% relative to a baseline measurement, such as at least about 10%, or about 20%, or about 60% or more. The baseline measurement can be derived from an untreated subject.

[0026] A therapeutically effective amount of a therapeutic compound can reduce the tumor size of a subject or improve the symptoms of a subject. One of ordinary skill in the art can determine these amounts based on factors such as the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration selected.

[0027] The terms "treatment" or "therapy" refer to the administration of an active agent for the purpose of curing, rehabilitating, slowing, alleviating, altering, remedying, improving, modifying, or affecting a condition (e.g., a disease) or the symptoms of a condition in a statistically significant manner, or preventing or delaying the occurrence of the symptoms, complications, or biochemical markers of a disease, or arresting or inhibiting the further development of a disease, condition, or disorder.

[0028] As used herein, the term "subject" is intended to include human and non-human animals. Preferred subjects include human patients in need of enhancing an immune response. The method is particularly suitable for treating human patients suffering from conditions that can be treated by enhancing an immune response. In certain embodiments, the method is particularly suitable for in vivo treatment of neoplastic or infectious diseases.

[0029] The use of alternatives (e.g., "or") should be understood to mean one, both, or any combination of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to refer to "one or more" of any recited or listed components.

[0030] As used herein, "about" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one standard deviation or greater than one standard deviation in accordance with the practice in the art. Alternatively, "about" can represent a range of up to 20%. When a particular value is provided in the application and claims, unless otherwise stated, the meaning of "about" should be considered to be within the acceptable error range of that particular value.

[0031] The inventors of the present invention unexpectedly found that the hlyCABD operon can be modified in such a way (as Figure 1 shown) that it enables the delivery of cargo molecules into the interstitial space between eukaryotic cells ( Figure 2 ). To provide more rational control and standardized application of this system, the inventors of the present invention performed a rational reconstruction of the hly genomic island. As a starting point, the hly operon was divided into two segments: a first segment ("cargo segment") and a second segment ("structural segment"). The process of separating the four genes of the hlyCABD operon into two segments transcriptionally separates the genes involved in cargo production and / or activation (hlyC, hlyA) from the genes involved in secretion (hlyB, hlyD). The toxin sequence of hlyA can be replaced with a reporter gene (e.g., mScarlet) or a heterologous nucleotide that actually encodes any other cargo molecule, while retaining the translocation peptide (HlyAs). The optimal transcriptional levels of the cargo and secretion modules were screened by manipulating the strength of the promoters upstream of each segment. The results described herein show that dividing the operon into two segments results in functional translocation, and that functional translocation increases as the transcriptional levels of the two segments increase. Accordingly, the present invention relates to an attenuated live Gram-negative bacterium comprising a modified hly operon.

[0032] Some Gram-negative bacteria use a type I secretion system (T1SS) to translocate proteins across their inner and outer membranes into the extracellular environment. Among these T1SSs, the Escherichia coli α-hemolysin (HlyA) secretion system has been the most fully characterized. Using the T1SS, proteins and other cargo molecules can be actively presented to eukaryotic cells of the host immune system by being exported from the bacterial cytoplasm, rather than being encountered by eukaryotic cells only after the bacteria are phagocytosed and degraded. HlyA is a bacterial toxin and virulence factor. The secretion and activation of HlyA are determined by the hlyCABD operon. Briefly, once HlyA is transcribed and translated, there are three components that regulate the export of HlyA: HlyB, HlyD, and TolC. HlyB and HlyD are inner membrane proteins (visible in the HlyB-HlyD complex anchored to the inner membrane of Gram-negative bacterial cells), while TolC is located in the outer membrane of Gram-negative bacterial cells. HlyA carries a translocation signal sequence at its C-terminus, called HlyAs. Recognition of HlyAs by the HlyB-HlyD complex induces contact with TolC, thereby forming a periplasmic export channel between the inner and outer membranes. HlyC plays a role in the activation of HlyA. Thus, replacing the HlyA toxin with a heterologous nucleotide encoding a protein or other cargo molecule enables the specific heterologous protein or other target molecule to be exported from the carrier bacterium into the extracellular environment via the C-terminal HlyAs sequence.

[0033] Accordingly, in a first aspect, the present invention provides an attenuated live Gram-negative bacterium comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is divided into a first segment and a second segment, each segment being operably linked to an independently controlled promoter, wherein the first segment comprises a heterologous polynucleotide encoding a cargo molecule upstream of the hlyAs translocation sequence, wherein the heterologous polynucleotide encoding the cargo molecule replaces the hlyA gene, and wherein the second segment comprises the hly genes involved in secretion.

[0034] Accordingly, it is contemplated that the attenuated live Gram-negative bacterium of the present invention can serve as an effective and reliable method for delivering or exporting cargo molecules from the bacterial cytoplasm into the extracellular environment, including the interstitial space between eukaryotic cells. Thus, the bacterial strains disclosed herein are recombinant strains comprising a modified hlyCABD operon that contains a heterologous polynucleotide encoding a cargo molecule. Accordingly, the heterologous polynucleotide has a nucleotide coding structure that permits its transcription and, if the cargo molecule is a protein, permits its subsequent translation into the encoded cargo molecule.

[0035] It is contemplated that the modified hlyCABD operon is divided into a first segment and a second segment, each segment being operably linked to an independently controlled promoter.

[0036] It is contemplated that a first segment (“cargo region”) contains a heterologous polynucleotide encoding a cargo molecule, the heterologous polynucleotide being upstream of the hlyA s translocation sequence.

[0037] As used herein, the term “cargo” is well known to those of skill in the art and refers to a specific target molecule that is intended to be translocated, delivered, transported, or exported from one location to another. Specifically, the cargo molecule can be translocated from the bacterial cytoplasm to the extracellular environment surrounding a eukaryotic cell. In a preferred embodiment, the cargo molecule is a protein and / or a peptide. The cargo molecule can be a heterologous protein that is not naturally present in the carrier bacterial cell. The cargo peptide and / or protein can be a therapeutic peptide and / or a therapeutic protein. While it is contemplated that the cargo molecule of the present invention can be a protein or a peptide, other cargo types include DNA and RNA molecules. Thus, in another embodiment, the cargo molecule is an RNA molecule. The terms “RNA” and “ribonucleic acid” as used herein are used interchangeably and refer to a nucleic acid composed of the ribonucleic acid bases uracil, adenine, guanine, and cytosine. These terms and concepts are well known to those of skill in the art. Types of RNA molecules include, for example, messenger RNA (mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), transfer RNA (tRNA), self-amplifying RNA (saRNA), and ribosomal RNA (rRNA). Thus, the RNA cargo molecule can be an mRNA molecule. The terms “mRNA” and “messenger RNA” as used herein are used interchangeably and refer to a single-stranded RNA molecule involved in protein synthesis. The mRNA molecule of a eukaryote is transcribed from DNA in the nucleus of the eukaryotic cell and subsequently exported from the nucleus to the cytoplasm of the eukaryotic cell, where the mRNA molecule is translated into a protein. The bacterial mRNA molecule is transcribed from non-compartmentalized DNA and is translated in a transcription-coupled manner in the cytosol. These terms and concepts are well known to those of skill in the art. The RNA molecule is transcribed and translated within the bacterium itself. For example, an attenuated live Gram-negative bacterium can encode up to 10 different heterologous mRNA molecules, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different heterologous mRNA molecules. The cargo mRNA molecule itself can encode a peptide and / or a protein, whereby the peptide and / or protein can be a therapeutic peptide and / or a therapeutic protein.

[0038] In one embodiment, when the cargo is a therapeutic peptide and / or a therapeutic protein, the therapeutic peptide and / or the therapeutic protein can be a cytokine, a chemokine, an antibody or a functional fragment thereof, a cytotoxic agent, an anti-cancer agent, or any combination thereof. The invention disclosed herein provides an attenuated live Gram-negative bacterium, in which the cargo molecule is expressed inside the bacterium itself before being exported. For example, the attenuated live Gram-negative bacterium can encode up to 10 different cargo (protein) molecules, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different cargo (protein) molecules.

[0039] It is also contemplated to fuse a heterologous polynucleotide encoding a cargo molecule with the hlyAs sequence encoding the translocation peptide HlyAs. In one embodiment, when the cargo molecule is a protein or a peptide, the HlyAs translocation peptide is located at the C-terminus of the cargo molecule. The length of the HlyAs protein can be about 50 to 220 amino acids. In some embodiments, the length of the HlyAs protein is 218 amino acids. Through site-directed mutagenesis, CD, and NMR spectroscopy studies, several structural and sequence motifs in HlyAs have been identified as being important for its signal function [Holland, I.B., et al., 1990; Koronakis, V., 1989; Jarchau, T., et al., 1994]. The terms "translocation sequence", "signal sequence", and "target sequence" as used herein can be used interchangeably and refer to a gene encoding a translocation peptide or protein that is recognized by the cellular export machinery and is targeted for export or secretion from the cell. Translocation peptides are typically found at the N-terminus or C-terminus of a peptide or protein that is intended to translocate from one location to another. The translocation sequence typically encodes a peptide with a specific amino acid sequence or motif that can be recognized by the cellular export machinery. For the hlyCABD operon, the HlyAs translocation peptide is recognized by the HlyB and HlyD structural proteins, which bind to TolC to form a periplasmic channel, enabling HlyAs (and any cargo fused thereto, possibly) to translocate through the inner and outer membranes of the attenuated live Gram-negative bacterium.

[0040] In one embodiment, the heterologous polynucleotide encoding the cargo molecule is located upstream of the HlyAs sequence. In another embodiment, the heterologous polynucleotide encoding the cargo molecule is located downstream of an independently controlled promoter. In yet another embodiment, the heterologous polynucleotide encoding the cargo molecule is located upstream of the HlyAs sequence and downstream of an independently controlled promoter.

[0041] In an alternative embodiment, the hlyCABD operon may further comprise the hlyC gene. HlyC is associated with the activation of HlyA. However, according to the literature, the region at the 3' end of HlyC can affect the secretion yield. Therefore, in another embodiment, the hlyCABD operon may further comprise a functional fragment or portion of the hlyC gene. In one embodiment, the hlyC gene or its functional fragment is located upstream of the heterologous polynucleotide encoding the cargo molecule, and the heterologous polynucleotide encoding the cargo molecule is encoded upstream of the translocation sequence of hlyAs. In another embodiment, the hlyC gene or its functional fragment is located downstream of an independently controlled promoter. In yet another embodiment, the hlyC gene or its functional fragment is located upstream of the heterologous polynucleotide encoding the cargo molecule, and the heterologous polynucleotide encoding the cargo molecule is encoded upstream of the translocation sequence of hlyAs and downstream of an independently controlled promoter.

[0042] In an alternative embodiment, after secretion, the cargo molecule retains the HlyAs translocation peptide. In an alternative embodiment, the HlyAs translocation peptide is removed or cleaved from the cargo molecule after secretion.

[0043] It is contemplated that the secretion of any given cargo molecule can be optimized. In one embodiment, when the cargo molecule is a peptide or protein, the folding rate of the cargo can be modified, as the literature indicates that cargo molecules exhibiting a lower folding rate experience a higher secretion rate. In another embodiment, the translation efficiency of the cargo molecule can be modified by adjusting the ribosome binding sequence. In yet another embodiment, the coding sequence can be engineered such that one or more codons within the heterologous polynucleotide encoding the cargo molecule can be changed without changing the encoded amino acid due to the redundancy of the genetic code (synonymous codon change). Those skilled in the art will recognize that the folding rate, translation efficiency, and coding sequence can be optimized for each peptide, protein, or gene involved and will depend on the nature of the cargo molecule (gene, peptide, or protein cargo molecule) and its intended use.

[0044] It is contemplated that the second segment (“structural segment”) contains the hly genes involved in secretion.

[0045] In the case of the hlyCABD operon, the hly genes involved in secretion are hlyB and hlyD. Therefore, in one embodiment, the second segment contains the hlyB gene and the hlyD gene. The hlyB gene and the hlyD gene are also referred to as T1SS structural genes. In one embodiment, the hlyB gene is located upstream of the hlyD gene. In another embodiment, the hlyB gene is located downstream of an independently controlled promoter. In yet another embodiment, the hlyB gene is located upstream of the hlyD gene and downstream of an independently controlled promoter.

[0046] The inventors of the present invention unexpectedly found that dividing the operon into two segments results in functional translocation, and that as the transcription levels of the two segments increase, the functional translocation also increases. Accordingly, the engineered hlyCABD operon of the present invention is divided into two segments: a first segment and a second segment, each segment being operably linked to its own independently controlled promoter, wherein the first segment comprises a heterologous polynucleotide encoding a cargo molecule upstream of the hlyA translocation sequence, wherein the heterologous polynucleotide encoding the cargo molecule replaces the hlyA gene, and wherein the second segment comprises the hly genes involved in secretion. To allow transcription and translation of the heterologous polynucleotide and production of the cargo molecule in the attenuated live Gram-negative bacterium, the heterologous polynucleotide is operably linked to an independently controlled promoter. In addition, to allow transcription and translation of the hly genes involved in secretion, the hly genes involved in secretion are operably linked to an independently controlled promoter. As used herein, the term "independently controlled promoter" refers to a promoter that is controlled by regulatory elements that are different from the regulatory elements of another promoter in the system. Specifically, the promoter controlling the expression of the first segment may be different from the promoter controlling the expression of the second segment, or may have a different regulatory mechanism therewith.

[0047] Temporal control of expression can be achieved by using different promoters, including invasion-related SPI-1, SPI-4 or flagella-related promoters. Suitable promoters can include, but are not limited to, invF, hilA, hilD, sicA, siiE, flhDC and fliC.

[0048] In a preferred embodiment, the independently controlled promoter is a strong promoter such that when a strong promoter is used, high transcription rates are initiated. The terms "strong promoter" or "active promoter" as used herein are used interchangeably and refer to a promoter that under its control produces a high gene transcription rate. Genes regulated by strong promoters recruit RNA polymerase more frequently and thus produce more mRNA than genes regulated by weak promoters and thus more product protein. While any strong promoter that achieves the functions disclosed herein is suitable, specific examples of suitable strong promoters include but are not limited to the ptrc promoter, the ptet promoter, the pcon5 promoter, the pBAD promoter, the placUV5 promoter, invasion-related promoters (such as SPI-1, SPI-4 or flagella), intracellular promoters (SPI-2), host cell cytosol promoters or the pTac promoter. Other suitable examples include but are not limited to uphT (glucose-6-phosphate), frubKA (fructose). SPI-1 is an invasion-related promoter that is active outside the host cell and when the bacterium attempts to invade the host cell. Additional promoters for use in the present invention may include but are not limited to the trc promoter, the tac promoter, the trp promoter, the lac operon promoter, the lac / tac promoter, the tac / trc promoter, the trp / lac promoter, the bad / ara promoter, the ssaG promoter, the pagC promoter, the nirB promoter, the dps promoter or the spv promoter. The term "cytosol promoter" as used herein refers to an intracellular promoter in bacteria. Host cell cytosol promoters for use in the present invention may include but are not limited to uhpT, mntH, entC, fhuE, iroN, fepB, fepA, fhuA, sitA, stn3250, sufA, yjjZ, soxS, sfbA. In one embodiment, the SPI-2 gene is an ssa gene. Suitable promoters may include but are not limited to ssaV, ssaJ, ssaU, ssaK, ssaL, ssaM, ssaO, ssaP, ssaQ, ssaR, ssaS, ssaT, ssaD, ssaE, ssaG, ssaI, ssaC and ssaH. Other vacuole promoters include PipB2, zinT, mtgC. In a preferred embodiment, PipB2 and ssaG can be used for intracellular delivery. PipB2 is a strong SPI-2-dependent promoter relative to other SPI-2 promoters. The SPI-2 promoter can also be used to activate the expression of the type 1 secretion system (T1SS) that promotes the export of hemolysin from Listeria. The strength of the promoter selected for each segment can be manipulated to enhance the secretion efficiency of the cargo molecule (Figures 3 and 4). Table 1 details the promoters used in the screening.

[0049]

[0050]

[0051] Table 1: Promoters used in the screening and their corresponding sequences. Promoter sequences with the -10 consensus region (variable) highlighted in bold are shown with double underlines, riboJ or vtmoJ ribozyme sequences are shown with dotted underlines. On the sequence of promoter 2, the BBa_B1006 terminator is shown with a single underline.

[0052] The bacterium of the present invention is an attenuated live Gram-negative bacterium. Examples of attenuated live Gram-negative bacteria for use in the present invention include, but are not limited to, Salmonella, Escherichia coli, Shigella, Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, Legionella, Chlamydia, and Yersinia. However, in one embodiment, the attenuated live Gram-negative bacterium is Salmonella. The Salmonella can be Salmonella Typhi or Salmonella Typhimurium.

[0053] In another embodiment of the present invention, the attenuated live Gram-negative bacterium is a genetically engineered non-natural bacterium.

[0054] Accordingly, the present invention discloses an attenuated live Gram-negative bacterium that has been genetically altered to produce a bacterial strain capable of effectively delivering various cargo molecules. Those skilled in the art will understand that genes can be mutated by many well-known methods in the art, such as homologous recombination with a recombinant plasmid targeting the target gene. In this case, the engineered gene homologous to the target gene is integrated into a suitable nucleic acid vehicle (such as a plasmid or phage), and then transfected into the target cell. Then the homologous engineered gene is recombined with the native gene to replace the native gene or mutate the native gene to achieve the desired inactivating mutation. Such modification can occur in the coding part or any regulatory part of the gene, such as the promoter region. Those skilled in the art will understand that any suitable genetic modification technique can be used to mutate the target gene, such as the CRISPR / Cas system, such as CRISPR / Cas9, to produce the bacterial strains disclosed herein. Table 2 details the sequences used for constructing the expression plasmid.

[0055]

[0056]

[0057]

[0058]

[0059]

[0060] Table 2: Sequences for constructing expression plasmids, including conserved regions for amplifying DNA blocks and BsaI-dependent cleavage sites for GoldenGate assembly.

[0061] Thus, a variety of methods and techniques for genetically engineering bacterial strains will be well-known to those skilled in the art. These techniques include those required for introducing heterologous genes into bacteria by chromosomal integration or by introducing stable autosomal self-replicating genetic elements. Exemplary methods for genetically modifying (also referred to as "transforming" or "engineering") bacterial cells include phage infection, transduction, conjugation, lipid transfection, or electroporation. General discussions of these and other methods in molecular and cell biochemistry can be found in standard textbooks such as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HarBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996), which are hereby incorporated by reference.

[0062] In a preferred embodiment, the attenuated live Gram-negative bacterium is Salmonella. The attenuated live Gram-negative bacterium can be selected from the group consisting of: Ty21a, CVD 908-htrA, CVD 909, Ty800, M01ZH09 (also known as ZH9), x9633, x639, x9640, x8444, DTY88, ZH9PA, MD58, WT05, ZH26, SL7838, SL7207, VNP20009, A1-R, or any combination thereof. In a more preferred embodiment, the attenuated live Gram-negative bacterium is M01ZH09 (also known as ZH9). These attenuated live strains are readily available and easily identifiable, and are widely used by those skilled in the art. For example, EP2801364A1 discloses Ty21a, CVD 908-htrA, CVD 909, Ty800, M01ZH09, χ9633, χ9640, and χ8444. In addition, EP3917565A1 discloses in detail the ZH9 strain and its derivatives, including ZH9PA. More references regarding these strains can be found in the literature, particularly Petrovska 2004, Hindle 2002, Lehouritis 2017, and Kimura 2010. Also intended to be included are any derivatives or variants of the strains, including genetically engineered or genetically modified strains.

[0063] The genetically engineered non-native bacterium can also contain one or more gene cassettes. Such gene cassettes can be used to deliver additional prokaryotic molecules to support the function of the genetically engineered non-native bacterium in modulating the immune system, or to support the activity of the cargo molecule.

[0064] The present invention provides a means of delivering cargo molecules to the extracellular space (including the interstitial space) between eukaryotic cells. Accordingly, the present invention provides a bacterial delivery system that has broad applicability in numerous disease areas. Those skilled in the art will appreciate that such a system has significant and broad therapeutic benefits. The heterologous polypeptide may encode cargo molecules that can be therapeutic peptides, therapeutic proteins, and / or heterologous antigens (depending on the indication to be treated). In a preferred embodiment, the therapeutic peptide or protein is a cytokine, chemokine, antibody or functional fragment thereof, cytotoxic agent, cancer agent, or any combination thereof. More preferably, the resulting therapeutic protein can be IL-15, IL-21, CXCL9, CXCL10, IL-18, IL-27, IFNγ, IFNα, IFNβ, IL-1, or any combination thereof. In one embodiment, the attenuated live Gram-negative bacterium contains a modified hlyCABD operon encoding a therapeutic cargo molecule that is designed to be secreted into the extracellular environment between eukaryotic cells. As used herein, the terms "extracellular environment", "extracellular space", "extracellular compartment", or "extracellular milieu" are used interchangeably and refer to the region within a multicellular organism that is external to cells, i.e., outside the plasma membrane, and occupied by the extracellular matrix. The extracellular environment includes three compartments: the interstitial compartment, the intravascular compartment, and the transcytotic compartment.

[0065] In one embodiment, the cargo molecules are designed to be secreted into the interstitial space. As used herein, the terms "interstitial compartment", "interstitial milieu", "interstitial space", "tissue space", or "interstitial milieu" are used interchangeably and refer to the space surrounding tissue cells (i.e., the space outside blood vessels, lymphatic vessels, and parenchymal cells), also known as the tissue microenvironment. The interstitial space consists of two main phases: interstitial fluid, which provides the direct microenvironment between eukaryotic cells, and structural molecules that include the extracellular matrix. The eukaryotic cells can be mammalian cells. In a preferred embodiment, the eukaryotic cells are human cells. When the eukaryotic cells are human cells, the target cells can be cancerous human cells or non-cancerous human cells.

[0066] The tissue microenvironment is associated with solid and hematological cancers. As used herein, the terms "tumor microenvironment" or "TME" are used interchangeably and refer to the local environment surrounding a tumor, the tumor interstitial space, and interstitial fluid. The TME is generated by the tumor and is dominated by tumor-induced interactions, but may also include immune effector cells that have been recruited to the tumor, fibroblasts, signaling molecules, and blood vessels. Accordingly, it is envisioned that attenuated live Gram-negative bacteria can be engineered to deliver therapeutically relevant proteins into the interstitial space of the TME of a subject with a tumor.

[0067] In one embodiment of the present invention, the attenuated live Gram-negative bacterium is administered intratumorally, peritumorally, intravenously, intraperitoneally, subcutaneously, intradermally, or orally. In a more preferred embodiment, the attenuated live Gram-negative bacterium is administered intratumorally. However, in certain cases, other administration methods may also be considered. Thus, in certain cases, the attenuated live Gram-negative bacterium of the present invention can be administered by injection, infusion, continuous infusion, intradermally, intraarterially, intralesionally, intravaginally, intrarectally, intramuscularly, subcutaneously, subconjunctivally, mucosally, intrapericardially, intraumbilically, intravitreally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, through a catheter, through lavage, or by other methods known to those of ordinary skill in the art or any combination of the foregoing methods (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990).

[0068] The amount of the attenuated live Gram-negative bacterium administered to the subject is sufficient to deliver the cargo molecule to the interstitial space at a high enough concentration to produce the desired effect. Those skilled in the art will readily understand that the precise amount to be administered will depend on many factors, such as the disease to be treated and the medical history of the subject to be treated.

[0069] The dosage of the attenuated live Gram-negative bacterium can be from 10 5 to 10 12 CFU, where CFU is colony forming unit. For example, suitable dosages can be from 10 5 to 10 6 CFU, 10 5 to 10 7 CFU, 10 5 to 10 8 CFU, 10 5 to 10 9 CFU, 10 5 to 10 10 CFU, 10 5 to 10 11 CFU, 10 6 to 10 7 CFU, 10 6 to 10 8 CFU, 10 6 to 10 9 CFU, 10 6 to 10 10 CFU, 10 6 to 10 11 CFU, 10 6 to 10 12 CFU, 10 7 to 108 CFU, 10 7 to 10 9 CFU, 10 7 to 10 10 CFU, 10 7 to 10 11 CFU, 10 7 to 10 12 CFU, 10 8 to 10 9 CFU, 10 8 to 10 10 CFU, 10 8 to 10 11 CFU, 10 8 to 10 12 CFU, 10 9 to 10 10 CFU, 10 9 to 10 11 CFU, 10 9 to 10 12 CFU, 10 10 to 10 11 CFU, 10 10 to 10 12 CFU or 10 11 to 10 12 CF. The attenuated live Gram - negative bacteria can be administered as a single dose or multiple doses. The specific dose to be administered should be understood to depend on the specific cargo molecule to be delivered and the specific indication to be treated.

[0070] In one embodiment, the attenuated live Gram - negative bacteria of the present invention are contemplated to allow delivery of a therapeutically relevant cargo molecule into the interstitial space between eukaryotic cells of the TME of a subject having a tumor. Thus, the attenuated live Gram - negative bacteria disclosed herein can be used for therapeutic purposes. For example, the attenuated live Gram - negative bacteria can be used to treat, reduce, inhibit, prevent, prevent recurrence or control a disease. In a preferred embodiment, the disease is a human disease. In a more preferred embodiment, the disease can be a neoplastic disease, an infectious disease, a cardiovascular disease, a neurodegenerative disease, a gastrointestinal disease, a respiratory disease, a kidney disease, a liver disease, an autoimmune disease, an inflammatory disease or a genetic disease. In a preferred embodiment, the attenuated live Gram - negative bacteria are used to treat, reduce, inhibit, prevent, prevent recurrence or control a neoplastic disease or an infectious disease.

[0071] When the disease to be treated is a neoplastic disease, the neoplastic disease can be related to a solid tumor or a hematological tumor. In a specific aspect, the neoplastic disease is related to a cancer selected from prostate cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, brain cancer, mesothelioma, hepatocellular carcinoma, lymphoma, leukemia, gastric cancer, endometrial cancer, vulvar cancer, vaginal cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer or sarcoma.

[0072] Neoplasms, tumors, and cancers include benign, malignant, metastatic, and non-metastatic types, and include neoplasms, tumors, or cancers at any stage (I, II, III, IV, or V) or grade (G1, G2, G3, etc.), or neoplasms, tumors, cancers, or metastases that are progressing, worsening, stable, or in remission. Cancers that can be treated according to the present invention include, but are not limited to, cells or neoplasms of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testicle, tongue, or uterus. In addition, cancers can specifically be the following histological types, but are not limited to the following: malignant neoplasms; carcinomas; undifferentiated carcinomas; giant cell and spindle cell carcinomas; small cell carcinomas; papillary carcinomas; squamous cell carcinomas; lymphoepithelial carcinomas; basal cell carcinomas; pilomatrix carcinomas; transitional cell carcinomas; papillary transitional cell carcinomas; adenocarcinomas; malignant gastrinomas; cholangiocarcinomas; hepatocellular carcinomas; combined hepatocellular and cholangiocarcinomas; trabecular adenocarcinomas; adenoid cystic carcinomas; adenocarcinoma in adenomatous polyps; familial adenocarcinoma of the colon; solid carcinomas; malignant carcinoid tumors; bronchioloalveolar adenocarcinomas; papillary adenocarcinomas; chromophobe cell carcinomas; eosinophilic cell carcinomas; eosinophilic adenocarcinomas; basophilic cell carcinomas; clear cell adenocarcinomas; granular cell carcinomas; follicular adenocarcinomas; papillary follicular adenocarcinomas; unencapsulated sclerosing carcinomas; adrenocortical carcinomas; endometrioid carcinomas; adnexal carcinomas; apocrine adenocarcinomas; sebaceous gland carcinomas; ceruminous gland carcinomas; mucoepidermoid carcinomas; cystadenocarcinomas; papillary cystadenocarcinomas; papillary serous cystadenocarcinomas; mucinous cystadenocarcinomas; mucinous adenocarcinomas; signet ring cell carcinomas; invasive ductal carcinomas; medullary carcinomas; lobular carcinomas; inflammatory carcinomas; Paget's disease of the breast; acinar cell carcinomas; adenosquamous carcinomas; adenocarcinomas with squamous metaplasia; malignant thymomas; malignant ovarian stromal tumors; malignant thecomas; malignant granulosa cell tumors; malignant arrhenoblastomas; sertoli cell carcinomas; malignant stromal cell tumors; malignant lipomas; malignant paragangliomas; malignant extra-mammary paragangliomas; pheochromocytomas; glomangiosarcomas; malignant melanomas; amelanotic melanomas; superficial spreading melanomas; malignant melanoma within giant pigmented nevi; epithelioid cell melanomas; malignant blue nevi; sarcomas; fibrosarcomas; malignant fibrous histiocytomas; myxosarcomas; liposarcomas; leiomyosarcomas; rhabdomyosarcomas; embryonal rhabdomyosarcomas; alveolar rhabdomyosarcomas; stromal sarcomas; mixed tumors; Mullerian duct mixed tumors; nephroblastomas; hepatoblastomas; carcinosarcomas; malignant mesenchymomas; malignant Brenner tumors; malignant phyllodes tumors; synovial sarcomas; malignant mesotheliomas; dysgerminomas; embryonal carcinomas; malignant teratomas; malignant struma ovarii; choriocarcinomas; malignant mesonephromas; angiosarcomas; malignant hemangioendotheliomas; Kaposi sarcomas; malignant hemangiopericytomas; lymphangiosarcomas; osteosarcomas; juxtacortical osteosarcomas; chondrosarcomas; malignant chondroblastomas; mesenchymal chondrosarcomas; giant cell tumors of bone; Ewing's sarcomas; malignant odontogenic tumors; ameloblastic odontosarcomas; malignant ameloblastomas; ameloblastic fibrosarcomas; malignant pinealomas; chordomas;Malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant schwannoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; small lymphocytic malignant lymphoma; diffuse large cell malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other specified non-Hodgkin lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocytic leukemia; myeloid sarcoma; and hairy cell leukemia. Preferably, the neoplastic disease can be a tumor associated with a cancer selected from the group consisting of prostate cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colorectal cancer, pancreatic cancer, brain cancer, hepatocellular carcinoma, lymphoma, leukemia, gastric cancer, endometrial cancer, vulvar cancer, vaginal cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, head and neck cancer, skin cancer and soft tissue sarcoma and / or other forms of cancer. The tumor can be a metastatic tumor or a malignant tumor.;

[0073] In a preferred embodiment, the neoplastic disease is associated with a cancer selected from bladder cancer, prostate cancer, lung cancer, mesothelioma, hepatocellular carcinoma, melanoma, esophageal cancer, gastric cancer, endometrial cancer, vulvar cancer, vaginal cancer, cervical cancer, ovarian cancer, colorectal cancer, head and neck cancer or breast cancer.

[0074] In a second aspect, the present invention provides a vaccine composition comprising an attenuated live Gram-negative bacterium, the attenuated live Gram-negative bacterium comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is divided into a first segment and a second segment, each segment operably linked to an independently controlled promoter, wherein the first segment comprises a heterologous polynucleotide encoding a cargo molecule upstream of the hlyA translocation sequence, wherein the heterologous polynucleotide encoding the cargo molecule replaces the hlyA gene, and wherein the second segment comprises hly genes involved in secretion.

[0075] In one embodiment, the vaccine composition of the present invention can be used for therapeutic purposes. For example, the attenuated live Gram-negative bacterium can be used to treat, reduce, inhibit, prevent, prevent recurrence or control a disease.

[0076] Specifically contemplated is that the vaccine compositions disclosed herein can be used to treat, reduce, inhibit, prevent recurrence or control infectious diseases, such as diseases caused by bacteria, viruses, parasites or fungi. In such cases, the heterologous polynucleotides of the present invention can encode antigens of pathogens of specific infectious diseases so as to generate an immune response in a host. Alternatively, it is contemplated that the vaccine compositions disclosed herein can be used as cancer vaccines. In such cases, the vaccine composition comprises a Gram-negative bacterium that contains a heterologous polynucleotide encoding a cancer antigen capable of generating an immune response in a host. Thus, it can be understood that various cancers and infectious diseases can be prevented / treated by using the bacteria and methods disclosed herein. In other cases, the heterologous polynucleotide can encode siRNA or shRNA molecules, which are designed to enhance immune anti-infection functions or tissue anti-infection defenses.

[0077] The vaccine compositions of the present invention may also comprise an adjuvant, a pharmaceutically acceptable carrier or an excipient.

[0078] As used herein, "pharmaceutically acceptable carrier / adjuvant / diluent / excipient" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (such as antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes and other substances and combinations thereof known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Examples include but are not limited to disodium hydrogen phosphate, soya peptone, potassium dihydrogen phosphate, ammonium chloride, sodium chloride, magnesium sulfate, calcium chloride, sucrose, borate buffer, sterile saline solution (0.9% NaCl) and sterile water.

[0079] Suitable aqueous and non-aqueous carriers that can be employed in the vaccine compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Appropriate fluidity can be maintained, for example, by using coating materials (such as lecithin), by maintaining the required particle size in the case of dispersions, and by using surfactants.

[0080] The vaccine composition disclosed by the present invention may further contain adjuvants such as preservatives, wetting agents, emulsifying agents, dispersing agents, etc. The prevention of the presence of unwanted microorganisms can be ensured by the above sterilization procedures and by including various antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, etc.). It may also be desirable to include isotonic agents in the composition, such as sugars, sodium chloride, etc. In addition, the absorption of injectable pharmaceutical forms can be prolonged by including agents that delay absorption, such as aluminum monostearate and gelatin. The vaccine composition may also optionally include additional therapeutic agents known to be effective against, for example, infectious diseases or neoplastic diseases. Therefore, the vaccine composition disclosed herein may also contain antiretroviral drugs, antibiotics, antifungal drugs, antiparasitic drugs, and anticancer agents.

[0081] The vaccine composition may also contain additional components for enhancing the immune response of the subject after administration. Examples of such additional components include, but are not limited to: aluminum salts such as aluminum hydroxide, aluminum oxide, and aluminum phosphate, oil-based adjuvants such as Freund's complete adjuvant and Freund's incomplete adjuvant, mycolate-based adjuvants (such as trehalose dimycolate), bacterial lipopolysaccharide (LPS), peptidoglycan (such as murein, mucopeptide, or glycoprotein, such as N-Opaca, muramyl dipeptide [MDP], or MDP analogs), proteoglycans (such as those extracted from Klebsiella pneumoniae), streptococcal preparations (such as OK432), muramyl dipeptide, immunostimulating complexes ("Iscoms" disclosed in EP109942, EP180564, and EP231039), saponins, DEAE-dextran, neutral oils (such as miglyol), vegetable oils (such as peanut oil), liposomes, polyols, Ribi adjuvant system (see, for example, GB-A-2189141), vitamin E, carbomer, interferons (such as IFN-α, IFN-γ, or IFN-β), or interleukins, especially those interleukins that stimulate cell-mediated immunity (such as IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, and IL-17).

[0082] The attenuated live Gram-negative bacterium of the vaccine composition disclosed herein may include any one or any combination of the characteristics of the attenuated live Gram-negative bacterium disclosed herein. In a third aspect, the present invention provides a method of treating, preventing, inhibiting, preventing recurrence or controlling a disease in a subject, the method comprising administering to the subject an attenuated live Gram-negative bacterium comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is divided into a first segment and a second segment, each segment being operably linked to an independently controlled promoter, wherein the first segment comprises a heterologous polynucleotide encoding a cargo molecule upstream of the hlyAs translocation sequence, wherein the heterologous polynucleotide encoding the cargo molecule replaces the hlyA gene, and wherein the second segment comprises hly genes involved in secretion.

[0083] The method of treating, preventing, inhibiting, preventing recurrence or controlling a disease in a third aspect may include one or more of the foregoing embodiments for any of the foregoing aspects.

[0084] In a fourth aspect, the present invention provides a method of delivering a therapeutic molecule to the interstitial space between eukaryotic cells in the tumor microenvironment of a subject having a tumor, the method comprising the steps of: i) modifying an attenuated live Gram-negative bacterium comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is divided into a first segment and a second segment, each segment being operably linked to an independently controlled promoter, wherein the first segment comprises a heterologous polynucleotide encoding a cargo molecule upstream of the hlyAs translocation sequence, wherein the heterologous polynucleotide encoding the cargo molecule replaces the hlyA gene, and wherein the second segment comprises hly genes involved in secretion, and ii) administering the modified Gram-negative bacterium to a subject in need thereof.

[0085] In one embodiment of the fourth aspect of the present invention, the therapeutic molecule may be a protein or a peptide. In another embodiment of the present invention, the therapeutic molecule may be an RNA molecule that is subsequently translated into a protein or a peptide. The term "therapeutic molecule" refers to any molecule that can cause a reduction, improvement, alleviation, mitigation, delay and / or slowdown of one or more signs, symptoms or causes of a disease, or any other desired change in a biological system. For example, in the context of cancer, the therapeutic molecule may be a molecule that causes a reduction in tumor size.

[0086] The method of delivering a therapeutic molecule to the interstitial space between eukaryotic cells in the tumor microenvironment of a subject having a tumor in a fourth aspect may include one or more of the foregoing embodiments described for any of the foregoing aspects.

[0087] The attenuated live Gram-negative bacteria containing the engineered hylCABD operon of the present invention may also have applications that allow for the safe, effective, and reliable delivery of RNA molecules into target eukaryotic cells. Thus, in one embodiment, a method of using the engineered attenuated live Gram-negative bacteria disclosed herein to deliver RNA molecules into eukaryotic cells is provided. As used herein, the term "bacterial transfection" refers to the process of transducing genetic material from bacteria (e.g., Salmonella) into mammalian cells. Specifically, the term "bacterial transfection" in the context of the present invention refers to the use of attenuated live Gram-negative bacteria to deliver RNA molecules into the cytosol of eukaryotic cells after delivering the attenuated live Gram-negative bacteria to the target eukaryotic cells. The SPI-2 promoter can be used to activate the expression of a type I secretion system (T1SS) that promotes the export of hemolysin from Listeria (e.g., releasing bacterial cells from the vacuole into the cytoplasm of eukaryotic cells, thereby enabling the bacteria to enter the cytoplasm of eukaryotic cells).

[0088] Examples

[0089] Construct a combinatorial library with different promoters controlling the expression of T1SS cargo and structural genes

[0090] To find the optimal expression ratio between the secretory element (e.g., mScarlet fused to the T1SS signal peptide hlyAs, fused at the C-terminus) and the structural elements (i.e., HlyB and HlyD), these two parts are separated by a bidirectional terminator (BBa_B1006) and independently controlled by promoters (pro1, proA, proB, proC) of different strengths. In addition, it has been reported that the gene hlyC encoded upstream of hlyA in the native genomic island contains coding regions that affect the secretion efficiency of hlyA or any other cargo. Therefore, two variants of the secretory part were generated: one containing hlyC and one without hlyC. Taking all these into account, a total of 32 plasmids were designed, each with a unique P1 / P2 and hlyC composition.

[0091] By integration DNA technology (IDT), the fragments are used as dsDNA blocks. The dsDNA blocks are cloned into pJET plasmids, verified by sequencing, and further used as reference materials. Golden Gate Assembly (GGA) fragments are generated by PCR from the verified plasmids.

[0092] The plasmid library was assembled via BsaI-dependent GGA and the reaction mixtures were made using the ECHO 525 liquid handling platform. On a 96-well PCR plate (reaction volume of 5 μL), the DNA volume of the mixture was adjusted to 3 μL and 2 μL of NEB Bridge + BsaI (1.667 μL of NEB Bridge and 0.333 μL of BsaI) was added to each reaction. To ensure successful reactions, the mixture samples were thoroughly mixed and centrifuged, with 30 cycles of 4 minutes at 37 °C and 2 minutes at 16 °C (running for approximately 4 hours). The resulting assemblies were transformed into DH5α (only 2 μL).

[0093] Plasmid assembly was first visually confirmed (no red-background colonies), and then two cultures of each colony were grown overnight. Minipreps of the plasmids were performed and verified by PCR using CloneAmp with primers T1SSVal_PF / R01 (CGACTGAGCCTTTCGTTTTATTTGATGCC (SEQ NO ID: 15), GGTCATTACTGGATCTATCAACAGGAGTCC AAG (SEQ NO ID: 16), T A = 58 °C, t ext = 35 s, amplicon = 6379 / 5866 bp). Successfully amplified plasmids were sent for sequencing (one clone per plasmid), using primers T1SSVal_PF01 and SQ_mscarlet_for (gcatggacgaactgtataagggatcc (SEQ ID NO17)).

[0094] Assessment of T1SS expression status

[0095] The plasmids in the library were transformed into DH5α cells by heat shock (standard protocol), inoculated into vLBA and cultured overnight at 37 °C. A total of 3 single colonies were picked for each construct, inoculated into 1 mL of vLBA in a 96-deep well plate and supplemented with 12.5 μg mL -1 chloramphenicol, and cultured overnight at 200 rpm and 37 °C. Then the samples were diluted 1:100 in 500 μL of vLBA supplemented with chloramphenicol. A 100 μL volume was transferred to a 96-well plate and shaken at 700 rpm at 37 °C, and growth over time was monitored on a ClarioStar. The remaining volume was grown on a deep well plate under the same conditions.

[0096] The supernatant of the bacterial cultures grown at 37 °C in the deep well plate was subjected to HiBit assay. The samples were brought to OD 600is approximately 0.5, then an aliquot is taken and processed as follows: (1) an aliquot is taken from the deep well plate (100 μL) and used to measure the cell density (OD 600 ), (2) the cells in all plates are pelleted at 4000 x g for 10 minutes at room temperature, (3) the supernatant is carefully transferred to a new plate, (4) 10 μL of the supernatant is mixed with the previously prepared HiBit MasterMix (10 μL), and the amount of HiBit tag in the supernatant is determined by measuring luminescence.

[0097] OD 600 There was no significant difference. When analyzed using the HiBit Assay, the results showed that an increase in the strength of P1 (cargo promoter) led to a higher amount of cargo present in the supernatant, independent of the expression of the structural gene of P2. This can be explained by the autocrine ability of the fluorescent protein and the proteins released by cell turnover. Therefore, the results were normalized to the P1 strength to evaluate the effect of increasing the P2 strength on secretion. In this case, secretion was enhanced at higher levels of the structural gene. The experiment was repeated using 3 additional independent clones different from the clones already tested. The results obtained were also consistent with the data obtained previously.

[0098] Replacing the mScarlet cargo with LLO resulted in reproducible secretion results

[0099] A plasmid library in which mScarlet was replaced with LLO (also known as Listeriolysin O) was obtained by performing BbsI-dependent Golden Gate Assembly on the previously generated library and the block encoding hly from Listeria monocytogenes (surrounded by appropriate BbsI sites) on a 96-well PCR plate (reaction volume 2 μL). The conditions used were standard conditions (0.67 μL NEB Bridge, 0.264 μL 10 μM hly insert, 0.5 μL 2.5 μM vector, 0.132 μL BbsI, 0.434 μL water). To ensure the success of the reaction, after the mixture sample was thoroughly mixed, it was centrifuged, and a total of 30 cycles were performed at 37 °C for 4 minutes and 16 °C for 2 minutes (running for approximately 4 hours). The resulting assembly was transformed into DH5α. The plasmid assembly was confirmed by sequencing.

[0100] The validated plasmids in the library were transformed into DH5alpha cells by heat shock (standard protocol), inoculated into vLBA, and cultured overnight at 37 °C. A total of 3 single colonies were picked for each construct and inoculated into 1 mL of vLBA in a 96-deep well plate and supplemented with 12.5 μg mL -1Chloramphenicol, cultured overnight at 200 rpm and 37 °C. Then the sample was diluted 1:100 in 500 μL vLBA supplemented with chloramphenicol. A 100 μL volume was transferred to a 96-well plate and shaken at 700 rpm at 37 °C, and growth over time was monitored on a ClarioStar. The remaining volume was grown on a deep-well plate under the same conditions.

[0101] Cells were treated as described previously (see previous section), and secretion in all samples was measured using HiBit-induced luminescence. Overall, the growth of all samples was similar (end-point OD 600 range was the same). As described previously, higher strength of p2 (T1SS gene) led to higher output rates. Interestingly, higher p1 also generally led to higher output, which contradicts the "less is more" hypothesis when expressing the cargo in the cytosol.

[0102] Finally, as observed elsewhere, replacing the composite cargo (hly) with a fluorescent protein (mScarlet) led to a reduction in the output cargo to approximately 1 / 10 - this may be due to the higher expression rate of the fluorescent protein and its inherent transmembrane ability.

[0103] Dual-plasmid expression system

[0104] To achieve a higher level of control over the expression levels of the two systems, a dual-plasmid expression system was designed and constructed. Here, the cargo-containing plasmid was integrated into a p15A ori plasmid (approximately 10 copies / cell), which allowed for in-frame cloning (BbsI-dependent) of any relevant cargo with the hlyAs sequence. Additional control over the expression level could be achieved by BsaI-dependent cloning of the target promoter. Then, output of such cargo was achieved by introducing a second plasmid with a compatible origin of replication (e.g., pSC101, approximately 5 copies / cell), which expressed the hlyBD operon under the control of a relevant promoter that had been introduced by BsaI-dependent Golden Gate, as previously done for the cargo-hlyAs fusion. Both plasmids were constructed using the same eBlocks previously used for the single-plasmid system.

[0105] To test the system, three cargo plasmids with increasing relative expression strengths were constructed by cloning the pro1 (hlyAs-1), proC (hlyAs-4), and J23199 (hlyAs-6) promoters upstream of the mscarlet-hlyAs fusion. At the same time, three plasmids containing hlyBD (hlyBD-1, hlyBD-4, hlyBD-6) were constructed under the control of the same promoters. Using standard protocols and 100 ng of each vector, they were co-transformed into Salmonella enterica serovar Typhimurium ZH9 bacteria by electroporation and selected on LB agar medium supplemented with the respective antibiotics (carbenicillin and kanamycin) at 37 °C for 16 h. A single plasmid (only cargo) control was also transformed and selected in carbenicillin. -1 Single colonies (3 per construct) were then picked and inoculated into LB medium supplemented with the appropriate antibiotic at the same concentration as the solid medium. The samples were grown in 96-deep well plates for 16 h and diluted 1:500 in 1 mL of fresh medium. Then, the growth of 100 μL aliquots was monitored for 16 h at 700 rpm and 37 °C, and the remaining cultures were grown in a shaking incubator under the same conditions. After growth, the samples were spun down at 4000 x g for 15 min, and then the supernatant was transferred to a new 96-well plate. Then, 10 μL aliquots of each supernatant were analyzed by HiBit as described in detail previously.

[0106] Therefore, the results presented herein support the use of a two-plasmid expression system that allows for optimized yields without overloading the bacterial vector.

[0107] The sequences that form part of the specification

[0108] SEQ ID NO:1 - hly(LLO) (DNA sequence)

[0109]

[0110] ​

[0111] SEQ ID NO:2 - LLO (Amino Acid Sequence)

[0112] MAKDASAFNKENSISSMAPPASPPASPKTPIEKKHADEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADIQVVNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVNTLVERWNEKYAQAYPNVSAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVIKNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEVNYDPEGNEIVQHKNWSENNKSKLAHFTSSIYLPGNARNINVYAKECTGLAWEWWRTVIDDRNLPLVKNRNISIWGTTLYPKYSNKVDNPIE

[0113] SEQ ID NO:3 - Pro1_1 (P1)

[0114] CTCGGTCCCCAGGCATTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAGCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTTACGGGCATGCATAAGGCTCGGTATCTATATTCAGGCACAGCACAACGGTTTCCTTTTAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAAAAG

[0115] SEQ ID NO:4 - PROA_1

[0116] CTCGGTCCCCAGGCATTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAGCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTTACGGGCATGCATAAGGCTCGTAGGCTATATTCAGGCACAGCACAACGGTTTCCTTTTAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAAAAG

[0117] SEQ ID NO:5-PROB-1

[0118] CTCGGTCCCCAGGCATTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAGCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTTACGGGCATGCATAAGGCTCGTAATATATATTCAGGCACAGCACAACGGTTTCCTTTTAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAAAAG

[0119] SEQ ID NO:6-PROC-1

[0120] CTCGGTCCCCAGGCATTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAGCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTTACGGGCATGCATAAGGCTCGTATGATATATTCAGGCACAGCACAACGGTTTCCTTTTAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAAAAG

[0121] SEQ ID NO:7-PRO1_2

[0122] GTAAGTCCCCAGGCATTACTAGAGTCACACTAAAAAAAAACCCCGCCCCTGACAGGGCGGGGTTTTTTTTTTTATAGCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTTACGGGCATGCATAAGGCTCGGTATCTATATTCAGGCACAGCACAACGGTTTCCTTTTAGTCCGTAGTGGATGTGTATCCACTCTGATGAGTCCGAAAGGACGAAACGGACCTCTACAAATAATTTTGTTTAACACA

[0123] SEQ ID NO:8 - PROA_2

[0124] GTAAGTCCCCAGGCATTACTAGAGTCACACTAAAAAAAAACCCCGCCCCTGACAGGGCGGGGTTTTTTTTTTTATAGCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTTACGGGCATGCATAAGGCTCGTAGGCTATATTCAGGCACAGCACAACGGTTTCCTTTTAGTCCGTAGTGGATGTGTATCCACTCTGATGAGTCCGAAAGGACGAAACGGACCTCTACAAATAATTTTGTTTAACACA

[0125] SEQ ID NO:9 - PROB_2

[0126] GTAAGTCCCCAGGCATTACTAGAGTCACACTAAAAAAAAACCCCGCCCCTGACAGGGCGGGGTTTTTTTTTTTATAGCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTTACGGGCATGCATAAGGCTCGTAATATATATTCAGGCACAGCACAACGGTTTCCTTTTAGTCCGTAGTGGATGTGTATCCACTCTGATGAGTCCGAAAGGACGAAACGGACCTCTACAAATAATTTTGTTTAACACA

[0127] SEQ ID NO:10 - PROC_2

[0128] GTAAGTCCCCAGGCATTACTAGAGTCACACTAAAAAAAAACCCCGCCCCTGACAGGGCGGGGTTTTTTTTTTTATAGCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTTACGGGCATGCATAAGGCTCGTATGATATATTCAGGCACAGCACAACGGTTTCCTTTTAGTCCGTAGTGGATGTGTATCCACTCTGATGAGTCCGAAAGGACGAAACGGACCTCTACAAATAATTTTGTTTAACACA

[0129] SEQ ID NO:11 - hlyA s

[0130]

[0131] SEQ ID NO:12 - hlyCA s

[0132]

[0133] SEQ ID NO:13 - hlyB

[0134]

[0135] SEQ ID NO:14 - hlyD

[0136]

[0137] SEQ ID NO:15 - Forward T1SS primer

[0138] CGACTGAGCCTTTCGTTTTATTTGATGCC

[0139] SEQ ID NO:16 - Reverse T1SS primer

[0140] GGTCATTACTGGATCTATCAACAGGAGTCCAAG

[0141] SEQ ID NO:17 - SQ_mscarlet primer

[0142] gcatggacgaactgtataagggatcc

[0143] SEQ ID NO:18 - riboJ

[0144] AGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAA

[0145] SEQ ID NO:19 - vtmoJ

[0146] AGTCCGTAGTGGATGTGTATCCACTCTGATGAGTCCGAAAGGACGAAACGGACCTCTACAAATAATTTTGTTTAA

[0147] SEQ ID NO:20 - BBA_B1006 terminator

[0148] AAAAAAAAACCCCGCCCCTGACAGGGCGGGGTTTTTTTTTTTATAGCACAGCTAACACCACGTCGTCCCTA

[0149] SEQ ID NO:21 - PROMOTER 6 (J23199)

[0150] CAGAGTCCCCAGGCATTACTAGAGTCACACTTTTATAGCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCAGGCACAGCACAACGGTTTCCTTTTAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGGCA

[0151] References

[0152] Carrier, M. J., et al., Expression of human IL-1beta in Salmonella typhimurium. A model system for the delivery of recombinant therapeutic proteins in vivo. J Immunol, 1992. 148(4): p. 1176 - 81.

[0153] Chen, J., et al., Salmonella flagella confer anti - tumor immunological effect via activating Flagellin / TLR5 signalling within tumor microenvironment. Acta Pharm Sin B, 2021. 11(10): p. 3165 - 3177.

[0154] Freudl, R., Signal peptides for recombinant protein secretion in bacterial expression systems. Microb Cell Fact, 2018. 17(1): p. 52.

[0155] Gentschev, I., G. Dietrich, and W. Goebel, The E. coli alpha-hemolysin secretion system and its use in vaccine development. Trends Microbiol, 2002. 10(1): p. 39-45.

[0156] Hoffman, R. M., Tumor-seeking Salmonella amino acid auxotrophs. Curr Opin Biotechnol, 2011. 22(6): p. 917-23.

[0157] Holland, I. B. et al. (1990) The mechanism of secretion of hemolysin and other polypeptides from Gram-negative bacteria. J. Bioenerg. Biomembr. 22, 473–491.

[0158] Jarchau, T. et al. (1994) Selection for transport competence of C-terminal polypeptides derived from Escherichia coli hemolysin: the shortest peptide capable of autonomous HlyB / HlyD-dependent secretion comprises the C-terminal 62 amino acids of HlyA. Mol. Gen. Genet. 245, 53–60.

[0159] Khosa, S., et al., An A / U-Rich Enhancer Region Is Required for High-Level Protein Secretion through the HlyA Type I Secretion System. Appl Environ Microbiol, 2018. 84(1).

[0160] Koronakis, V. (1989) Isolation and analysis of the C-terminal signal directing export of Escherichia coli hemolysin protein across both bacterial membranes. EMBO J. 8, 595–605.

[0161] Lhocine, N., et al., Apical invasion of intestinal epithelial cells by Salmonella typhimurium requires villin to remodel the brush border actin cytoskeleton. Cell Host Microbe, 2015. 17(2): p. 164-77.

[0162] Madrid, C., et al., Temperature- and H-NS-dependent regulation of a plasmid-encoded virulence operon expressing Escherichia coli hemolysin. J Bacteriol, 2002. 184(18): p. 5058-66.

[0163] Nagamatsu, K., et al., Dysregulation of Escherichia coli alpha-hemolysin expression alters the course of acute and persistent urinary tract infection. Proc Natl Acad Sci U S A, 2015. 112(8): p. E871-80.

[0164] Nieto, J.M., et al., Expression of the hemolysin operon in Escherichia coli is modulated by a nucleoid-protein complex that includes the proteins Hha and H-NS. Mol Gen Genet, 2000. 263(2): p. 349-58.

[0165] Park, D., et al., Visualization of the type III secretion mediated Salmonella-host cell interface using cryo-electron tomography. Elife, 2018. 7.

[0166] Pourhassan, N.Z., et al., Optimized Hemolysin Type 1 Secretion System in Escherichia coli by Directed Evolution of the Hly Enhancer Fragment and Including a Terminator Region. Chembiochem, 2022. 23(6): p. e202100702.

[0167] Ruano-Gallego, D., et al., Screening and purification of nanobodies from E. coli culture supernatants using the hemolysin secretion system. Microbial Cell Factories, 2019. 18(1): p. 47.

[0168] Thomas, S., I.B. Holland, and L. Schmitt, The Type 1 secretion pathway - the hemolysin system and beyond. Biochim Biophys Acta, 2014. 1843(8): p. 1629-41.

[0169] Wang, B., M. Mittermeier, and I. Artsimovitch, RfaH May Oppose Silencing by H-NS and YmoA Proteins during Transcription Elongation. J Bacteriol, 2022. 204(4): p.e0059921.

[0170] Yang, E.Y. and K. Shah, Nanobodies: Next Generation of Cancer Diagnostics and Therapeutics. Front Oncol, 2020. 10: p.1182.

Claims

1. An attenuated live Gram-negative bacterium comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is divided into a first segment and a second segment, each segment being operably linked to an independently controlled promoter, wherein the first segment comprises a heterologous polynucleotide encoding a cargo molecule upstream of the hlyA translocation sequence, wherein the heterologous polynucleotide encoding the cargo molecule replaces the hlyA gene, and wherein the second segment comprises hly genes involved in secretion.

2. The attenuated live Gram-negative bacterium according to claim 1, wherein the independently controlled promoter operably linked to the first segment is located upstream of the heterologous polynucleotide encoding the cargo molecule.

3. The attenuated live Gram-negative bacterium according to claim 1 or 2, wherein the first segment further comprises the hlyC gene or a fragment thereof located upstream of the heterologous polynucleotide encoding the cargo molecule.

4. The attenuated live Gram-negative bacterium according to any one of the preceding claims, wherein the independently controlled promoter operably linked to the first segment is located upstream of the hlyC gene or a fragment thereof.

5. The attenuated live Gram-negative bacterium according to any one of the preceding claims, wherein the hly genes involved in secretion are hlyB and hlyD.

6. The attenuated live Gram-negative bacterium according to any one of the preceding claims, wherein the independently controlled promoter operably linked to the second segment is located upstream of the hlyB gene.

7. The attenuated live Gram-negative bacterium according to any one of the preceding claims, wherein the cargo molecule is a peptide and / or a protein.

8. The attenuated live Gram-negative bacterium according to claim 7, wherein the peptide and / or protein is a therapeutic peptide and / or a therapeutic protein.

9. The attenuated live Gram-negative bacterium according to claims 1 to 6, wherein the cargo molecule is an RNA molecule.

10. The attenuated live Gram-negative bacterium according to claim 9, wherein the RNA molecule is an mRNA molecule.

11. The attenuated live Gram-negative bacterium according to claim 10, wherein the mRNA molecule encodes a therapeutic protein and / or a therapeutic peptide.

12. The attenuated live Gram-negative bacterium according to any one of the preceding claims, wherein the independently controlled promoter operably linked to the first segment is a strong promoter.

13. The attenuated live Gram-negative bacterium according to any one of the preceding claims, wherein the independently controlled promoter operably linked to the first segment comprises the PipB2, ssaG or proC promoter.

14. The attenuated live Gram-negative bacterium according to claim 13, wherein the independently controlled promoter operably linked to the first segment is the proC promoter.

15. The attenuated live Gram-negative bacterium according to any one of the preceding claims, wherein the independently controlled promoter operably linked to the second segment is a strong promoter.

16. An attenuated live Gram-negative bacterium according to any one of the preceding claims, wherein the independently controlled promoter operably linked to the second comprises the PipB2, ssaG or proC promoter.

17. The attenuated live Gram-negative bacterium according to claim 16, wherein the independently controlled promoter operably linked to the first segment is the proC promoter.

18. An attenuated live Gram-negative bacterium according to any one of the preceding claims for therapeutic use.

19. The attenuated live Gram-negative bacterium for use according to claim 18, wherein the attenuated live Gram-negative bacterium is for treating, reducing, inhibiting, preventing or controlling a neoplastic disease, an infectious disease, a cardiovascular disease, a neurodegenerative disease, a gastrointestinal disease, a respiratory disease, a kidney disease, a liver disease, an autoimmune disease, an inflammatory disease or a genetic disease, preferably, the attenuated live Gram-negative bacterium is for treating, reducing, inhibiting, preventing recurrence or controlling a neoplastic disease or an infectious disease.

20. The attenuated live Gram-negative bacterium for use according to claim 19, wherein the neoplastic disease is a solid cancer and / or a hematological malignancy.

21. The attenuated live Gram-negative bacterium for use according to claim 20, wherein the solid cancer and / or hematological malignancy is a cancer selected from the following: prostate cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, brain cancer, mesothelioma, hepatocellular carcinoma, lymphoma, leukemia, gastric cancer, prostate cancer, endometrial cancer, endometrial cancer, vulvar / vaginal cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer or sarcoma, preferably wherein the neoplastic disease is associated with a cancer selected from bladder cancer, lung cancer, mesothelioma, hepatocellular carcinoma, melanoma, esophageal cancer, gastric cancer, ovarian cancer, colorectal cancer, head and neck cancer, prostate cancer, endometrial cancer, cervical cancer or breast cancer.

22. A vaccine composition comprising an attenuated live Gram-negative bacterium, the attenuated live Gram-negative bacterium comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is divided into a first segment and a second segment, each segment being operably linked to an independently controlled promoter, wherein the first segment comprises a heterologous polynucleotide encoding a cargo molecule upstream of the hlyAs translocation sequence, wherein the heterologous polynucleotide encoding the cargo molecule replaces the hlyA gene, and wherein the second segment comprises hly genes involved in secretion.

23. The vaccine composition according to claim 22, wherein the vaccine composition further comprises an adjuvant, a pharmaceutically acceptable carrier or an excipient.

24. The vaccine composition according to claim 22 or 23, wherein the vaccine composition comprises an attenuated live Gram-negative bacterium according to any one of claims 2 to 17.

25. A method of treating, preventing, suppressing, preventing recurrence or controlling a disease in a subject, wherein the method comprises administering to the subject an attenuated live Gram-negative bacterium, the attenuated live Gram-negative bacterium comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is divided into a first segment and a second segment, each segment being operably linked to an independently controlled promoter, wherein the first segment comprises a heterologous polynucleotide encoding a cargo molecule upstream of the hlyAs translocation sequence, wherein the heterologous polynucleotide encoding the cargo molecule replaces the hlyA gene, and wherein the second segment comprises hly genes involved in secretion.

26. The method of treating, preventing, suppressing, preventing recurrence or controlling a disease in a subject according to claim 25, wherein the method comprises the attenuated live Gram-negative bacterium according to any one of claims 2 to 17.

27. A method of delivering a therapeutic molecule to the interstitial space between eukaryotic cells in the tumor microenvironment of a subject having a tumor, the method comprising the steps of: i) modifying an attenuated live Gram-negative bacterium, the attenuated live Gram-negative bacterium comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is divided into a first segment and a second segment, each segment being operably linked to an independently controlled promoter, wherein the first segment comprises a heterologous polynucleotide encoding a cargo molecule upstream of the hlyAs translocation sequence, wherein the heterologous polynucleotide encoding the cargo molecule replaces the hlyA gene, and wherein the second segment comprises hly genes involved in secretion, and ii) administering the modified Gram-negative bacterium to a subject in need thereof.

28. The method of delivering a therapeutic molecule to the interstitial space between eukaryotic cells in the tumor microenvironment of a subject having a tumor according to claim 27, wherein the method comprises the live attenuated bacterium according to any one of claims 2 to 17.

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