Bacterial transfection

Abstract

The present invention relates to a modified live attenuated Gram-negative bacterium wherein the bacterium has been modified such that an RNA molecule can be safely and efficiently delivered to a target eukaryotic cell. Thus, the present invention relates to a bacterial delivery system and various uses and methods thereof.

Description

Technical Field

[0001] The present invention relates to a modified attenuated live Gram-negative bacterium and use thereof. Background Art

[0002] Gene therapy has been used to transfer genetic material into cancer cells to inactivate oncogenes or temporarily change their phenotype, thereby reducing or eliminating tumor growth [Molecular Cell Therapies (2014) 2:27; Journal of Vascular and Interventional Radiotherapy (2013) 24(8):1115; Journal of Immunotherapy of Cancer (2020) 8:e001]. Oncolytic virotherapy (OV), which uses genetically modified viruses to infect cancer cells and replicate specifically in them, has shown great promise, with three OVs approved for treatment worldwide in 2020 [Journal of Immunotherapy of Cancer (2020) 8:e001]. These therapies have been shown to be safe, with mild side effects and minimal shedding.

[0003] However, there are several limitations to the use of these agents [Journal of Gene Medicine (2005) 7:1380]: (1) patients develop antiviral antibodies that eliminate the vector before it reaches its target; (2) high doses of viral particles can lead to a cytokine cascade that affects the patient's health; (3) unforeseen mutations can lead to safety concerns; and (4) tumor delivery is limited [Oncolytic virotherapy (2017) 6:39]. In addition, there are discrepancies between the results obtained in preclinical studies and the antitumor effects observed in clinical trials [Oncolytic virotherapy (2017) 6:39].

[0004] Bacterial transfection is the process of transferring genetic material from bacteria (e.g., Salmonella) into mammalian cells. An early example of bacterial transfection using Salmonella [Blood (1998) 92:3172] employed strain SL7207 (aroA-), which spontaneously and passively transferred plasmid material into spleen cells—preferentially into macrophages (F4 / 80 +), with an efficiency of ~19%. Byrne et al. 2014 also confirmed that when Escherichia coli (E. Coli) MG1655 was used as a vector, the bacterial transfection efficiency was approximately 20% [Journal of Controlled Release (2014) 196: 384]. They also confirmed that preferential bacterial transfection was performed in macrophages. This limited efficiency is attributed not only to the instability of the vector strain, which is mainly due to the use of a plasmid system, which is inherently variable and causes a metabolic burden [Gene Therapy (2005) 12: 364], but also because the plasmid may need to enter the cell nucleus due to unmethylated CpG regions [Molecular Therapy (2009) 17: 767]. In addition, the transfer of unmethylated CpG islands may help trigger the TLR9 response (which is known to be involved in cancer regulation in a complex manner). A relevant example demonstrating the use of bacterial transfection in immune system modulation is the publication by Shen et al. [Microbiology and Immunology (2004) 48:329], who used Listeria monocytogenes to "transfect" mice (BALB / c) to produce IL-10, IL-12, or IFNγ.

[0005] There remains a need for new delivery systems and methods for delivering heterologous polypeptides to target eukaryotic cells. Summary of the Invention

[0006] The inventors of the present invention unexpectedly discovered that Gram-negative bacteria can be modified to enable safe, efficient, and effective delivery of RNA molecules to target eukaryotic cells. Therefore, the present invention provides a bacterial delivery system with broad applicability across numerous disease areas.

[0007] In order to successfully achieve bacterial transfection of RNA, RNA polymerase is required to synthesize RNA from a DNA template. However, the inventors of the present invention have confirmed that typical phage RNA polymerases have significant shortcomings, which have hindered the successful use of bacterial transfection. First, the inventors of the present invention unexpectedly discovered that T7 RNA polymerase does not contribute to the safe, efficient and effective delivery of RNA molecules to target eukaryotic cells, and may cause unnecessary toxicity in certain bacterial strains. When Gram-negative bacteria are subjected to bacterial transfection, the inventors of the present invention realize that it is necessary to modify to improve efficiency, and find that traditional strong promoters (such as T7 promoters) used to drive DNA template expression will produce unwanted toxicity due to the exhaustion of host bacterial cell resources. Secondly, the inventors determine that the presence of T7 promoters in the ZH9 Salmonella chromosome may drive the expression of unknown genes, some of which may be toxic, thereby leading to specificity problems.

[0008] Therefore, the inventors have unexpectedly discovered that the choice of promoter is important for successful bacterial transfection and any subsequent downstream applications. Specifically, the inventors have identified two solutions to the above-mentioned toxicity and specificity problems. The first is to use an RNA polymerase encoded by a heterologous split-type RNA polymerase plasmid, thereby maximizing RNA production and minimizing toxicity. The second is to use an RNA polymerase and promoter pairing that is orthogonal to any components required for endogenous gene expression in Gram-negative bacteria, thereby avoiding the specificity problems described previously.

[0009] Thus, in a first aspect, the present invention provides a live attenuated Gram-negative bacterium comprising: i) a heterologous polynucleotide encoding an RNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter; and ii) a polynucleotide encoding an RNA polymerase, wherein the RNA polymerase is associated with the promoter, wherein the RNA polymerase is encoded by a heterologous split-type RNA polymerase plasmid, and wherein following invasion of the eukaryotic cell by the Gram-negative bacterium, the RNA molecule is transcribed and is capable of translocating into the cytoplasm of the eukaryotic cell.

[0010] In a second aspect, the present invention provides a live, attenuated Gram-negative bacterium comprising: i) a heterologous polynucleotide encoding an RNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter; and ii) a polynucleotide encoding an RNA polymerase that is orthogonal to components required for endogenous gene expression in Gram-negative bacteria, wherein the RNA polymerase binds to the promoter, and wherein following invasion of the eukaryotic cell by the Gram-negative bacterium, the RNA molecule is transcribed and capable of translocating into the cytoplasm of the eukaryotic cell.

[0011] In a third aspect, the present invention provides a vaccine composition comprising a live attenuated Gram-negative bacterium, wherein the Gram-negative bacterium comprises: i) a heterologous polynucleotide encoding a functional mRNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter and the mRNA molecule encodes a therapeutic protein and / or therapeutic peptide; and ii) a polynucleotide encoding an RNA polymerase that is orthogonal to components required for endogenous gene expression in the Gram-negative bacterium, wherein the RNA polymerase binds to the promoter, and wherein following invasion of the eukaryotic cell by the Gram-negative bacterium, the mRNA molecule is transcribed and capable of translocating into the cytoplasm of the eukaryotic cell.

[0012] In a fourth aspect, the present invention provides a vaccine composition comprising a live attenuated Gram-negative bacterium, wherein the Gram-negative bacterium comprises: i) a heterologous polynucleotide encoding a functional mRNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter and the mRNA molecule encodes a therapeutic protein and / or therapeutic peptide; and ii) a polynucleotide encoding an RNA polymerase, wherein the RNA polymerase is associated with the promoter, wherein the RNA polymerase is encoded by a heterologous split-type RNA polymerase plasmid, and wherein after the Gram-negative bacterium invades the eukaryotic cell, the mRNA molecule is transcribed and can be translocated into the cytoplasm of the eukaryotic cell.

[0013] In a fifth aspect, the present invention provides a method for treating, inhibiting, preventing recurrence of, or managing a disease in a subject, wherein the method comprises administering to the subject a live attenuated Gram-negative bacterium comprising: i) a heterologous polynucleotide encoding an RNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter; and ii) a polynucleotide encoding RNA, wherein an RNA polymerase is associated with the promoter, wherein the RNA polymerase is encoded by a heterologous split-type RNA polymerase plasmid, and wherein following invasion of the eukaryotic cell by the Gram-negative bacterium, the RNA molecule is transcribed and capable of translocating into the cytoplasm of the eukaryotic cell.

[0014] In a sixth aspect, the present invention provides a method for treating, inhibiting, preventing recurrence of, or managing a disease in a subject, wherein the method comprises administering to the subject a live, attenuated Gram-negative bacterium comprising: i) a heterologous polynucleotide encoding an RNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter; and ii) a polynucleotide encoding an RNA polymerase that is orthogonal to components required for endogenous gene expression in the Gram-negative bacterium, wherein the RNA polymerase binds to the promoter, and wherein following invasion of the eukaryotic cell by the Gram-negative bacterium, the RNA molecule is transcribed and capable of translocating into the cytoplasm of the eukaryotic cell.

[0015] In a seventh aspect, the present invention provides a method for delivering an RNA molecule into a eukaryotic cell, the method comprising the steps of: i) modifying a Gram-negative bacterium such that a heterologous polynucleotide encoding the RNA molecule is integrated into the bacterial genome, wherein the heterologous polynucleotide is operably linked to a promoter; ii) contacting the Gram-negative bacterium with a eukaryotic cell such that the Gram-negative bacterium replicates within the eukaryotic cell, whereby the heterologous polynucleotide is transcribed and subsequently transferred from the Gram-negative bacterium to the cytoplasm of the eukaryotic cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 shows how T7 RNA polymerase (RNAP) can be toxic to Salmonella enterica Typhi. Figure 1A A schematic diagram of the experimental setup is shown. Salmonella enterica ZH9 was transformed with two plasmids, one containing T7 RNA polymerase under the control of an SPI-2 inducible promoter and the other containing mScarlet (a red fluorescent protein) under the control of a T7 promoter (T7p). Putatively active T7p was found within the Salmonella genome, downstream of a prophage integrase-like gene and upstream of four uncharacterized open reading frames (ORFs, one of which had homology to a phage regulatory protein). BLASTp searches showed that these proteins can be found in different enteric bacteria such as Salmonella, Escherichia, or Shigella (data not shown). Figure 1B The results show an evaluation of the effects of temperature and promoter strength on T7 RNAP-dependent toxicity. Toxicity was found to be more pronounced under strong promoters, especially near the optimal growth temperature (37°C).

[0017] Figure 2 A schematic diagram of the split RNAP system is shown. In this design, the expression level of the core RNAP determines the total polymerase activity and, therefore, the toxicity. Careful choice of promoter 1 (e.g., a constitutive promoter or an SPI-2-dependent promoter) allows the generation of strains with maximal polymerase activity at the lowest possible toxicity level. A second (or more) promoters control the expression of the "σ" region (or DNA binding region) of the RNAP. After σ binds to the core, polymerase activity is restored and is specific for the cognate promoter of σ. This allows the generation of a platform in which different σ factors control different bacterial transfection circuits (e.g., σ1 controls the production of the therapeutic RNA, while σ2 controls the synthesis of lysin, which releases the therapeutic RNA into the cytosol of eukaryotic cells).

[0018] Figure 3Results of expressing the split RNAP system in Escherichia coli (E. coli) and Salmonella enterica ZH9 are shown. On the left, E. coli DH5α cells were transformed with a medium-copy number expression plasmid containing a polymerase phage promoter (e.g., T7, T3, K1F, or CGG) upstream of the mScarlet reporter gene, and with a low-copy number plasmid containing a split RNAP with a specific σ factor (shown on the left) or superfolded green fluorescent protein (sfGFP) (negative). mScarlet was expressed only in the correctly paired σ promoter strain. On the right, Salmonella enterica ZH9 was transformed with the above expression vectors and a split RNAP plasmid with a cognate σ factor or sfGFP, using strong (H) or weak (L) ribosome binding sites (RBS) to control the level of the RNAP core (middle) and the σ factor controlled by PuhpT (right) for evaluation of the system in liquid culture.

[0019] Figure 4 shows the results of in vitro evaluation of different cytosol promoters. Figure 4A Shown are plasmids generated for expression of constitutive mScarlet encoding red fluorescent protein, and inducible sfGFP encoding green fluorescent protein under the control of any of the indicated promoters. Figure 4B As shown, Salmonella enterica ZH9 cells carrying these plasmids were grown in M9 medium supplemented with 1 mM Fe 3+ Mg 2+ (PmtgC only) or Zn 2+ (PzinT only) and fluorescence was monitored over time. In the absence of inducer, cells showed different expression levels.

[0020] Figure 5 Shown are visualization results of an invasion assay using Salmonella enterica ZH9 containing a reporter plasmid that constitutively expresses mScarlet and sfGFP under a cytosol-inducible promoter under a microscope 24 hours after invasion. All tested promoters (specified above each micrograph), except PyjjZ, are red, indicating the lack of sfGFP production by SKOV-3 cells (an ovarian cancer cell line).

[0021] Figure 6 shows the evaluation of cytosolic promoters in vacuolar release. Figure 6AThe activity of the promoter puhpT, which is reported to be responsive to cytosolic glucose-6-phosphate, was demonstrated. PuhpT was assessed in cells by invading SK-OV-3 cells with Salmonella Typhimurium CD12 carrying a reporter plasmid that constitutively expresses mScarlet (a red fluorescent protein) and sfGFP (a green fluorescent protein) in response to glucose-6-phosphate. Figure 6B Representative examples (two each) of cells in the vacuole (small number, red only) and in the cytosol (highly replicated, yellow) are shown.

[0022] Figure 7 We show that σ factors are responsible for spatiotemporal control within host cells. σ factor expression can be selectively controlled using either vacuole-inducible (ssaG, sseJ, sseA, or sifA) or cytosol-inducible (fhuA, iroN, mntH, or sitA) promoters. Furthermore, differential expression levels from each of these promoters (as indicated by fluorescence from sfGFP downstream of each tagged promoter) provide an additional layer of control for split-RNAP activity. DETAILED DESCRIPTION

[0023] In order to more readily understand the present invention, some terms are first defined. Additional definitions are set forth throughout the detailed description.

[0024] In the context of the present invention, the term "attenuation" as used herein refers to altering a microorganism to reduce its pathogenicity, making it harmless to the host while maintaining its viability. This method is frequently used due to its ability to elicit a highly specific immune response while maintaining acceptable safety. Methods for obtaining attenuated microorganisms may include, but are not limited to, passage the pathogen under in vitro conditions until it loses toxicity, chemical mutagenesis, and genetic engineering techniques. Such attenuated microorganisms are preferably attenuated live microorganisms, although attenuated non-live microorganisms are also disclosed.

[0025] As used herein, the term "inactivating mutation" refers to a modification of the native genetic code of a specific gene or a gene promoter associated with that gene, such as by changing the nucleotide code, deleting nucleotide fragments, adding non-coding nucleotides or non-natural nucleotides, etc., so that the specific gene cannot be properly transcribed or translated, or is expressed as an inactive protein, thereby eliminating or reducing the native function of the gene to an unmeasurable level. Therefore, a gene mutation inactivates the function of the gene or the function of the protein encoded by the gene.

[0026] The term "non-natural bacteria" as used herein refers to a bacterial (prokaryotic) cell that has been genetically modified or "engineered" to be altered relative to a naturally occurring cell. This genetic modification can, for example, be the incorporation of additional genetic information into the cell, the modification of existing genetic information, or the actual 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. Therefore, the term "non-natural bacteria" can refer to both recombinantly modified and non-recombinantly modified bacterial strains.

[0027] As used herein, the term "bacterial transfection" refers to the process of transducing genetic material from bacteria (e.g., Salmonella) into eukaryotic cells. Preferably, the eukaryotic cells are mammalian cells. More preferably, the eukaryotic cells are human cells. Specifically, in the context of the present invention, the term "bacterial transfection" refers to the delivery of Gram-negative bacteria to target eukaryotic cells, followed by the use of Gram-negative bacteria to deliver RNA molecules transcribed in the Gram-negative bacteria into the cytosol of the eukaryotic cells.

[0028] As used herein, the term "split RNA polymerase" or "RNAP" refers to a system in which the different components of an RNA polymerase are encoded on separate genes and assemble to form a functional RNA polymerase during transcription and translation. Specifically, RNA polymerase can be divided into a "core" component and a DNA-binding component, referred to as "σ" or "σ factor."

[0029] As used herein, the terms "orthogonal" or "orthogonality" are used interchangeably and refer to the biological process of orthogonalization. Orthogonalization refers to the purposeful inability of two or more biological molecules with similar structure and / or function to interact or affect their respective substrates. Thus, in the context of the present invention, the term "orthogonal" refers to the absence of components required for expression of a polynucleotide encoding an RNA polymerase in Gram-negative bacteria. Preferably, the term "orthogonal" refers to the absence of components required for expression of a polynucleotide encoding an RNA polymerase in the genome of Salmonella enterica.

[0030] As used herein, the term "prophylactic treatment" refers to a medical procedure intended to prevent, rather than treat or cure, an infection or disease. In the context of the present invention, this particularly applies to vaccine compositions. As used herein, the term "prevention" is not intended to be absolute and may also include partial prevention of an infection or disease and / or one or more symptoms of said infection or disease. In contrast, the term "curative treatment" refers to a medical procedure, as understood in the art, intended to treat or cure an infection or disease or its associated symptoms.

[0031] The present invention relates to the modification of Gram-negative bacteria so that RNA molecules can be safely, efficiently and effectively delivered to target eukaryotic cells. The terms "RNA" and "ribonucleic acid" used herein can be used interchangeably and refer to nucleic acids consisting of uracil, adenine, guanine and cytosine ribonucleic acid bases. These terms and concepts are well known to those skilled in the art. The types of RNA molecules include, for example, messenger RNA (mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), transfer RNA (tRNA), ribosomal RNA (rRNA) and self-amplification (or self-replication) RNA (saRNA). In a preferred embodiment, the RNA molecule to be encoded is an mRNA, siRNA or shRNA molecule.

[0032] As used herein, the terms "mRNA" and "messenger RNA" are used interchangeably and refer to single-stranded RNA molecules involved in protein synthesis. Eukaryotic mRNA molecules are transcribed from DNA in the nucleus of eukaryotic cells and subsequently exported from the nucleus to the cytoplasm of the eukaryotic cell, where translation of the mRNA into protein occurs. Bacterial mRNA molecules are transcribed from non-compartmentalized DNA and translated in the cytosol in conjunction with transcription. These terms and concepts are well known to those skilled in the art.

[0033] The terms "siRNA" and "short interfering RNA" used herein can be used interchangeably and refer to a specific method of RNA interference (RNAi). RNAi is a sequence-specific RNA degradation process that can theoretically provide a direct way to knock out or silence any gene. In naturally occurring RNAi, double-stranded RNA (dsRNA) is cut into siRNA molecules by RNaseIII / helicase protein Dicer. These siRNA molecules are dsRNAs with 19 to 27 nucleotides (nt) with 2-nt overhangs at the 3' end. These siRNAs are incorporated into a multicomponent ribonuclease known as RNA-induced silencing complex (RISC). One chain of the siRNA remains associated with RISC and guides the complex toward a target RNA with a sequence complementary to the guide single-stranded siRNA in RISC. The endonuclease guided by this siRNA digests RNA, thereby inactivating it.

[0034] The terms "shRNA" and "short hairpin RNA" used herein can be used interchangeably and refer to a specific method of RNA interference (RNAi). RNAi is a sequence-specific RNA degradation process that provides a theoretically relatively simple and direct method to knock out or silence any gene. shRNA consists of a stem-loop structure. The stem-loop structure consists of a stem portion comprising a double-stranded sequence. The double-stranded stem portion consists of an antisense (guide) strand on one side of the stem and a sense (follower) strand on the other side of the stem. The stem-loop structure also includes a single-stranded loop portion at one end of the stem. After processing by Drosha and Dicer, shRNA is loaded into the RNA-induced silencing complex (RISC). The sense strand (follower strand) degrades, and the antisense strand (guide strand) guides RISC to an mRNA with a complementary sequence. In the case of complete complementarity, RISC cuts the mRNA. In the case of incomplete complementarity, RISC inhibits the translation of mRNA. In both cases, shRNA can cause target gene silencing.

[0035] As used herein, the terms "saRNA," "self-amplifying," and "self-replicating RNA" are used interchangeably and refer to a class of mRNAs that encode a replicase that copies the original strand of RNA once the RNA is delivered to the desired location (i.e., in the context of the present invention, to the cytosol of the host cell). The use of saRNA improves the efficiency of RNA delivery and the expression of RNA molecules.

[0036] As used herein, a "heterologous polynucleotide" refers to a polynucleotide that has been introduced into a Gram-negative bacterium, i.e., a polynucleotide that was not previously present. A heterologous polynucleotide in the context of the present invention will encode an RNA molecule for delivery to a eukaryotic cell. The resulting RNA molecule is also referred to herein as a "cargo" or "cargo molecule." In a particularly preferred embodiment, the RNA molecule to be encoded is a mammalian RNA molecule.

[0037] The terms "vaccine composition" or "vaccine" are interchangeably referred to herein as "compositions" and refer to biological preparations that provide active acquired immunity against a specific disease. Typically, vaccines contain an agent or "foreign" agent that is similar to the causative pathogen and, in the context of the present invention, is encoded by an mRNA molecule (or cargo) for delivery to target eukaryotic cells. The vaccine recipient's immune system recognizes this foreign agent, destroys it, and develops a "memory" against the causative pathogen, resulting in a level of lasting protection against future infections or diseases caused by the same or similar pathogens. Through vaccination routes, including those of the present invention, it is conceivable that once a vaccinated subject encounters the same pathogen or pathogen isolate against which the subject was vaccinated, the individual's immune system can recognize the pathogen or pathogen isolate and trigger a more effective defense against infection or disease. The active acquired immunity induced in a subject due to the vaccine can be humoral and / or cellular in nature. In the context of the present invention, various vaccine antigens can be delivered to the subject's target eukaryotic cells using the Gram-negative bacteria disclosed herein as a delivery method, thereby priming the subject's immune system against the antigen.

[0038] The terms "tumor," "cancer," "malignancy," and "neoplasia" are used interchangeably and refer to a cell or group of cells that grows, proliferates, or survives at a rate greater than that of a corresponding normal cell (e.g., a disorder of cell proliferation or differentiation). Typically, growth is uncontrolled. The term "malignancy" refers to the invasion of nearby tissues. The term "metastasis" refers to the spread or dissemination of a tumor, cancer, or neoplasia to other sites, locations, or areas in a subject's body that are different from the primary tumor or cancer. In one embodiment, the cancer is malignant. In alternative embodiments, the cancer is non-malignant.

[0039] The term "effective amount" or "pharmaceutically effective amount" refers to a sufficient amount of an agent to provide a desired biological or therapeutic result. The result can be a reduction, improvement, alleviation, reduction, delay and / or postponement 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 a cancer or tumor or prolong survival or induce stability.

[0040] 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 result in one or more of the following: (i) reducing the number of cancer cells; (ii) reducing tumor size; (iii) inhibiting, slowing, slowing to some extent, or preferably preventing cancer cells from infiltrating peripheral organs; (iv) inhibiting (i.e., slowing to some extent, preferably preventing) tumor metastasis; (v) inhibiting tumor growth; (vi) preventing or delaying tumor development and / or recurrence; and / or (vii) alleviating to some extent one or more symptoms associated with cancer.

[0041] For example, for the treatment of a tumor, a "therapeutically effective dose" can induce a tumor to shrink by at least about 5%, such as at least about 10%, or about 20%, or about 60% or more relative to a baseline measurement. The baseline measurement may be derived from an untreated subject.

[0042] A therapeutically effective amount of a therapeutic compound can reduce the size of a tumor in a subject, or improve symptoms. One of ordinary skill in the art can determine such an amount 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.

[0043] The terms "treat" or "treatment" refer to the administration of an active agent with the purpose of curing, rehabilitating, delaying, alleviating, altering, remedying, improving, improving or affecting a condition (e.g., a disease) or symptoms of a condition, or preventing or delaying the onset of symptoms, complications, biochemical markers of a disease, or arresting or inhibiting in a statistically significant manner the further development of a disease, condition or disorder.

[0044] As used herein, the term "subject" is intended to include humans and non-human animals. Preferred subjects include human patients in need of an enhanced immune response. The method is particularly suitable for treating human patients suffering from conditions that can be treated by enhancing the immune response. In specific embodiments, the method is particularly suitable for treating neoplastic diseases or infectious diseases in vivo.

[0045] The use of alternatives (eg, "or") should be understood to mean one, two, or any combination of the alternatives. The indefinite articles "a" or "an" used herein should be understood to refer to "one or more" of any recited or listed components.

[0046] As used herein, "about" means within an acceptable error range for 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 1 standard deviation or greater than 1 standard deviation, as practiced in the art. Alternatively, "about" can mean a range of up to 20%. When a particular value is provided in the application and claims, unless otherwise indicated, the meaning of "about" should be considered to be within an acceptable error range for that particular value.

[0047] Previous strategies to increase bacterial transfection levels of DNA have been to induce vector lysis after cell invasion. This has been shown in Listeria monocytogenes [Cellular Microbiology (2005) 7: 709; Journal of Gene Med (2002) 4: 655], Escherichia coli (reportedly 90% transduction) [Journal of Controlled Release (2021) 332: 233], and Salmonella Typhimurium [Material Horizons (2020) 8: 1454]. Others [Cellular Microbiology (2005) 7 (5): 709] co-express hemolysin from Listeria or phospholipase C from Clostridium spp. to increase cleavage of endosomal membranes. However, due to the low transfer rate of plasmids from the cytosol to the nucleus, the above methods still lead to a bottleneck effect.

[0048] To address the above problems, bacteria have been used to release RNA rather than DNA directly into the cytosol. Bacteria have previously been used as delivery vectors, using T7 polymerase (which is a common method for producing large amounts of RNA) or other strong promoters (WO2020245093; Schoen et al., 2005, Cellular Microbiology, 7 (5), 709-724) to transfer RNA. The translation of bacterial RNA is ensured by adding an IRES sequence at the 5' end. The main limitation of this method is the instability of bacterial RNA expression, which may degrade before transfection. The presence of an IRES sequence may also enhance the stability of RNA in the bacterial cytosol. Like DNA, the release of RNA after intracellular bacterial lysis is also enhanced.

[0049] However, the present inventors unexpectedly discovered that the use of high expression levels of T7 RNA polymerase (and corresponding T7 promoter) is not conducive to the safe, efficient and effective delivery of RNA molecules when targeting eukaryotic cells, and may lead to unwanted toxicity and specificity problems. Therefore, the present inventors have identified the need to modify the promoter systems previously used in the art. As mentioned above, the inventors have overcome the problems of the promoter systems previously used by the following two solutions. The first is to use an RNA polymerase encoded by a heterologous split RNA polymerase plasmid, and the second is to use an RNA polymerase and promoter pairing that is orthogonal to any component of the Gram-negative bacteria required for endogenous gene expression.

[0050] Thus, in a first aspect, the present invention provides a live attenuated Gram-negative bacterium comprising: i) a heterologous polynucleotide encoding an RNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter; and ii) a polynucleotide encoding an RNA polymerase, wherein the RNA polymerase is bound to the promoter, wherein the RNA polymerase is encoded by a heterologous split-type RNA polymerase plasmid, and wherein following invasion of the eukaryotic cell by the Gram-negative bacterium, the RNA molecule is transcribed and is capable of translocating into the cytoplasm of the eukaryotic cell.

[0051] The inventors of the present invention unexpectedly discovered that the toxicity and specificity problems previously observed when using the T7 promoter can be overcome by using a split RNA polymerase system. In such a system, the RNA polymerase is separated into a "core" component and a DNA-binding component, called "σ" or "σ factor." These separate components of the RNA polymerase are encoded on separate genes and can therefore be controlled separately.

[0052] Figure 2A schematic diagram of a split RNA polymerase system is shown. Taking T7 RNA polymerase as an example, the "core" component of the RNA polymerase consists of amino acids 1 to 601 of T7 RNA polymerase. The DNA binding component of the RNA polymerase, hereafter referred to as "σ" or "σ" factor", consists of amino acids 600 to 883 of T7 RNA polymerase. The σ element can contain variants, which allow it to bind to different promoter sequences. To ensure the binding of both partners, a synthetic coiled-coil peptide (SYNZIP) that guides protein binding is added. In addition, two circuit variants were designed, in which the core element is expressed from a low-strength promoter (proA) and a strong (H) or weak (L) RBS. The use of split RNA polymerases offers the possibility of using two or more different σ factors to control the transcription of two or more elements. Different σ factors have different efficiencies, opening up the possibility of expressing the promoter at different ratios. In one embodiment, the present invention provides a method for producing a lysin by splitting RNAP into two fragments, wherein the expression level of the core fragment is fine-tuned, thereby obtaining the maximum RNA yield with minimum toxicity. In addition, multiple σ elements targeting different promoters can be expressed simultaneously, and these σ elements will be combined with the optimal pool of expressed core elements to perform independent functions (e.g., RNA generation and lysin expression) at different expression levels. The inventors unexpectedly found that the use of split RNAP as described above not only allows better control of the expression level of the final product, but also causes reduced toxicity compared to the use of the promoter system previously described.

[0053] In a second aspect, the present invention provides a live attenuated Gram-negative bacterium comprising: i) a heterologous polynucleotide encoding an RNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter; and ii) a polynucleotide encoding an RNA polymerase that is orthogonal to components required for endogenous gene expression in Gram-negative bacteria, wherein the RNA polymerase binds to the promoter, and wherein following invasion of a eukaryotic cell by the Gram-negative bacterium, the RNA molecule is transcribed and capable of translocating into the cytoplasm of the eukaryotic cell.

[0054] The heterologous polynucleotide of the second aspect may be operably connected to a promoter that can be combined by RNA polymerase, and the component required for endogenous gene expression in this RNA polymerase and host Gram-negative bacteria is orthogonal. Therefore, in one embodiment, the promoter / RNA polymerase pairing is orthogonal to the component required for endogenous gene expression in the host Gram-negative bacteria. In this case, the component required for endogenous gene expression in the host Gram-negative bacteria can comprise transcription and translation system. By effectively isolating some system (for example, non-endogenous RNA polymerase / promoter pairing) and host component (for example, component required for endogenous gene expression in the host Gram-negative bacteria), can orthogonally regulate gene expression. In host Gram-negative bacteria, the component required for endogenous gene expression in the orthogonal RNA polymerase and host Gram-negative bacteria is incompatible and cannot interact. Orthogonal RNA polymerase can be non-endogenous for host Gram-negative bacteria, and only interacts with its compatible promoter, therefore only starts transcription from these promoters. Non-endogenous (or orthogonal) RNA polymerases do not interact with endogenous components of the host Gram-negative bacteria (e.g., endogenous promoters and gene sequences). Likewise, orthogonal promoters do not interact with endogenous RNA polymerases.

[0055] In some embodiments, the present invention discloses a live attenuated Gram-negative bacterium comprising: i) a heterologous polynucleotide encoding an RNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter; and ii) a polynucleotide encoding an RNA polymerase that is orthogonal to components required for endogenous gene expression in the Gram-negative bacterium, wherein the RNA polymerase is bound to the promoter, wherein the RNA polymerase is encoded by a heterologous split-type RNA polymerase plasmid, and wherein following invasion of the eukaryotic cell by the Gram-negative bacterium, the RNA molecule is transcribed and capable of translocating into the cytoplasm of the eukaryotic cell.

[0056] In order for RNA polymerase in Gram-negative bacteria within a target eukaryotic cell to successfully transcribe an RNA molecule, the heterologous polynucleotide encoding the RNA molecule is linked to a promoter to which RNA polymerase binds to initiate transcription of the RNA molecule.

[0057] In a preferred embodiment of the first and second aspects, the promoter that is operably connected to the heterologous polynucleotide encoding the RNA molecule and is combined with RNA polymerase is a phage promoter. In a more preferred embodiment, the promoter that is operably connected to the heterologous polynucleotide encoding the RNA molecule and is combined with RNA polymerase is a strong phage promoter, so that when using a strong phage promoter, a high transcription rate is started. Therefore, in a preferred embodiment of the first aspect of the present invention, the phage promoter is orthogonal to the component required for endogenous gene expression in gram-negative bacteria. With regard to a second aspect of the present invention, the phage promoter is orthogonal to the component required for endogenous gene expression in gram-negative bacteria.

[0058] The term "bacteriophage promoter" as used herein refers to a promoter derived from a virus (particularly a bacteriophage virus) that infects and replicates only in bacterial cells. Examples of bacteriophage promoters include, but are not limited to, T7p, T3p, K1Fp, and CGGp. In a preferred embodiment, the bacteriophage promoter is CGGp. In this case, a polymerase derived from the virus (i.e., a bacteriophage RNA polymerase) is paired with the bacteriophage promoter. In a preferred embodiment of the second aspect of the present invention, the best performing system may comprise a CGGp bacteriophage orthogonal promoter paired with a split RNA polymerase (CGG).

[0059] Examples of possible promoter / RNA polymerase pairings of the first aspect of the present invention are shown in Table 1. Therefore, the following RNA polymerases are split-type RNA polymerases. In one embodiment, the promoter that is operably connected to the heterologous polynucleotide encoding the RNA molecule and bound to the RNA polymerase is a T7 promoter, and the RNA polymerase is a T7 split-type RNA polymerase. In one embodiment, the promoter that is operably connected to the heterologous polynucleotide encoding the RNA molecule and bound to the RNA polymerase is a T3 promoter, and the RNA polymerase is a T3 split-type RNA polymerase. In one embodiment, the promoter that is operably connected to the heterologous polynucleotide encoding the RNA molecule and bound to the RNA polymerase is a K1F promoter, and the RNA polymerase is a K1F split-type RNA polymerase. In a preferred embodiment, the promoter that is operably connected to the heterologous polynucleotide encoding the RNA molecule and bound to the RNA polymerase is a CGG promoter, and the RNA polymerase is a CGG split-type RNA polymerase. In one embodiment, the above-mentioned promoter / RNA polymerase pairings are all orthogonal to the components required for endogenous gene expression in the host Gram-negative bacteria.

[0060] The present inventors unexpectedly showed that the T7 polymerase (using the T7 promotor) of high expression levels is toxic. In addition, although the inventors show that the T7 polymerase of low expression levels allows cell growth, even the T7 polymerase of low expression levels also can cause loop output efficiency to be low. Therefore, in one embodiment, the promoter that is operably connected with the heterologous polynucleotides encoding RNA molecules and is combined with RNA polymerase is not the T7 promotor, and RNA polymerase is not the T7 split-type RNA polymerase. In another embodiment, the promoter that is operably connected with the heterologous polynucleotides encoding RNA molecules and is combined with RNA polymerase is not the T7 promotor, and RNA polymerase is not the T7 RNA polymerase.

[0061] In one embodiment, the T7 promoter and RNA polymerase pairing are not orthogonal. In another embodiment, the T7 promoter and split RNA polymerase pairing are not orthogonal.

[0062]

[0063] Table 1: Examples of possible promoter / RNA polymerase pairings. A tick symbol (√) indicates a pairing between a promoter and the sigma factor of an RNA polymerase.

[0064] In one embodiment, the sigma factor RNA polymerase can have a polynucleotide sequence according to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or a sequence having 90% sequence identity thereto. For example, the sigma factor RNA polymerase can have a polynucleotide sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0065] In one embodiment, the promoter operably linked to the heterologous polynucleotide encoding the RNA molecule and bound to the RNA polymerase can have a polynucleotide sequence according to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or a sequence having 90% sequence identity thereto. For example, the promoter operably linked to the heterologous polynucleotide encoding the RNA molecule and bound to the RNA polymerase can have a polynucleotide sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0066] In one embodiment, the sigma factor RNA polymerase has a polynucleotide sequence according to SEQ ID NO: 3, or a sequence having 90% sequence identity thereto, and the promoter operably linked to the heterologous polynucleotide encoding the RNA molecule and to which the RNA polymerase binds has a polynucleotide sequence according to SEQ ID NO: 11, or a sequence having 90% sequence identity thereto. In another embodiment, the sigma factor RNA polymerase has a polynucleotide sequence according to SEQ ID NO: 5, or a sequence having 90% sequence identity thereto, and the promoter operably linked to the heterologous polynucleotide encoding the RNA molecule and to which the RNA polymerase binds has a polynucleotide sequence according to SEQ ID NO: 12, or a sequence having 90% sequence identity thereto. In another embodiment, the sigma factor RNA polymerase has a polynucleotide sequence according to SEQ ID NO: 7, or a sequence having 90% sequence identity thereto, and the promoter operably linked to the heterologous polynucleotide encoding the RNA molecule and to which the RNA polymerase binds has a polynucleotide sequence according to SEQ ID NO: 13, or a sequence having 90% sequence identity thereto. In a preferred embodiment, the sigma factor RNA polymerase has a polynucleotide sequence according to SEQ ID NO: 9, or a sequence having 90% sequence identity thereto, and the promoter operably linked to the heterologous polynucleotide encoding the RNA molecule and to which the RNA polymerase binds has a polynucleotide sequence according to SEQ ID NO: 14, or a sequence having 90% sequence identity thereto.

[0067] As used herein, the terms "sequence identity" and "sequence homology" are interchangeable and refer to the number of identical residues over a specified length in a given DNA sequence or amino acid sequence alignment. To calculate the percent sequence identity of any sequence disclosed herein, sequence comparison software can be used, for example, using the default settings on the BLAST package (V2.10.1).

[0068] To drive expression of the RNA polymerases of the first and second aspects of the invention (ie, the split-type RNA polymerase system and the non-split-type RNA polymerase system), intracellular promoters, such as cytosol- and vacuole-dependent promoters, are used.

[0069] In the context of the present invention, these promoters can be used to activate the sigma factors of the above-mentioned RNA polymerase systems (e.g., the sigma factors disclosed in Table 1) under specific conditions, such as at a specific time or at a specific time in the infection cycle. The promoters are dispersed throughout the genome of Gram-negative bacteria (e.g., Salmonella enterica), are located upstream of the relevant genes, and are activated by a large number of signals. For example, vacuolar promoters typically respond to acidification and hypotonicity, while cytosol promoters typically respond to iron, manganese, hexose, and oxidative stress. In the context of a split RNA polymerase system, multiple sigma factors may be driven by the above-mentioned intracellular promoters. For example, the expression of one sigma factor may be driven by a cytosol promoter, while the expression of a different second sigma factor may be driven by a vacuole-dependent promoter. Alternatively, the expression of two different sigma factors may be driven by two cytosol promoters or two vacuole-dependent promoters.

[0070] Therefore, in a particularly preferred embodiment, the promoter used to activate the RNA polymerase system of the first and second aspects of the invention is a cytosol promoter or a vacuole-dependent promoter. The term "cytosol promoter" as used herein refers to an intracellular promoter in bacteria. In a more preferred embodiment, the cytosolic promoter used in the present invention may include, but is not limited to, uhpT (SEQ ID NO: 23), mntH (SEQ ID NO: 31), entC (SEQ ID NO: 25), fhuE (SEQ ID NO: 26), iRON (SEQ ID NO: 35), fepB (SEQ ID NO: 36), fepA (SEQ ID NO: 32), fhuA (SEQ ID NO: 29), sitA (SEQ ID NO: 30), stn3250 (SEQ ID NO: 45), sufA (SEQ ID NO: 33), yjjZ (SEQ ID NO: 28), soxS (SEQ ID NO: 34), sfbA (SEQ ID NO: 27), and the vacuole-dependent promoter is selected from zinT (SEQ ID NO: 22), mtgC (SEQ ID NO: 47), ssaG (SEQ ID NO: 48), sseJ (SEQ ID NO: 47), sseA (SEQ ID NO: 49), or sifA (SEQ ID NO: 50). NO:50).

[0071] The use of such promoters allows precise control of the activation of the RNA polymerase system, for example, activation can be initiated at a specific location or time of the infection cycle. In addition, temporal control of expression can be achieved by using intracellular promoters (SPI-2), oxidative stress responsive promoters (e.g., soxP, grxA), sugar sensing promoters (e.g., uphT (glucose-6-phosphate), frubKA (fructose) promoters) or metal dependent promoters (e.g., iroN, fhuE, mntH, entC, fepB). These cytosol promoters can be used to drive differential expression of lysins and RNA polymerases. The SPI-2 promoter can also be used to activate the expression of the type 1 secretion system (T1SS), which helps hemolysins to be exported from Listeria and, for example, enables bacterial cells to be released from the vacuole into the cytoplasm of eukaryotic cells, thereby enabling the bacteria to enter the cytoplasm of eukaryotic cells.

[0072] In order to regulate the total amount of polymerase activity, an in vivo promoter or constitutive promoter can be utilized. As used herein, the term "constitutive promoter" refers to a promoter that initiates transcription only when exposed to a specific stimulus. In the context of the split-type RNA polymerase of the present invention, this type of promoter is responsible for generating the "core" of the RNA polymerase, thereby regulating the extent to which the "sigma factor" can bind.

[0073] The invention disclosed herein relates to a method for transferring RNA molecules transcribed in bacteria to the intracellular sol of eukaryotic host cells. In order to start the transcription of RNA molecules (such as mRNA, siRNA or shRNA molecules), Gram-negative bacteria must first invade the target eukaryotic cell. The RNA molecule can be transferred to the cytoplasm (for example, in the vacuole of the target eukaryotic cell) in a variety of ways, or the Gram-negative bacteria can be encoded to self-cleave by comprising a lytic protein, thereby releasing the transcribed RNA molecule. It is ideal to release DNA or RNA molecules from the phagocytic vacuole of the bacteria, as demonstrated by the higher in vitro bacterial transfection efficiency of the cytosol pathogen Listeria. Therefore, in one embodiment, the bacteria is designed to allow escape from the phagocytic vacuole. For example, escaping from the vacuole (SCV) containing Salmonella can be achieved in at least two ways. First, by adding exogenous genes. For example, in Salmonella, expressing hly from Listeria results in the use of a type 1 secretion system to export from the cytosol of the bacteria to the SCV. Secondly, the SCV is destabilized and released into the cytosol by manipulating the SPI-1 (or SPI-2) effector proteins (sopF and sopE) of Salmonella. The effector proteins are injected into the cytosol of invaded mammalian cells and participate in the maintenance of early vacuoles. Literature shows that invasion with strains lacking sopF or overexpressing sopE gene products leads to early vacuolar escape and establishment in the host cytosol or re-encapsulation through autophagy.

[0074] As described above, the Gram-negative bacteria of the present invention can be encoded to undergo self-cleavage by containing a lytic protein to release the transcribed RNA molecule into the cytoplasm of the eukaryotic host cell. One method for achieving this goal is to use a cytosolic promoter. Upon entering the cytoplasm of a eukaryotic cell, bacteria carrying a cytosolic promoter will induce their own cleavage and produce RNA that enters directly into the cytosol, thereby avoiding the transfer of genetic material into the nucleus of the target eukaryotic cell.

[0075] In the context of Listeria, the present inventors have discovered that the use of a cytosolic promoter can increase the release of RNA molecules from Listeria by approximately tenfold. Therefore, in a preferred embodiment, bacterial cell lysis is achieved by using a cytosolic promoter. Bacterial cell lysis can also be achieved by using lysins or bacterially released proteins, such as lysE, kilR, kil, or BRP.

[0076] In addition to the promoter and the RNA polymerase bound to the promoter, the attenuated live Gram-negative bacteria may also include many other auxiliary proteins that optimize the delivery of RNA molecules to target eukaryotic cells. For example, the attenuated live Gram-negative bacteria may also include one or more of the following auxiliary proteins integrated into the Gram-negative bacterial genome: i) a polynucleotide encoding an RNA stability enhancing component, preferably (in the case of an mRNA molecule) wherein the RNA stability enhancing component is an IRES sequence; ii) a polynucleotide encoding a lytic protein, preferably wherein the lytic protein is a hemolysin; iii) a polynucleotide encoding a phospholipase, preferably wherein the phospholipase is a phospholipase C; iv) a polynucleotide encoding an invasion factor; and / or v) a bacteriocin release protein, a bacteriophage lambda lysozyme, and a perforin. In a preferred embodiment, the lytic protein is derived from Listeria spp., the phospholipase is derived from Clostridium spp. or Listeria spp., and the invasion factor is derived from Yersinia spp. The polynucleotides encoding bacteriocin release protein, bacteriophage lambda lysozyme and perforin enable the bacteria to self-lyse after entering the target eukaryotic cell.

[0077] The invention provides a kind of mode that can silence target gene, prevent or reduce protein synthesis or synthesize new protein.As those skilled in the art will appreciate, this method has significant and extensive therapeutic benefit.In order to allow heterologous polynucleotide to be transcribed and produce RNA molecule in Gram-negative bacteria, heterologous polynucleotide is operably connected to functional promoter, wherein Gram-negative bacteria also comprises the heterologous polynucleotide of encoding suitable RNA polymerase.These components act together so that RNA molecule (being intended for being delivered to eukaryotic cells) can be synthesized in Gram-negative bacteria.The method that heterologous genetic material is incorporated into bacterial genome is well known to those skilled in the art, and will be described in further detail below.

[0078] The attenuated live Gram-negative bacteria of the present invention can be used as an effective and reliable method for delivering heterologous RNA to target eukaryotic cells. Therefore, the bacterial strains disclosed herein are recombinant strains comprising polynucleotides encoding RNA molecules (i.e., RNA molecules such as mRNA, siRNA, shRNA, saRNA or miRNA).

[0079] The functional RNA molecule delivered to eukaryotic cells can be messenger RNA (mRNA). Messenger RNA is transcribed from DNA and contains the genetic blueprint for protein production. Prokaryotic mRNA does not require processing and can immediately synthesize proteins. In eukaryotes, newly transcribed RNA transcripts are considered pre-mRNA and require maturation to form mRNA. Pre-mRNA contains non-coding and coding regions, known as introns and exons, respectively. During pre-mRNA processing, introns are spliced ​​and exons are joined together. A 5' cap, called 7-methylguanosine, is added to the 5' end of the RNA transcript, and the 3' end is polyadenylated. Polyadenylation refers to the process by which a poly(A) tail (i.e., a sequence of adenine nucleotides) is added to the transcript. The 5' cap protects the mRNA from degradation, while the 3' poly(A) tail contributes to the stability of the mRNA and aids its transport. The live attenuated Gram-negative bacterium may 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.

[0080] The mRNA of the present invention is transcribed in Gram-negative bacteria and subsequently transferred to the target eukaryotic cell for translation using the translation system of the host eukaryotic cell. Therefore, the invention disclosed herein provides an mRNA molecule with the above-mentioned modification. In another embodiment, the heterologous polynucleotide encodes a mammalian mRNA molecule. In one embodiment, the mRNA is a self-amplifying RNA (saRNA). The use of saRNA may lead to enhanced RNA delivery efficiency and increased expression of RNA molecules. The saRNA molecule is capable of "self-amplification" because the polynucleotide sequence encoding the RNA molecule also encodes a replicase that enables the amplification of the original chain of the RNA after delivery to the host cell. Therefore, the use of saRNA can achieve the above-mentioned advantages while requiring only a minimum dose of RNA.

[0081] The polypeptide encoded by the mRNA molecule can be any therapeutic protein for treating or preventing a disease. It is readily understood that the specific therapeutic protein will depend on a variety of factors, particularly the specific disease to be treated. In some embodiments, the attenuated live Gram-negative bacteria do not encode heterologous polypeptides that modify the genome of the target eukaryotic cell, for example, the Gram-negative bacteria of the present invention do not encode components of the CRISPR / Cas9 gene editing system, zinc finger nucleases, or RNA encoding transcription activator-like effector nucleases (TALENs).

[0082] In another embodiment, the functional RNA molecule is an siRNA or shRNA molecule that targets a specific mRNA molecule present in eukaryotic cells. After transcription of the siRNA or shRNA molecule in bacteria, the transcribed siRNA or shRNA molecule is transferred to the cytoplasm of the eukaryotic cell, where it localizes and binds to the target mRNA. Therefore, the invention disclosed herein also provides a method for effectively silencing a target gene by degrading the corresponding mRNA through siRNA or shRNA molecules.

[0083] The present invention can target a single mRNA or multiple mRNAs using siRNA and / or shRNA molecules. Thus, the attenuated live Gram-negative bacteria of the present invention can deliver a single siRNA molecule or multiple siRNA molecules, each targeting a different target mRNA. For example, the attenuated live Gram-negative bacteria can encode up to 10 different heterologous siRNA and shRNA molecules, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 different heterologous siRNA or shRNA molecules.

[0084] In some embodiments, the target mRNA can be any mRNA encoding a protein of interest, for example, encoding a protein mRNA relevant to a disease state. For example, the target mRNA can include mRNAs that participate in proliferation / cell cycle, migration, angiogenesis, immune activation / inhibition, cell death, and fibrosis. In addition, the target mRNA can include but is not limited to the various transcription factors of the HIF1 protein family, the TGFb receptor protein family, and driver cancer cell imbalance. It is readily understood that the targeted specific mRNA will depend on various factors, especially the specific disease to be treated. In some embodiments, attenuated live gram-negative bacteria do not encode heterologous siRNA or shRNAs that modify the target eukaryotic cell genome.

[0085] The attenuated live bacteria of the present invention are gram-negative bacteria. Examples of gram-negative bacteria used in the present invention include, but are not limited to, Escherichia coli, Salmonella, Shigella, Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, Legionella, Chlamydia, and Yersinia.

[0086] Preferably, the attenuated live Gram-negative bacteria are Salmonella species. Examples of Salmonella species used in the present invention are Salmonella enterica and Salmonella bongori. Salmonella enterica can be further divided into different serotypes or serovars. Examples of serotypes or serovars used in the present invention are Salmonella enterica Typhi, Salmonella enterica Paratyphi A, Salmonella enterica Paratyphi B, Salmonella enterica Paratyphi C, Salmonella enterica Typhimurium, and Salmonella enterica Enteritidis. In a preferred embodiment, the attenuated live Gram-negative bacteria are Salmonella enterica Typhi and / or Salmonella enterica Typhimurium. In a most preferred embodiment, the attenuated live Gram-negative bacteria are Salmonella enterica Typhi.

[0087] In another embodiment of the present invention, the live attenuated Gram-negative bacteria are genetically engineered non-natural bacteria.

[0088] Therefore, the present invention discloses genetically altered Gram-negative bacteria to produce attenuated bacterial strains that can effectively deliver RNA molecules as their goods. It will be appreciated by those skilled in the art that genes can be mutated by many well-known methods in the art, such as homologous recombination with the recombinant plasmid of the gene of interest targeted, in which case the engineered gene with homology to the target gene is incorporated into an appropriate nucleic acid vector (such as a plasmid or phage), which is then transfected into the target cell. The homologous engineered gene is then recombined with the native gene to replace the native gene or to mutate the native gene, thereby achieving desired inactivation mutations. This modification may be in the coding portion or any regulatory portion of the gene, such as a promoter region. It will be appreciated by those skilled in the art that any appropriate genetic modification technology (such as CRISPR / Cas systems, such as CRISPR / Cas9) can be used to mutate the gene of interest, thereby producing bacterial strains disclosed herein.

[0089] Therefore, the various methods and techniques for genetically engineered bacterial strains are well known to those skilled in the art. These techniques include the technology required for heterologous genes to be introduced into bacteria by chromosome integration or by introducing stable autosome self-replicating genetic elements. Exemplary methods for genetically modifying bacterial cells (also referred to as "conversion" or "engineered") include phage infection, transduction, conjugation, lipofection or electroporation. A general discussion of these methods and other methods in molecule and cell biochemistry can be found in the following 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); These standard textbooks are incorporated herein by reference.

[0090] The attenuated live Gram-negative bacteria can be selected from Ty21a, CVD 908-htrA, CVD 909, Ty800, M01ZH09, x9633, x639, x9640, x8444, DTY88, ZH9PA, MD58, WT05, ZH26, SL7838, SL7207, VNP20009, A1-R or any combination thereof. In a preferred embodiment, the attenuated live Gram-negative bacteria is M01ZH09 (also referred to as ZH9).

[0091] In one embodiment, the non-natural bacteria of genetic engineering can be derived from Salmonella species, which can include an attenuating mutation in the Salmonella pathogenicity island 2 (SPI-2) gene and an attenuating mutation in a second gene. Suitable genes and details of such attenuated live bacteria are described in WO2000 / 68261, which is incorporated herein by reference in its entirety.

[0092] In one embodiment, the SPI-2 gene is an ssa gene. For example, the present invention includes attenuating mutations in one or more of ssaV, ssaJ, ssaU, ssaK, ssaL, ssaM, ssaO, ssaP, ssaQ, ssaR, ssaS, ssaT, ssaD, ssaE, ssaG, ssaI, ssaC, and ssaH. Preferably, the attenuating mutation is in the ssaV or ssaJ gene. Even more preferably, the attenuating mutation is in the ssaV gene.

[0093] The non-natural bacteria of genetic engineering can also be included in the attenuation mutation in the second gene, and this gene may be located in or not located in the SPI-2 zone.This mutation may be outside the SPI-2 zone, and participates in the biosynthesis of aromatic compounds.For example, the present invention is included in the attenuation mutation in the aro gene.In a preferred embodiment, the aro gene is aroA or aroC.Even more preferably, the aro gene is aroC.

[0094] The genetically engineered non-natural bacteria may also contain one or more gene cassettes that can be used to deliver additional prokaryotic molecules to support the function of the genetically engineered non-natural bacteria in modulating the immune system or supporting the activity of a therapeutic protein encoded by an mRNA molecule.

[0095] In another embodiment, the genetically engineered non-natural bacteria can be derived from a Salmonella species and can comprise an inactivating mutation in one or more genes selected from pltA, pltB, cdtB, and ttsA, and further comprise an attenuating mutation in one or more genes selected from aroA and / or aroC and / or ssaV. Details of the genes and mutations are as described in WO2019 / 110819, which is incorporated herein by reference in its entirety.

[0096] It is envisaged that an inactivating mutation (e.g., deletion) in the genes pltA, pltB, and cdtB will prevent the Salmonella species from producing typhoid toxin, and an inactivating mutation (e.g., deletion) in ttsA will prevent the Salmonella species from secreting typhoid toxin. It is envisaged that such a non-natural bacterium may specifically be derived from Salmonella enterica.

[0097] The present invention allows safe, efficient and effective delivery of heterologous RNA molecules to target eukaryotic cells. In the case where the RNA molecule is an mRNA molecule, the resulting heterologous polypeptide can be a therapeutic protein and / or a heterologous antigen (depending on the indication to be treated, for example, in the context of a vaccine composition, the heterologous polypeptide can be a heterologous antigen). In a preferred embodiment, the resulting therapeutic protein is a cytokine, a chemokine, an antibody or a fragment thereof, a cytotoxic agent, an anticancer agent, or any combination thereof. Even more preferably, the resulting therapeutic protein can be IL-15, IL-21, CXCL9, IL-18, IL-27, IFNγ, IL-1, or any combination thereof. In another embodiment, the heterologous siRNA or shRNA molecule is delivered to the target eukaryotic cell using the siRNA or shRNA delivery method disclosed herein. Using this method, any mRNA molecule can be effectively targeted, and in fact the related gene can be silenced. Therefore, the present invention has special uses in disease states where preventing the production of a specific protein would be beneficial.

[0098] In a preferred embodiment of the present invention, attenuated live gram-negative bacteria is used, intravenously, intraperitoneally or orally by tumor.In a most preferred embodiment, attenuated live gram-negative bacteria is used by tumor.But, in some cases, also consider that other methods of administration can be used.Therefore, in some cases, attenuated live gram-negative bacteria of the present invention can be by injection, infusion, continuous infusion, intradermal, intraarterial, intralesional, intravaginal, intrarectal, intramuscular, subcutaneous, subconjunctival, mucosa, pericardium, umbilical, intraocular, intracranial, intraarticular, prostate, intrapleural, intratracheal, intranasal, suction (for example, atomized inhalation), via conduit, via lavage or by any combination of additive method or aforementioned approach, as known to those of ordinary skill in the art (referring to for example, Remington's Pharmaceutical Sciences, 18th Ed.Mack Printing Company, 1990).

[0099] The amount of attenuated live Gram-negative bacteria administered to a subject is sufficient to deliver the heterologous RNA molecule to the target eukaryotic cells at a sufficiently high concentration so that the heterologous RNA molecule, once translated in the eukaryotic cells, can produce the desired effect. Those skilled in the art will readily appreciate that the precise amount to be administered will depend on many factors, such as the disease to be treated, the desired protein to be translated (in the case where the RNA molecule is an mRNA molecule), the translation level of the target mRNA present in the target eukaryotic cells (in the case where the RNA molecule is an siRNA or shRNA molecule), and the medical history of the subject to be treated.

[0100] The dosage of attenuated live Gram-negative bacteria can be 10 5 to 1012 During CFU, among them, CFU is the place where bacterial drop formation occurs. For example, it is possible to exceed the amount of the match 10 5 CFU up to 10 6 CFU, 10 5 CFU up to 10 7 CFU, 10 5 CFU up to 10 8 CFU, 10 5 CFU up to 10 9 CFU, 10 5 CFU up to 10 10 CFU, 10 5 CFU up to 10 11 CFU, 10 6 CFU up to 10 7 CFU, 10 6 CFU up to 10 8 CFU, 10 6 CFU up to 10 9 CFU, 10 6 CFU and 10 10 CFU, 10 6 CFU and 10 11 CFU, 10 6 CFU and 10 12 CFU, 10 7 CFU and 10 8 CFU, 10 7 CFU and 10 9 CFU, 10 7 CFU and 10 10 CFU, 10 7 CFU and 10 11 CFU, 10 7 CFU and 10 12 CFU, 10 8 CFU and 10 9 CFU, 10 8 CFU and 10 10 CFU, 10 8 CFU and 10 11 CFU, 10 8 CFU and 10 12 CFU, 10 9 CFU and 10 10 CFU, 10 9 CFU and 10 11 CFU, 10 9 CFU and 10 12 CFU, 10 10 CFU and 10 11 CFU, 1010 CFU and 10 12 CFU, or 10 11 CFU and 10 12 CFU. The live attenuated Gram-negative bacteria can be administered in a single dose or multiple doses. The specific number of doses to be administered depends on the specific RNA molecule to be delivered or targeted, as well as the specific indication to be treated.

[0101] The present invention aims to provide an effective and efficient method for delivering RNA molecules to target eukaryotic cells for translation into desired therapeutic proteins (when the RNA molecules are mRNA molecules), or delivering iRNA molecules to target eukaryotic cells (when the RNA molecules are siRNA or shRNA molecules). The target eukaryotic cells can be mammalian cells. In a preferred embodiment, the target eukaryotic cells are human cells. When the eukaryotic cells are human cells, the target cells can be human cancerous cells or human non-cancerous cells.

[0102] The attenuated live Gram-negative bacteria of the first or second aspects of the present invention 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 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.

[0103] When the disease to be treated is a neoplastic disease, the neoplastic disease can be relevant to solid tumors and / or hematologic malignancies. These diseases include sarcoma, carcinoma, adenocarcinoma, melanoma, myeloma, blastoma, glioma, lymphoma or leukemia. In a preferred embodiment, the neoplastic disease is relevant to solid tumors. In a specific aspect, the neoplastic disease is relevant 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, cervical cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer or sarcoma.

[0104] Neoplasias, tumors, and cancers include benign, malignant, metastatic, and non-metastatic types, and include any stage (I, II, III, IV, or V) or grade (G1, G2, G3, etc.), or a neoplasia, tumor, cancer, or metastasis that is progressing, worsening, stable, or in remission. Cancers that can be treated according to the present invention include, but are not limited to, cells or tumors of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testicles, tongue, or uterus. Furthermore, the cancer may be specifically of the following but not limited to the following histological types: malignant tumor; carcinoma; undifferentiated; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatricoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; bile duct carcinoma; hepatocellular carcinoma; mixed hepatocellular and bile duct carcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial polyposis coli; solid carcinoma; malignant carcinoid tumor; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe cell carcinoma; oncocytic carcinoma; oncocytic adenocarcinoma; basophilic Granulocyte carcinoma; clear cell adenocarcinoma; granulosa cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinomas; nonencapsulated sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; carcinoma of the skin appendages; apocrine gland carcinoma; sebaceous gland carcinoma; cerumenic gland carcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant theca cell tumor Malignant granulosa cell tumor of the ovary; Malignant androblastoma; Sertoli cell carcinoma; Malignant Leydig cell tumor; Malignant lipid cell tumor; Malignant paraganglioma; Malignant extramammary paraganglioma; Pheochromocytoma; Glomus sarcoma; Malignant melanoma; Amelanotic melanoma; Superficial spreading melanoma; Malignant melanoma in giant pigmented nevus; Epithelioid cell melanoma; Malignant blue nevus; Sarcoma; Fibrosarcoma; Malignant fibrous histiocytoma; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonic rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Mesenchymal sarcoma; mixed tumor; Mullerian mixed tumor; Wilms' tumor; hepatoblastoma; carcinosarcoma; malignant mesenchymal tumor; malignant Brenner's tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant ovarian goiter; choriocarcinoma; malignant mesonephroma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone;Ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontosarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; 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 neurilemoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's Hodgkin's disease; Hodgkin's; paragranulomatosis; small lymphocytic lymphoma; diffuse large cell lymphoma; follicular lymphoma; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small bowel disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocyte leukemia; myeloid cell sarcoma; and hairy cell leukemia. Preferably, the neoplastic disease may be a tumor associated with a cancer selected from prostate cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colorectal cancer, pancreatic cancer, brain cancer, hepatocellular carcinoma, lymphoma, leukemia, gastric 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 may be a metastatic tumor or a malignant tumor.

[0105] In a preferred embodiment, the neoplastic disease is associated with a cancer selected from the group consisting of bladder cancer, lung cancer, mesothelioma, hepatocellular carcinoma, melanoma, esophageal cancer, gastric cancer, ovarian cancer, colorectal cancer, head and neck cancer, or breast cancer.

[0106] In a third aspect, the present invention provides a vaccine composition comprising a live attenuated Gram-negative bacterium, wherein the Gram-negative bacterium comprises: i) a heterologous polynucleotide encoding a functional mRNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter and the mRNA molecule encodes a therapeutic protein and / or therapeutic peptide; and ii) a polynucleotide encoding an RNA polymerase that is orthogonal to components required for endogenous gene expression in the Gram-negative bacterium, wherein the RNA polymerase binds to the promoter, and wherein following invasion of the eukaryotic cell by the Gram-negative bacterium, the mRNA molecule is transcribed and capable of translocating into the cytoplasm of the eukaryotic cell.

[0107] In a fourth aspect, the present invention provides a vaccine composition comprising a live attenuated Gram-negative bacterium, wherein the Gram-negative bacterium comprises: i) a heterologous polynucleotide encoding a functional mRNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter and the mRNA molecule encodes a therapeutic protein and / or therapeutic peptide; and ii) a polynucleotide encoding an RNA polymerase, wherein the RNA polymerase is associated with the promoter, wherein the RNA polymerase is encoded by a heterologous split-type RNA polymerase plasmid, and wherein after the Gram-negative bacterium invades the eukaryotic cell, the mRNA molecule is transcribed and can be translocated into the cytoplasm of the eukaryotic cell.

[0108] In one embodiment, the present invention provides a vaccine composition comprising a live attenuated Gram-negative bacterium, wherein the Gram-negative bacterium comprises: i) a heterologous polynucleotide encoding a functional mRNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter and the mRNA molecule encodes a therapeutic protein and / or therapeutic peptide; and ii) a polynucleotide encoding an RNA polymerase, which is orthogonal to components required for endogenous gene expression in the Gram-negative bacterium, wherein the RNA polymerase is associated with the promoter, wherein the RNA polymerase is encoded by a heterologous split-type RNA polymerase plasmid, and wherein following invasion of the eukaryotic cell by the Gram-negative bacterium, the mRNA molecule is transcribed and capable of translocating into the cytoplasm of the eukaryotic cell.

[0109] In one embodiment, the vaccine composition of the present invention 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.

[0110] Thus, the vaccine composition of the third aspect or the fourth aspect may comprise one or more of the aforementioned embodiments for any of the aforementioned aspects.

[0111] The phage promoter of the vaccine composition of the third or fourth aspect of the present invention may be T3p, K1Fp or CGGp.

[0112] It is particularly contemplated that the vaccine compositions disclosed herein can be used for the treatment, reduction, suppression, prevention of recurrence, or control of infectious diseases, such as diseases caused by bacteria, viruses, parasites, or fungi. In this case, the heterologous polynucleotide to be transcribed into a functional mRNA molecule may be an antigen of the causative agent of a specific infectious disease to generate an immune response in the host. Alternatively, it is contemplated that the vaccine compositions disclosed herein can be used as cancer vaccines. In this case, the vaccine composition comprises a Gram-negative bacterium comprising a heterologous polynucleotide encoding a cancer antigen that is capable of generating an immune response in the host. Therefore, it can be appreciated that a wide range of cancers and infectious diseases can be prevented / treated using the bacteria and methods disclosed herein. In other cases, the heterologous polynucleotide will be transcribed into siRNA or shRNA molecules that are intended to enhance anti-infective immune function or tissue anti-infective defense.

[0113] The vaccine composition of the present invention may further comprise an adjuvant, a pharmaceutically acceptable carrier or an excipient.

[0114] As used herein, "pharmaceutically acceptable carriers / adjuvants / diluents / excipients" include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, and the like, and combinations thereof, known to those of ordinary skill in the art (e.g., see Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Examples include, but are not limited to, disodium hydrogen phosphate, soy peptone, potassium dihydrogen phosphate, ammonium chloride, sodium chloride, magnesium sulfate, calcium chloride, sucrose, borate buffer, sterile saline solution (0.9% NaCl), and sterile water.

[0115] Suitable aqueous and non-aqueous carriers that can be used in the vaccine compositions of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (e.g., olive oil) and injectable organic esters (e.g., ethyl oleate). Appropriate fluidity can be maintained, for example, by using coating materials (e.g., lecithin), by maintaining the desired particle size in the case of dispersions, and by using surfactants.

[0116] The vaccine composition disclosed in the present invention may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. The presence of unwanted microorganisms can be ensured by the above-mentioned sterilization procedures and by including various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol sorbic acid, etc.). It may also be desirable to include isotonic agents such as sugars, sodium chloride, etc. in the composition. In addition, by including agents that delay absorption (e.g., aluminum monostearate and gelatin), the absorption of injectable drug forms can be prolonged. The vaccine composition may also optionally include known additional therapeutic agents that are effective for, for example, infectious diseases or tumor diseases. Therefore, the vaccine composition disclosed herein may also include antiretroviral drugs, antibiotics, antifungals, antiparasitic drugs, and anticancer drugs.

[0117] 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 (e.g., trehalose dimycolate), bacterial lipopolysaccharide (LPS), peptidoglycans (e.g., mureins, mucopeptides, or glycoproteins such as N-Opaca, muramyl dipeptide [MDP], or MDP analogs), proteoglycans (e.g., from Klebsiella pneumoniae), and proteoglycans. pneumoniae), streptococcal preparations (e.g., OK432), muramyl dipeptide, immunostimulatory complexes ("Iscoms" disclosed in EP 109942, EP 180564, and EP 231039), saponins, DEAE-dextran, neutral oils (e.g., miglyol), vegetable oils (e.g., peanut oil), liposomes, polyols, Ribi adjuvant system (see, e.g., GB-A-2189141), vitamin E, Carbopol, interferons (e.g., IFN-α, IFN-γ, or IFN-β), or interleukins (particularly those that stimulate cell-mediated immunity (e.g., 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)).

[0118] The live attenuated Gram-negative bacteria of the vaccine compositions disclosed herein can include any one or any combination of the characteristics of the live attenuated Gram-negative bacteria disclosed herein.

[0119] A fifth aspect of the present invention provides a method for treating, inhibiting, preventing recurrence or managing a disease in a subject, wherein the method comprises administering to the subject a live attenuated Gram-negative bacterium comprising: i) a heterologous polynucleotide encoding an RNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter; and ii) a polynucleotide encoding an RNA polymerase that is orthogonal to components required for endogenous gene expression in the Gram-negative bacterium, wherein the RNA polymerase is bound to the promoter, wherein the RNA polymerase is encoded by a heterologous split-type RNA polymerase plasmid, and wherein after the Gram-negative bacterium invades the eukaryotic cell, the RNA molecule is transcribed and capable of being translocated into the cytoplasm of the eukaryotic cell.

[0120] Therefore, the method of treating, inhibiting, preventing recurrence or managing a disease in a subject in the fifth aspect may include one or more of the aforementioned embodiments of any of the aforementioned aspects.

[0121] In one embodiment, a split-type RNA polymerase plasmid may be incorporated into the fifth aspect of the invention to enable safe, efficient, and effective delivery of RNA molecules to target eukaryotic cells.

[0122] In a sixth aspect, the present invention provides a method for treating, inhibiting, preventing relapse, or managing a disease in a subject, wherein the method comprises administering to the subject a live attenuated Gram-negative bacterium for treating, reducing, inhibiting, preventing relapse, or managing the disease, wherein the Gram-negative bacterium comprises i) a heterologous polynucleotide encoding an RNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter; and ii) a polynucleotide encoding an RNA polymerase that is orthogonal to components required for endogenous gene expression in the Gram-negative bacterium, wherein the RNA polymerase binds to the promoter, and wherein following invasion of the eukaryotic cell by the Gram-negative bacterium, the RNA molecule is transcribed and capable of translocating into the cytoplasm of the eukaryotic cell.

[0123] Therefore, the method of treating, inhibiting, preventing recurrence or controlling a disease in a subject in the sixth aspect may include one or more of the aforementioned embodiments of any of the aforementioned aspects.

[0124] In one embodiment, a cytosolic promoter may be incorporated into the sixth aspect of the invention to enable safe, efficient, and effective delivery of RNA molecules to target eukaryotic cells.

[0125] In a preferred embodiment, the live attenuated Gram-negative bacterium has any one or any combination of the characteristics of the live attenuated Gram-negative bacteria disclosed herein.

[0126] In a further preferred embodiment, the disease to be treated is a neoplastic disease or an infectious disease.

[0127] In a seventh aspect of the present invention, a method for delivering an RNA molecule into a eukaryotic cell is provided, the method comprising the following steps: i) modifying a Gram-negative bacterium such that a heterologous polynucleotide encoding the RNA molecule is integrated into the bacterial genome, wherein the heterologous polynucleotide is operably linked to a promoter; ii) contacting the Gram-negative bacterium with a eukaryotic cell such that the Gram-negative bacterium replicates within the eukaryotic cell such that the heterologous polynucleotide is transcribed and subsequently transferred from the Gram-negative bacterium to the cytoplasm of the eukaryotic cell.

[0128] Thus, the method of delivering an RNA molecule into a eukaryotic cell of the seventh aspect may include one or more of the aforementioned embodiments for any of the aforementioned aspects.

[0129] In a preferred embodiment, the live attenuated Gram-negative bacteria has any one or any combination of the characteristics of the live attenuated Gram-negative bacteria disclosed herein.In a further preferred embodiment, the method of delivering RNA molecules into eukaryotic cells is an in vivo method.

[0130] Example

[0131] The present invention is further described with reference to the following non-limiting examples:

[0132] Example 1: Construction and characterization of T7 RNAP expression in Salmonella enterica

[0133] Plasmids containing the T7 RNA polymerase (T7 RNAP) gene downstream of the SPI-2 inducible promoter (i.e., a promoter activated in the Salmonella-containing vacuole (SCV) during mid- to late-stage invasion) were assembled into the chloramphenicol-resistant pSC101 plasmid (~5 copies / cell) by Gibson assembly of DNA fragments containing appropriate overhangs synthesized by Integrated DNA Technologies (IDT). The assembled plasmids contained either the pipB, ssaG, sseJ, or ssrA promoters.

[0134] In parallel, a carbenicillin-resistant p15A plasmid containing mScarlet downstream of T7p (T7-responsive promoter) was assembled by inverse PCR and downgrading of pSEVA-16 (from the Standard European Vector Construction Collection) using appropriate phosphorylated primers.

[0135] After verification, the plasmids were co-transformed into Salmonella enterica Typhi ZH9 by electroporation and recovered in the presence of two antibiotics. Single colonies were isolated in a 4% culture medium supplemented with 25 μg mL of each antibiotic. -1The cells were grown in vLB liquid medium (Formedium) at 37°C and 200 rpm for 16 to 18 h, then diluted 1:10 in 100 μL PCN medium in a 96-well plate and grown in a Clariostar (BMG Labtech) at 37°C for 6 to 8 h, while growth and mScarlet fluorescence were recorded every 15 min. Toxicity and lack of growth (as OD 600 Additional toxicity verification was performed by plating 50 μL of the final culture in fresh vLBA medium (Formedium) containing the appropriate antibiotics and growing at 37° C. for 16 to 18 h (see FIG1 ).

[0136] Example 2: Construction of split RNAP circuit and expression plasmid

[0137] The RNA polymerase (RNAP) entry pSC101-ori plasmid containing the core RNAP sequence downstream of a weak promoter (proA) and a strong (H) or weak (L) ribosome binding site (RBS) was generated. A double terminator and two placeholder sequences were placed upstream of proA: (1) a promoter cloning site with two unique BsaI restriction sequences that allow cloning of any desired promoter by Golden Gate assembly; and (2) a sigma factor cloning site with an ultra-folded gfp sequence between two unique BbsI restriction sequences that allow cloning of any desired sigma factor by Golden Gate Assembly (see ). Figure 2 ).

[0138] Bacterial transfection expression plasmids were constructed based on plasmids with a p15A ori containing a phage-dependent promoter (e.g., T7, T3, K1F, or CGG) surrounded by a BbsI site upstream of a placeholder mScarlet sequence. This sequence can be replaced with any desired cargo for transfection via Golden Gate Assembly.

[0139] Example 3: Verification of the split RNAP circuit

[0140] Plasmids containing split RNAP circuits with σT7, T3, K1F or CGG σ factors were generated as described above. A first validation test was performed by cloning the promoter zinT (zinc-responsive) upstream of the σ factor region.

[0141] E. coli DH5α cells were co-transformed with the expression plasmid and all combinations of σ factors (see Example 2) and cultured overnight in vLB (Formedium) medium. Fluorescence was measured and observed only in samples containing the correctly paired promoter-σ system (see Figure 3 ).

[0142] The correctly paired promoter-σ plasmids were then co-transformed into Salmonella enterica ZH9 (no σ was used as a negative control) and grown in vLB medium at 37°C for 16 to 18 hours. The plasmid containing the CGG σ factor was the most fluorescent sample, while the plasmid expressing the strong T7 σ factor was toxic to ZH9 (see Figure 3 ).

[0143] To verify these results, the cytosolic promoter puhpT was cloned upstream of each σ factor element by BbsI-dependent Golden Gate reaction (using NEBridge as buffer and standard reaction conditions). As described above, these variants were co-transformed into Salmonella enterica ZH9 and grown in vLBA at 37°C for 16 to 18 hours. Single colonies were picked and grown in 100 μL vLB medium (supplemented with 0.4% glucose-6-phosphate (σ)) at 37°C and 700 rpm for 48 hours, while growth and mScarlet fluorescence were recorded. The results showed that ( Figure 3 ), after induction with glucose-6-phosphate and incubation at 37°C for 48 h, the results in liquid medium (vLB) were similar to those previously obtained in solid medium.

[0144] Example 4: Construction of promoter reporter plasmid

[0145] Promoter reporter plasmid vectors were constructed containing the constitutively expressed mScarlet gene under the control of the proB promoter and the inducible sfgfp gene encoded in various ways. A placeholder sequence was placed upstream of sfgfp to include two unique BsaI restriction sites that allow for the one-step introduction of any desired promoter via Golden Gate Assembly. Furthermore, once the promoter is characterized, sfgfp can be replaced with any gene of interest via BbsI-dependent Golden Gate Assembly.

[0146] Example 5: In vitro characterization of cytosolic plasmids

[0147] A promoter reporter plasmid constructed by BsaI-dependent Golden Gate Assembly (NEBridge as buffer, standard conditions) was used as a template to introduce a series of promoters that are assumed to be active when ZH9 is present in the cytosol or vacuole of mammalian cells (see Figure 4). Most promoters are metal-dependent and are active in response to different concentrations of Fe. 3+ (cytoplasm), Zn 2+ (vacuole), Mg 2+ (vacuole) or sugar (glucose-6-phosphate, cytosol) to respond. The resulting plasmid was transformed into ZH9, and the resulting strain was grown in vLBA at 37 ° C for 16 h to 18 h. Single colonies were picked and grown in vLB at 37 ° C for 16 h to 18 h, and then diluted 1:100 in 100 μL M9 minimal medium, with or without the corresponding induced metabolites (except uhpT (glucose-6-phosphate), zinT (Zn 2+ )、mtgC(Mg 2+ ), soxS (unknown), sfbA (unknown), all others are Fe 3+ The samples were then grown in a Clariostar at 37°C for 16 to 18 hours while recording the growth (OD 600 ) and sfGFP fluorescence.

[0148] All metal-dependent promoters showed repression of sfGFP after addition of the corresponding metabolites (see Figure 4). In the unknown cases, no significant changes were observed.

[0149] Example 6: Validation of inhibition of cytosolic promoters in the vacuole

[0150] Salmonella ZH9 previously transformed with a promoter reporter plasmid (see above) were prepared and subjected to invasion assays on SKOV-3 cells using standard protocols.

[0151] Briefly, a single colony was grown in vLB at 37°C for 16 to 18 h, then diluted into fresh vLB and grown at 37°C for approximately 5 h until the OD 600 The cell density was ~1.5. The cells were then pelleted by gentle centrifugation at 2000 x g for 15 min and washed twice with ice-cold PBS. After the final wash, the cells were resuspended in 10% glycerol PBS at a ratio of 1:10 of the initial culture volume, aliquoted in 200 μL of invasion stock solution, and stored at -80°C until needed. An aliquot was thawed and the cell concentration (CFU) was calculated by serially diluting the stock solution in PBS, plating it in vLB and growing it at 37°C for 16 to 18 hours. The CFU was determined by counting the number of viable colonies in the dilution series, usually at 10% of the stock solution. 10CFU / mL to 10 12 CFU / mL range.

[0152] On the day of the experiment, circulating SKOV-3 cells were incubated with previously thawed invasion stock at a multiplicity of infection (MOI) of 50 bacterial cells per mammalian cell. The two cell types were incubated together for 1 hour, after which uninvaded bacteria were washed away and killed with gentamicin. Samples were visualized 24 hours later, showing bacteria in the vacuole as red (i.e., only constitutive mScarlet expression), except in the case of the yjjZ promoter (see Figure 5 ).

[0153] Example 7: Verification of expression of the cytosol promoter puhpT in the cytosol

[0154] A promoter-reporter plasmid containing puhpT (see above) was transformed into the CD12 strain of Salmonella enterica (Salmonella enterica TMLΔaroCΔsifA), which is defective in the SifA SPI-2 effector, potentially allowing escape by vacuole rupture during the late stages of invasion. Bacterial invasion stocks were prepared as previously described and co-cultured with SKOV-3 cells at an MOI of 50, as previously described. Bacteria were imaged by microscopy 3, 6, and 24 hours after invasion. Escape was confirmed at 6 hours post-infection (see Figure 6), with bacteria showing signs of overreplication (outgrowing the host cells) and exhibiting green fluorescence.

[0155] Sequence forming part of the specification

[0156] SEQ ID NO:1-T7 core (DNA sequence)

[0157]

[0158] SEQ ID NO: 2 - T7 Core (Amino Acid Sequence)

[0159] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDEN

[0160] SEQ ID NO: 3 - T7 σ (DNA Sequence)

[0161] AAGAACACTGGTGAAATCTCTGAGAAAGTCAAGCTGGGCACTAAGGCACTGGCTGGTCAATGGCTGGCTTACGGTGTTACTCGCAGTGTGACTAAGAGTTCAGTCATGACGCTGGCTTACGGGTCCAAAGAGTTCGGCTTCCGTCAACAAGTGCTGGAAGATACCATTCAGCCAGCTATTGATTCCGGCAAGGGTCTGATGTTCACTCAGCCGAATCAGGCTGCTGGATACATGGCTAAGCTGATTTGGGAATCTGTGAGCGTGACGGTGGTAGCTGCGGTTGAAGCAATGAACTGGCTTAAGTCTGCTGCTAAGCTGCTGGCTGCTGAGGTCAAAGATAAAAAGACTGGAGAGATTCTTCGCAAGCGTTGCGCTGTGCATTGGGTAACTCCTGATGGTTTCCCTGTGTGGCAGGAATACAAGAAGCCTATTCAGACGCGCTTGAACCTGATGTTCCTCGGTCAGTTCCGCTTACAGCCTACCATTAACACCAACAAAGATAGCGAGATTGATGCACACAAACAGGAGTCTGGTATCGCTCCTAACTTTGTACACAGCCAAGACGGTAGCCACCTTCGTAAGACTGTAGTGTGGGCACACGAGAAGTACGGAATCGAATCTTTTGCACTGATTCACGACTCCTTCGGTACGATTCCGGCTGACGCTGCGAACCTGTTCAAAGCAGTGCGCGAAACTATGGTTGACACATATGAGTCTTGTGATGTACTGGCTGATTTCTACGACCAGTTCGCTGACCAGTTGCACGAGTCTCAATTGGACAAAATGCCAGCACTTCCGGCTAAAGGTAACTTGAACCTCCGTGACATCTTAGAGTCGGACTTCGCGTTCGCGTA

[0162] SEQ ID NO:4 - T7σ (Amino acid sequence)

[0163] KNTGEISEKVKLGTKALAGQWLAYGVTRSVTKSSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA

[0164] SEQ ID NO:5 - T3σ (DNA sequence)

[0165] AAGAACACTGGTGAAATCTCTGAGAAAGTCAAGCTGGGCACTAAGGCACTGGCTGGTCAATGGCTGGCTTACGGTGTTACTCGCAGTGTGACTAAGCGTTCAGTCATGACGCTGGCTTACGGGTCCAAAGAGTTCGGCTTCCGTCAACAAGTGCTGGAAGATACCATTCAGCCAGCTATTGATTCCGGCAAGGGTCTGATGTTCACTCAGCCGAATCAGGCTGCTGGATACATGGCTAAGCTGATTTGGGAATCTGTGAGCGTGACGGTGGTAGCTGCGGTTGAAGCAATGAACTGGCTTAAGTCTGCTGCTAAGCTGCTGGCTGCTGAGGTCAAAGATAAAAAGACTGGAGAGATTCTTCGCAAGCGTTGCGCTGTGCATTGGGTAACTCCTGATGGTTTCCCTGTGTGGCAGGAATACAAGAAGCCTATTCAGAAGCGCCTGGACATGATTTTCTTGGGTCAATTTCGCTTGCAACCTACCATTAACACCAACAAAGATAGCGAGATTGATGCACACAAACAGGAGTCTGGTATCGCTCCTAACTTTGTACACAGCCAAGACGGTAGCCACCTTCGTAAGACTGTAGTGTGGGCACACGAGAAGTACGGAATCGAATCTTTTGCACTGATTCACGACTCCTTCGGTACGATTCCGGCTGACGCTGCGAACCTGTTCAAAGCAGTGCGCGAAACTATGGTTGACACATATGAGTCTTGTGATGTACTGGCTGATTTCTACGACCAGTTCGCTGACCAGTTGCACGAGTCTCAATTGGACAAAATGCCAGCACTTCCGGCTAAAGGTAACTTGAACCTCCGTGACATCTTAGAGTCGGACTTCGCGTTCGCGTA

[0166] SEQ ID NO:6 - T3σ (Amino acid sequence)

[0167] KNTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAAKLLAAEVKDKKTGEIRLRKRCAVHWVTPDGFPVWQEYKK PIQKRLDMIFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA

[0168] SEQ ID NO:7-K1Fσ(DNA sequence)

[0169] AAGAACACTGGTGAAATCTCTGAGAAAGTCAAGCTGGGCACTAAGGCACTGGCTGGTCAATGGCTGGCTTACGGTGTTACTCGCAGTGTGACTAAGCGTTCAGTCATGACGCTGGCTTACGGGTCCAAAGAGTTCGGCTTCCGTCAACAAGTGCTGGAAGATACCATTCAGCCAGCTATTGATTCCGGCAAGGGTCTGATGTTCACTCAGCCGAATCAGGCTGCTGGATACATGGCTAAGCTGATTTGGGAATCTGTGAGCGTGACGGTGGTAGCTGCGGTTGAAGCAATGAACTGGCTTAAGTCTGCTGCTAAGCTGCTGGCTGCTGAGGTCAAAGATAAAAAGACTGGAGAGATTCTTCGCAAGCGTTGCGCTGTGCATTGGGTAACTCCTGATGGTTTCCCTGTGTGGCAGGAATACAAGAAGCCTATTCAGACGCGCTTGAACCTGAGGTTCCTCGGTTCGTTCAACCTCCAGCCGACCGTCAACACCAACAAAGATAGCGAGATTGATGCACACAAACAGGAGTCTGGTATCGCTCCTAACTTTGTACACAGCCAAGACGGTAGCCACCTTCGTAAGACTGTAGTGTGGGCACACGAGAAGTACGGAATCGAATCTTTTGCACTGATTCACGACTCCTTCGGTACGATTCCGGCTGACGCTGCGAACCTGTTCAAAGCAGTGCGCGAAACTATGGTTGACACATATGAGTCTTGTGATGTACTGGCTGATTTCTACGACCAGTTCGCTGACCAGTTGCACGAGTCTCAATTGGACAAAATGCCAGCACTTCCGGCTAAAGGTAACTTGAACCTCCGTGACATCTTAGAGTCGGACTTCGCGTTCGCGTA

[0170] SEQ ID NO:8 - K1Fσ (Amino acid sequence)

[0171] KNTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAAKLLAAEVKDKKTGEIRLRKRCAVHWVTPDGFPVWQEYKK PIQTRNLRFLGSFNLQPTVNTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA

[0172] SEQ ID NO:9-CGGσ(DNA sequence)

[0173] AAGAACACTGGTGAAATCTCTGAGAAAGTCAAGCTGGGCACTAAGGCACTGGCTGGTCAATGGCTGGCTTACGGTGTTACTCGCAGTGTGACTAAGCGTTCAGTCATGACGCTGGCTTACGGGTCCAAAGAGTTCGGCTTCCGTCAACAAGTGCTGGAAGATACCATTCAGCCAGCTATTGATTCCGGCAAGGGTCTGATGTTCACTCAGCCGAATCAGGCTGCTGGATACATGGCTAAGCTGATTTGGGAATCTGTGAGCGTGACGGTGGTAGCTGCGGTTGAAGCAATGAACTGGCTTAAGTCTGCTGCTAAGCTGCTGGCTGCTGAGGTCAAAGATAAGAAAACTGGAGAGATTCTTCGCAAGCGTTGCGCTGTGCATTGGGTAACTCCTGATGGTTTCCCTGTGTGGCAGGAATACAAGAAGCCTATTAAAACGCGCGTGCATATTATGTTCCTCGGTCAGTTCGAAATGCAGCCTACCATTAACACCAACAAAGATAGCGAGATTGATGCACGCAAACAGGAGTCTGGTATCGCTCCTAACTTTGTACACAGCCAAGACGGTAGCCACCTTCGTAAGACTGTAGTGTGGGCACACGAGAAGTACGGAATCGAATCTTTTGCACTGATTCACGACTCCTTCGGTACGATTCCGGCTGACGCTGCGAACCTGTTCAAAGCAGTGCGCGAAACTATGGTTGACACATATGAGTCTTGTGATGTACTGGCTGATTTCTACGACCAGTTCGCTGACCAGTTGCACGAGTCTCAATTGGACAAAATGCCAGCACTTCCGGCTAAAGGTAACTTGAACCTCCGTGACATCTTAGAGTCGGACTTCGCGTTCGCGTA

[0174] SEQ ID NO:10 - CGGσ (Amino acid sequence)

[0175] KNTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIKTRVHIMFLGQFEMQPTINTNKDSEIDARKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA

[0176] SEQ ID NO:11 - T7p (DNA sequence)

[0177] TAATACGACTCACTATAGG

[0178] SEQ ID NO:12 - T3p (DNA sequence)

[0179] TAATAACCCTCACTATAGG

[0180] SEQ ID NO:13 - K1Fp (DNA sequence)

[0181] TAATAACTATCACTATAGG

[0182] SEQ ID NO:14 - CGGp (DNA sequence)

[0183] TAATACCGGTCACTATAGG

[0184] SEQ ID NO:15 - SYNZIP18 (DNA sequence)

[0185] GGAGGTTCAGGTGGTGGATCCAACGAAAAAGAAGAACTGAAATCCAAAAAAGCGGAACTGCGCAACCGTATCGAACAGCTGAAACAGAAACGTGAACAACTGAAGCAGAAAATCGCGAACCTGCGTAAAGAAATCGAAGCTTACAAATAAT

[0186] SEQ ID NO:16 - SYNZIP18 (amino acid sequence, C - terminus)

[0187] GGSGGGSNEKEELKSKKAELRNRIEQLKQKREQLKQKIANLRKEIEAYK

[0188] SEQ ID NO:17-SYNZIP17 (DNA sequence)

[0189] ATGAGCATCGCGGCGACCCTGGAGAACGATCTGGCGCGTCTGGAAAACGAAAACGCTCGTCTCGAAAAAGACATCGCGAACCTGGAACGTGACCTGGCGAAACTGGAGCGTGAAGAAGCGTACTTC

[0190] SEQ ID NO:18-SYNZIP17 (amino acid sequence, N-terminus)

[0191] MSIAATLENDLARLENENARLEKDIANLERDLAKLEREEAYF

[0192] SEQ ID NO:19-proA (DNA sequence)

[0193] TTTACGGGCATGCATAAGGCTCGTAGGCTATATTC

[0194] SEQ ID NO:20-Strong RBS (H) (DNA sequence)

[0195] TACTAGAGTCATTTATGAAAGTACTAG

[0196] SEQ ID NO:21-Weak RBS(L) (DNA sequence)

[0197] TACTAGAGTCAGCCAAGAAAGTACTAG

[0198] SEQ ID NO:22-PzinT (DNA sequence)

[0199] AAGCGAGTAGTCACAAAAATTATGCCGCCTGTGCGCGGATATCTGCAAAGCCTGTGCCGAAGAGTGTGCAAGGCACGATCACGACCATTGCCAGAATTGCGCGCGGGCATGCAGCCAATGCGCAGACGCCTGCCTTAAAATGGCCGCGTAATTTTTCTTCCGCCATTAGCTCAACCGGATAGAGCATAGAGCTTCTACCTCTAAGGTTCGGGGTTCAATTCCTCGATGGCGGACCAGTTGATATCAAAAAAGGCCACCTGCGCGGTGGCCGCTGAGTTTCTGTTGAAATAAATGCAATGTTATAATATAACAATCATCTTTCTAAGAAAGATGAGGGTAACGTTTTGGTGATTCATTTAAAAAAACTGACAATGCTTCTGGGAATGCTGTTGGTAAATAGT

[0200] SEQ ID NO:23 - PuhpT (DNA sequence)

[0201] GCACTGGACCGGTTTTTTTGCGGTCATCGCCATCGCGGCGGGGATCTCCGCGCTATTGCTGTTGCCATTTCTGAACGCTCAGGCCCCACGCGAAACCCACGAAGCGTGATACACCTCACCTTTTTGCGCTGAATGGGGCAAAACTAAGAAATTTTCCCGGTTTTGCCTGGACGCTGTCGCAGGCCACTTTTCCTGTGGATTTTTACAATGCCTGCCATTCGCAGGTATAAAAATTAGCTCAGGAGTAATCC

[0202] SEQ ID NO:24 - PfhuA (DNA sequence)

[0203] TACGCTGTGCCAGCAGGGCGAGATGATGCAGCAGCAACAGCAGCCGTCAGGCAATCCGTTCGATCAGTCGTCTCAGCCGCAGCAGCCTGCGCAGCAACAGCCGCCGAAAGAAGAGAAGAGCGACGGCGTTGCCGGCTGGATTAAGGAGATGTTTGGCGGCAATTAATCACGGTAATAGTGCCGGGTGGCGCTGTGCTTACTCGGTCCACACCGTTACGACCCCATTATGTGCGACGTAGGCCGAATAAGACGCTTACATCGCCATCCGGCAAATCCTCCATAAATAACATTTCAGTCTAATTTATTAACCCTTCCTTTTCATCTGGTTGTTTCTTAACCCCTTAGTTTTCGTAGGGCCGCGTATCGCTTGCCATTGCGACGATATTTCGCCTATCATGCTGCGGTTATAATAATAATTATCGTTTACGTTATCATTCACTTTCATCAGAGATATACCAATGGCGCGTCTTAAAATTGCTCAGCCAAACTCCTCACTGCGTAAAATCGCAGTTGTAGTAGCCAC

[0204] SEQ ID NO:25 - PentC (DNA sequence)

[0205] TTTGCCGGGGCCAACCGGCGTCCTGGGCTAAGGATATTCCTGAAATTGATAAACCAACCACTAAAAGCAGCCAACGATAAAAGGCGGAGAGTCTCACAATAGCGTCCTGTTATTAATAAAGTTAATGCTTCTCATTTTCATGTCAGCGGCAGCGAGATGCAAGCCTTAGTGCCATTTAACTCATGACCAGAGTTGACAGAGCGACGTTTTACTCTTAGGTTAGCGCACTAAAAATAGAAATAATAATCATTATTATACACAAAATCATTCAAGAAGCATCGCGACGGCAAGGGAAGAATCCCCGCGGGCATAGATAACTGTGTGACCGGGGTTTCTGATCGCAGCCAACAAAGAGGCAGCTTGAAAGATGAAGTGTATATAAGCCTTTATCATTGGAGGATGATATGGATATGTCACTGGCCGAGGACGCTCAGGAGACAATGGCAACGCTTGCT

[0206] SEQ ID NO:26 - PfhuE (DNA sequence)

[0207] CATTGACGGTTGGGATCAGGATATTGGGAATAATCAAGATATGCGTCGGCGCCTGCGGTGAAATATCACGAAAGGCGGTGACCAGCTCATCCTGATAAACGATATCCGAAGGGATTTCACGACGAATAATTTTGCTGAATATAGTTTCTTCTGCCACGACGTTTTCCTTTTTCATAATAGCCCTTGCAGCGCACATGCTACGCCGAGCCATACTGCGAGTATAGAGTATGAGCGAGTTACCGACGCTCTTTCAACTTTAACCCACGATTTATTAAGCGAAAAATGACTACATGCTGGACTTACCGCCATATCCCCCTGCCATGACGCCTTTGTACGCTTAAAAAATATTTCTAGTTTCCCTGGTTATACCGTTTTACACATTTAATACAAATGCGTATATTTCTCATTTGCATTTTTATACGCATTAACTAGCAAAGAATGAAAAGGTTCAACGTCATACGTCCTGAACTTACCCCAATAACAAGCAAGGATTTTCAGATGTCTTTCATTCAATA

[0208] SEQ ID NO:27 - PsfbA (DNA sequence)

[0209] GGATGTTGGCGTTAAGACGTCTTATCCGGCATTTCCCTTTACGCCATTCCCGCCTGACACACGCCTGAGCGCCTGCCTCGTACATTTAACGACACACCAGGAAACATCATGAAATAATTTCAAGGACAGCAGGCTGTGATCTGTGTCATGTTAAGAAATAGCCTTTCGTTTGGGCCAAAACAGACGATGCCGCATGAACGGCATCCGGCACAGCATCACACTATTTAAAATGGAGAAATTATGGGATTGCGTCAGAGTTTACGCATT

[0210] SEQ ID NO:28 - PyjjZ (DNA sequence)

[0211] GCCGACACGGCGTTGAGAAACAAGAAAAGACGTAAAGAAAACTGATACTTCTTAATACGAAGCGACCGCCAGGATGGGGTTGTCATGGGTAATTGTCGTTATTTATCGGTGATATACACGGAATCGGGCGCCAACATGAAAATAACGTATGAGAAAAGGTCGCCTAAAGCGAGGTGTTGTTGTTTTTACGTTAACAGTCGGACAATTTATCACCTTACTGAATACGTGTCATCAACCGTTAAGTAAAACTCATCTCTTTAGCTTTCTCCCTGGCTGACAAATGAGAAAATATATCATATGATATTGGTTATCATTATCAATTCCAGAGGTGAAACCATGTTGCAGCGGACGTTAGGCAGCGG

[0212] SEQ ID NO:29 - Pstnc3080 (DNA sequence)

[0213] CGCCCGCCATCACCACCGGTAATTGACGAAACCCTTGCGCCCGGAGCGTATCCGCTGCATCCGGCACCAGATCGACGTTCACCATCTCAAATTCAAATCCACGGCTTTCCATCGCCCGCTTTGTGGCGTGGCACTGAACACAGTTATTGCGAGTGTAAATAGTAATGCTCATGATTCGTATTTCCATTTAAAATGAGAAA

[0214] SEQ ID NO:30 - PsitA (DNA sequence)

[0215] CGGCTGATAGCAGTGCACCGGGCACTGCTTACAGGCGGGTTTTTCCTCGCCAAATACGCATTTATCAAGACGTTTTTGCGCGTAGGCGAACAGCGCGTCGTAATGCCCCTGCACCGCTGACGCCTGTGGACACTGGCTTTCATACAGCGCGATCATTTTTTTGATCGTCAGTTTTTCACGAGCGATACGTTTACCAGGCATCGTGCTCTCTCCGAACATTAAGATGCATTTATTTTACACCTTATCCCTCTTTAGCACTATCACTGCATATCGTCGCCATTACGCAAATAAGAATTATTTTCATTTATTCATGCCTTGTGCTATATAACATAGCAAAGGCTATATTCGATGATTAATTAACCACATTGTTGCGAGGGATACTATGACGAATCTACATCGTCTAAAAACACTCCTGATTGCCGGTATTGTCGCGATACT

[0216] SEQ ID NO:31 - PmntH (DNA sequence)

[0217] TTTGCTCCAAATATGAGGCAGGTTTAATTTTCGTGCACATTCTATGCAACAGCTGTAAAGAAAACGAGATCCAACACACACTATAATAAGGACCTGTGACGAGATTCAAAATTAGTGATCTGTAATACACTTTTACTGTACTGAATATGAAAATGAAAAGTTATATCAGTGTGCTAATCTTGTAATGTTAAGCCAAACTGTTCTGATACAGGTCGCCATCGTATCGGTCTATCGTTTCACACTATCAAAGTAATCACCCGTACCCATTGAAATGCACTTGATAATCATTATCAATGAACATAGCATGAAACATAGCAAAGGCTATGTTTTTGAGGCAAAAGATGACTGACAATCGCGTAGAGAATAG

[0218] SEQ ID NO:32 - PfepA (DNA sequence)

[0219] AATAAAACAGTAGCTGCCGCGCCAGTTAGCGCTAAGCGCCGTGCTCCAGCGCCAGACATCCGTTCCGGCAATACGCGCCATCGTCTGAGGCTGCGCGTTTTGATGATGATCGGTTACGCCAGTGATGTAGACCCAGACACGCCGTATCGGGGAGTGTGTTTCGTTTCCCTGCGGGTCGCGCCACCAAAAAGTGACCCGATAATTTCCGTCTTTTTCCCGTATCCATTCCGGCCCGGTTTTCGTCCTCCACCAGGCCTCACTTCCCGTTGCCAGCGCCTCTTTCATTATAACCCTGTGTTTATTATGAATTTTGTATATAAAAGGTGAAATATATTGATAATATTATTGATAACTATTTGCATTTGCAATAGCGTATTGTAGCGCTATGGGACGCGCGAACACAATTTCACCACCCGGCCAATGCCTTTGACGGGCGCTTTGGCTTATGTGGCTAAAGAAAAGCAGGATATACAATGAACAAGAAGATTCATTC

[0220] SEQ ID NO:33 - PsufA (DNA sequence)

[0221] CTCATTCAGCACCTGAAATGCCGGAAAGAGTTTACATAAACCTATAGCTCAAACTGAGTTATAGAACCGCAGCGGATTATAAAGAGCGCAACGCCAGGTATCCATACAAAAAATGGGGTTCTGACCTCGCCGCCCGGCAATGTCGACAGCCTATTAATTAAATAGTCATTTTCTATACATCTTTTCGTTTTTGACCTGCCAGAACGGTTAATGTCTTATAAATCATTACTTATCAAAAAGTTAAGTGGTTTTTTGTCTGTCGTATGACCTGGCGGACAGGGTCTATGCTTAATAAAAGGCGCTCAATATGACCATTTGTTGGAAAGCCCCTGCGGTTAAGGGGTTGAAGTGATAATCATTATCACTAACATGCTGTTATATCCTGGTGATTTAGAACGCGAGGTAACTCTATGGAATTGCATTCAGGCACGTTTAACCCGGAGGACTTC

[0222] SEQ ID NO:34 - PsoxS (DNA sequence)

[0223] TTTCGCAGCGGACAGTCGCTACGCGATAAACAGCCGCAGCCGATACAACCGTCCAGCTCATCGCGCAACGCCACCAGCGTATGAATACGTCGGTCTAACTCTTCGCGCCACTGCGAGGAGAGCTGCTTCCACTCTTTCGCGCTTAACGTATGCCCTTCCGGCAAGATACCAAACGCGTCGCCGATAGTTGCCAGCGGGATGCCGATACGCTGGGCAATCTTGATAATCGCGACATAACGCAACACGTCACGCTTGTATCGCCGTTGGTTACCGCTATTACGGATACTGGTAATTAGCCCTTTGCTTTCATAGAAGTGCAGGGCGGACACAGCAACACCGCTACGTTTCGCAACTTCCCCCGGCGTCAGTAAGGCTTTTAAACGGGGAGATTTTTTTTCCATAAATCGCTTTACCTCAAGTTAACTTGAGGAATTATACTCGCCCGCAGACAAAACGACGAATCGAATACTGTTTAAGAGGCAACAATATGTCGCATCAGCAGATAATTCAGACCCTTAT

[0224] SEQ ID NO:35 - PiroN (DNA sequence)

[0225] AAGCGCCTGATAAATATTACCAGGCGCTTTGTATGTTGGTGCCAACATCACTTTCATCATCAAATATCGAATGGCTACAATCGTATCCGATCCCGCCATTACCCAGACGGAAAGTCGCTGGCAAACTGTAAGAATGGTTCGCCGTCGGCAGGGAAGCGGCGGTGAACCCTGAACCGTGCTATACCATCTTACCTGGGTGTTTCTTGTGATTAACGATCTGAAAAATAGTTTTATTTTATCTATTTCTGTTTTGTAAAACCTCCGTTCAGTAGGCGCATTCTGCCCCCCTTCCCGGATTTACTGGCAAAGCGGAGCCCGGACAGAGAGTCATATTGCAAAATCCCGTTTCCGTTTTTTTATTACCAGATTTTTGTGGTCGAAAGATTGCCTTTTCCTTAATTGAATGATAATTATTATCATTAGCATATGATAATAATTACTATATAGACGTAACCTGGCAAGGATGTGAGCTTGAGGGCAACAGCGCTACTTTAGACATTATTTAGGGAATGGGTATGAGAGTTAAGAAGTTC

[0226] SEQ ID NO:36 - PfepB (DNA sequence)

[0227] CAAACTGCTGGCGCAATTTCTGCTGGAAAGGGCTATCCGGCGAATCTCCGGCAACCGCAGGCTCGGTATAACGGGCGAAGCACCCGGAAGTGGTAAAACTGCGATACGGCGACATGAAGAAAAAGCGATCGGGAGCAAGCGTTGCCATTGTCTCCTGAGCGTCCTCGGCCAGTGACATATCCATATCATCCTCCAATGATAAAGGCTTATATACACTTCATCTTTCAAGCTGCCTCTTTGTTGGCTGCGATCAGAAACCCCGGTCACACAGTTATCTATGCCCGCGGGGATTCTTCCCTTGCCGTCGCGATGCTTCTTGAATGATTTTGTGTATAATAATGATTATTATTTCTATTTTTAGTGCGCTAACCTAAGAGTAAAACGTCGCTCTGTCAACTCTGGTCATGAGTTAAATGGCACTAAGGCTTGCATCTCGCTGCCGCTGACATGAAAATGAGAAGCATTAACTTTATTAATAACAGGACGCTATTGTGAGACT

[0228] SEQ ID NO:37 - LysE (Amino acid sequence)

[0229] MVRWTLWDTLAFLLLLSLLLPSLLIMFIPSTFKRPVSSWKALNLRKTLLMASSVRLKPLNCSRLPCVYAQETLTFLLTQKKTCVKNYVRKE

[0230] SEQ ID NO:38 - LysE (DNA sequence)

[0231] ATGGTACGCTGGACTTTGTGGGATACCCTCGCTTTCCTGCTCCTGTTGAGTTTATTGCTGCCGTCATTGCTTATTATGTTCATCCCGTCAACATTCAAACGGCCTGTCTCATCATGGAAGGCGCTGAATTTACGGAAAACATTATTAATGGCGTCGAGCGTCCGGTTAAAGCCGCTGAATTGTTCGCGTTTACCTTGCGTGTACGCGCAGGAAACACTGACGTTCTTACTGACGCAGAAGAAAACGTGCGTCAAAAATTACGTGCGGAAGGAG

[0232] SEQ ID NO:39 - kilR (Amino acid sequence)

[0233] MIAHHFGTDEIPRQCVTPGDYVLHEGRTYIASANNIKKRKLYIRNLTTKTFITDRMIKVF

[0234] SEQ ID NO:40 - kilR (DNA sequence)

[0235] ATGATAGCACATCATTTCGGCACCGATGAAATCCCCCGCCAGTGCGTTACCCCTGGTGATTACGTTCTGCATGAAGGTCGCACGTATATCGCGTCCGCCAACAACATCAAAAAACGTAAACTTTACATCCGTAATCTTACAACAAAGACGTTCATCACTGATCGTATGATTAAAGTATTT

[0236] SEQ ID NO:41 - Kil (Amino acid sequence)

[0237] MRKRFFVGIFAINLLVGCQANYIRDVQGGTIAPSSSSKLTGIAVQ*

[0238] SEQ ID NO:42 - Kil (DNA sequence)

[0239] ATGCGCAAAAGGTTCTTCGTAGGTATTTTCGCCATCAACCTGTTGGTGGGATGTCAAGCAAATTATATCCGTGACGTACAAGGTGGCACAATCGCGCCATCGAGTTCGAGTAAATTAACCGGAATCGCTGTTCAATAG

[0240] SEQ ID NO:43 - BRP (Amino acid sequence)

[0241] MKATKLVLGAVILGSTLLAGCQANYIRDVQGGTVAPSSSSELTGIAVN

[0242] SEQ ID NO:44 - BRP (DNA sequence)

[0243] ATGAAAGCGACCAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGCTGCCAGGCAAACTATATCCGGGATGTTCAGGGTGGAACGGTGGCACCATCGTCCTCCTCTGAACTGACGGGGATCGCGGTTAAC

[0244] SEQ ID NO:45 - (stn3250) (DNA sequence)

[0245] CAGAAGTCATAGGTATTGGAAGCGCCGCGACTGCTTACAGTTACGCCCGGCGTGTAACCCAACGCTTCTTTTACTGACTGGAATTGATGCATCTGCATCTCTTCGTTAGTGACCACCGAAACCGACTGTGGCGTTTTTTCGATAGATGTATCAGTTTTGGTGGTAGTGGCGGAACGCTTCGCGGCGATGGTCGGAGCCGGTCCCCAGGCACTTTCCTGCGGCGCAGGCGCTGCGGTTACGGTAATGGTTTCTTCTTTCGGTTGAACCGCCGCCTGTGCATAGACAGACATGCCGCTAACCGCTGTGGCTACTACAACTGCGATTTTACGCAGTGAGGAGTTTGGCTGAGCAATTTTAAGACGCGCCATTGGTATATCTCTGATGAAAGTGAATGATAACGTAAACGATAATTATTATTATAACCGCAGCATGATAGGCACAGCACAACGG

[0246] SEQ ID NO:46 - (mtgC) (DNA sequence)

[0247] CGTTTAGCATCCCTTTTCTGGTGGAACCCATTTTTTCCTCGTCATGTTGTTTTATTTTTTTACGTGCAGGCATCATAACAGAGCTATCGCCGGCATTAAGCAGGAATTTATTGTTTAATGATTTCAGACGAGCCTGTTATTGACATAATATTGTCATTTTTTTGTCACGGGAAATATCAAACAAACTTAAACAAATCGTCACTATCCCCGCCTTTGCACTTTACAGAACATATTGACTGACTATAATAAGCGCAAATTCATGCAGGAGTAATATGTTGGACAGTCACTTTTACGTAAATCATCTGGCAAGTTAACGCACGCTATTCCTGCGCTGCTTGCCGAACCGGTGGGCAGCAATCTCCCCTTGTGACGATTGTCATCCCAATAATGTTACAACACGCGCATTGTCGCGAGGTAATCGTCATGTTCATGTTTAAACACGCTTTATTTCCTCCGCCGTTAACACGACGCTAATTGCCTCAGGGCAGAAATTTGTCGTGTGCTAAATATAGCACGTACTTATTCTTCCAGAAAAAATGGAGGAACGTATGTTAATGTTTCCTTATATTTTAAATTTACTGGCCGCTATGCT

[0248] SEQ ID NO:47 - (sseJ) (DNA sequence)

[0249] CACATAAAACACTAGCACTTTAGCAATAATAGTCGGATGATAAGTTTGTCTGTTTTTCCTGAGTATCAAGCCAGCTCATACTCACGCCAGCACACTAAAATCAGGAGTGGCTTCTTTTTTAGATCTTTGCCTTAGCCAGGCGCACACTCAATAATGATAGCAGTCAGATAATATGTACCAGGCATTAACCTCACGTTGTTGATGATATATTTACTTCGTTGAAAAACAATAAACATTGTATGTATTTTATTGGCGACGAAAAACTGTTAAAGAAGCGTAATTCCATATACACCATTTACCTGATTACTTTTCTTGCTAATATTTGCTAATTAATTATTTGCTAAAGCGTGTTTAATAAAGTAAGGAGGAGGCACAGCACAACGG

[0250] SEQ ID NO:48 - (ssaG)(DNA sequence)

[0251] TATTGCCATCGCGGATGTCGCCTGTCTTATCTACCATCATAAACATCATTTGCCTATGGCTCACGACAGTATAGGCAATGCCGTTTTTTATATTGCTAATTGTTTCGCCAATCAACGCAAAAGTATGGCGATTGCTAAAGCCGTCTCCCTGGGCGGTAGATTAGCCTTAACCGCGACGGTAATGACTCATTCATACTGGAGTGGTAGTTTGGGACTACAGCCTCATTTATTAGAGCGTCTTAATGATATTACCTATGGACTAATGAGTTTTACTCGCTTCGGTATGGATGGGATGGCAATGACCGGTATGCAGGTCAGCAGCCCATTATATCGTTTGCTGGCTCAGGTAACGCCAGAACAACGTGCGCCGGAGTAATCGTTTTCAGGTATATACCGGATGTTCATTGCTTTCTAAATTTTGCTATGTTGCCAGTATCCTTACGATGTATTTATTTTAAGGAAAAGC

[0252] SEQ ID NO:49 - (sseA)(DNA sequence)

[0253]

[0254] SEQ ID NO:50 - (sifA) (DNA sequence)

[0255] ATAAGCGATTAATTGCGCAACGCTAACAAATCCACACGCATCCAGGCATGAAGTTTATTCAAGGGTAAACTTCATGCCTTCGGCATAAAAAACGCATGAAAGAAGTTGCCGCCAGTATTGCAAATCTACAACATCATCCGCGGTAGTCCTTCTTTTATTTTTACCTGTAGCGACGCTATCACAGACAGTAATGCGTTTATACGCGAAGCTCTCAGGTTTTATACTGATTGCCAGTCTCTTTTAAAAATTATATTACATCCGATGCGCCCGCAGTTGAGATAAAAAGGGTCGATTTAATCAATTATGTAGTCATTTTTACTCCAGTATAAGTGAGATTAAG

Claims

1. A live attenuated Gram-negative bacterium, comprising: i) a heterologous polynucleotide encoding an RNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter; and ii) a polynucleotide encoding an RNA polymerase, wherein the RNA polymerase binds to the promoter, wherein the RNA polymerase is encoded by a heterologous split RNA polymerase plasmid, and wherein after the Gram-negative bacterium invades a eukaryotic cell, the RNA molecule is transcribed and capable of being transferred into the cytoplasm of the eukaryotic cell.

2. A live attenuated Gram-negative bacterium, comprising: i) a heterologous polynucleotide encoding an RNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter; and ii) a polynucleotide encoding an RNA polymerase that is orthogonal to the components required for the expression of endogenous genes in the Gram-negative bacterium, wherein the RNA polymerase binds to the promoter, and wherein after the Gram-negative bacterium invades a eukaryotic cell, the RNA molecule is transcribed and capable of being transferred into the cytoplasm of the eukaryotic cell.

3. The live attenuated Gram-negative bacterium according to claim 1, wherein, the RNA polymerase is orthogonal to the components required for the expression of endogenous genes in the Gram-negative bacterium.

4. The live attenuated Gram-negative bacterium according to claim 2, wherein, the RNA polymerase is encoded by a heterologous split RNA polymerase plasmid contained within the Gram-negative bacterium.

5. The live attenuated Gram-negative bacterium according to any one of claims 1 to 4, wherein, the polynucleotide encoding the RNA polymerase is operably linked to a cytosolic promoter or a vacuole-dependent promoter, preferably wherein the cytosolic promoter is selected from uhpT, mntH, entC, fhuE, iroN, fepB, fepA, fhuA, sitA, stn3250, sufA, yjjZ, soxS, sfbA or any combination thereof, and wherein the vacuole-dependent promoter is selected from zinT, mtgC, ssaG, sseJ or any combination thereof.

6. The live attenuated Gram-negative bacterium according to any one of the preceding claims, wherein, the σ factor of the RNA polymerase binds to the promoter operably linked to the heterologous polynucleotide.

7. The live attenuated Gram-negative bacterium according to claim 6, wherein, the σ factor of the RNA polymerase is a CGG σ factor RNA polymerase, a T3 σ factor RNA polymerase or a K1F σ factor RNA polymerase.

8. The live attenuated Gram-negative bacterium according to claim 6, wherein, the σ factor of the RNA polymerase is a CGG σ factor RNA polymerase, and the promoter operably linked to the heterologous polynucleotide is a CGG promoter.

9. The live attenuated Gram-negative bacterium according to any one of the preceding claims, wherein, the heterologous polynucleotide encoding the RNA molecule is operably linked to a phage promoter.

10. The live attenuated Gram-negative bacterium according to claim 9, wherein, The phage promoter is not a T7 phage promoter.

11. The attenuated live Gram-negative bacterium according to any one of the preceding claims, wherein, the RNA molecule is an mRNA molecule, an siRNA molecule or an shRNA molecule.

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

13. The attenuated live Gram-negative bacterium according to claim 11, wherein, the siRNA molecule or the shRNA molecule targets an mRNA molecule for degradation.

14. The attenuated live Gram-negative bacterium according to any one of the preceding claims, wherein, the heterologous polynucleotide encoding the RNA molecule does not modify the genome of the eukaryotic cell.

15. The attenuated live Gram-negative bacterium according to any one of the preceding claims, wherein, the attenuated live Gram-negative bacterium is of the genus Salmonella.

16. The attenuated live Gram-negative bacterium according to any one of the preceding claims, wherein, the attenuated live Gram-negative bacterium is Salmonella enterica, preferably wherein the attenuated live Gram-negative bacterium is Salmonella enterica serovar Typhi and / or Salmonella enterica serovar Typhimurium.

17. The attenuated live Gram-negative bacterium according to any one of the preceding claims, wherein, the attenuated live Gram-negative bacterium is a genetically modified non-natural bacterium.

18. The attenuated live Gram-negative bacterium according to any one of the preceding claims, wherein, the attenuated live Gram-negative bacterium is selected from Ty21a, CVD 908-htrA, CVD 909, Ty800, M01ZH09, ZH9PA, x9633, x639, x9640, x8444, DTY88, MD58, WT05, ZH26, SL7838, SL7207, VNP20009, A1-R or any combination thereof, preferably wherein the attenuated live Gram-negative bacterium is M01ZH09.

19. The attenuated live Gram-negative bacterium according to any one of the preceding claims, wherein, the RNA molecule is mammalian.

20. The attenuated live Gram-negative bacterium according to claim 5, wherein, the cytosolic promoter is derived from SPI-2.

21. The attenuated live Gram-negative bacterium according to claim 12, wherein, the therapeutic protein is a cytokine, a chemokine, an antibody or a fragment thereof, a cytotoxic agent, a cancer antigen or any combination thereof, preferably wherein the resulting therapeutic protein is IL-15, IL-21, CXCL9, IL-18, IL-27, IFNγ, IL-1 or any combination thereof.

22. The attenuated live Gram-negative bacterium according to any one of the preceding claims, wherein, The attenuated live Gram-negative bacterium further comprises one or more of the following accessory proteins: i) a polynucleotide encoding an RNA stability enhancing component, preferably wherein the RNA stability enhancing component is an IRES sequence; ii) a polynucleotide encoding a lysis protein, preferably wherein the lysis protein is hemolysin; iii) a polynucleotide encoding a phospholipase, preferably wherein the phospholipase is phospholipase C; iv) a polynucleotide encoding an invasion factor; and / or v) bacteriocin release protein, bacteriophage λ lysozyme, and perforin.

23. The attenuated live Gram-negative bacterium according to any one of the preceding claims, wherein, the attenuated live Gram-negative bacterium is administered intratumorally, intravenously, intraperitoneally, or orally, preferably wherein the Gram-negative bacterium is administered intratumorally.

24. The attenuated live Gram-negative bacterium according to any one of the preceding claims, wherein, the eukaryotic cell is a mammalian cell, preferably wherein the eukaryotic cell is a human cell.

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

26. The attenuated live Gram-negative bacterium for use according to claim 23, wherein, the attenuated live Gram-negative bacterium is used for treating, reducing, inhibiting, preventing, or controlling neoplastic diseases, infectious diseases, cardiovascular diseases, neurodegenerative diseases, gastrointestinal diseases, respiratory diseases, kidney diseases, liver diseases, autoimmune diseases, inflammatory diseases, or genetic diseases, preferably, the attenuated live Gram-negative bacterium is used for treating, reducing, inhibiting, preventing recurrence, or controlling neoplastic diseases or infectious diseases.

27. The use of the attenuated live Gram-negative bacterium according to claim 24, wherein, the neoplastic disease is solid cancer and / or hematological malignancy.

28. The attenuated live Gram-negative bacterium according to claim 25, 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.

29. A vaccine composition comprising an attenuated live Gram-negative bacterium, wherein the Gram-negative bacterium comprises: i) a heterologous polynucleotide encoding a functional mRNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter and the mRNA molecule encodes a therapeutic protein and / or a therapeutic peptide; and ii) a polynucleotide encoding an RNA polymerase, the RNA polymerase being orthogonal to the components required for endogenous gene expression in the Gram-negative bacterium, wherein the RNA polymerase binds to the promoter, and wherein after the Gram-negative bacterium invades a eukaryotic cell, the mRNA molecule is transcribed and capable of being transferred into the cytoplasm of the eukaryotic cell.

30. A vaccine composition comprising an attenuated live Gram-negative bacterium, wherein the Gram-negative bacterium comprises: i) a heterologous polynucleotide encoding a functional mRNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter and the mRNA molecule encodes a therapeutic protein and / or a therapeutic peptide; and ii) a polynucleotide encoding an RNA polymerase, wherein the RNA polymerase binds to the promoter, wherein the RNA polymerase is encoded by a heterologous split RNA polymerase plasmid, and wherein after the Gram-negative bacterium invades the eukaryotic cell, the mRNA molecule is transcribed and capable of being transferred into the cytoplasm of the eukaryotic cell.

31. The vaccine composition according to claim 29 or 30, wherein, the vaccine composition further comprises an adjuvant, a pharmaceutically acceptable carrier or an excipient.

32. The vaccine composition according to claims 29 to 31, wherein, the vaccine composition comprises the attenuated live Gram-negative bacterium according to any one of claims 1 to 24.

33. A method for 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, the attenuated live Gram-negative bacterium comprising: i) a heterologous polynucleotide encoding an RNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter; and ii) a polynucleotide encoding an RNA polymerase, wherein the RNA polymerase binds to the promoter, wherein the RNA polymerase is encoded by a heterologous split RNA polymerase plasmid, and wherein after the Gram-negative bacterium invades the eukaryotic cell, the RNA molecule is transcribed and capable of being transferred into the cytoplasm of the eukaryotic cell.

34. A method for treating, preventing, suppressing, preventing recurrence or controlling a disease of an object, wherein the method comprises administering to the object an attenuated live Gram-negative bacterium, the attenuated live Gram-negative bacterium comprising: i) a heterologous polynucleotide encoding an RNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter; and ii) a polynucleotide encoding an RNA polymerase, the RNA polymerase being orthogonal to the components required for the expression of endogenous genes in the Gram-negative bacterium, wherein the RNA polymerase binds to the promoter, and wherein after the Gram-negative bacterium invades the eukaryotic cell, the RNA molecule is transcribed and capable of transferring into the cytoplasm of the eukaryotic cell.

35. The method for treating, preventing, suppressing, preventing recurrence or controlling a disease of an object according to claim 33 or 34, wherein, the method comprises the use of the attenuated live Gram-negative bacterium according to any one of claims 1 to 24.

36. The method for treating, preventing, suppressing, preventing recurrence or controlling a disease of an object according to any one of claims 33 to 35, wherein, the disease is a neoplastic disease or an infectious disease.

37. A method for delivering an RNA molecule into a eukaryotic cell, the method comprising the following steps: i) modifying a Gram-negative bacterium such that a heterologous polynucleotide encoding an RNA molecule is integrated into the bacterial genome, wherein the heterologous polynucleotide is operably linked to a promoter; ii) contacting the Gram-negative bacterium with the eukaryotic cell to allow the Gram-negative bacterium to replicate within the eukaryotic cell such that the heterologous polynucleotide is transcribed and subsequently transferred from the Gram-negative bacterium into the cytoplasm of the eukaryotic cell.

38. The method for delivering an RNA molecule into a eukaryotic cell according to claim 36, wherein, the method comprises the use of the attenuated live Gram-negative bacterium according to any one of claims 1 to 24.

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