Bactofection

Abstract

The present invention relates to modified live attenuated Gram-negative bacteria, which target RNA molecules. It has been modified for safe and efficient delivery into eukaryotic cells.The present invention also relates to bacterial delivery systems and various uses and methods thereof.

Description

[Technical Field]

[0001] Technical field to which the invention belongs The present invention relates to modified live attenuated Gram-negative bacteria and uses thereof. [Background technology]

[0002] background Gene therapy has been used to suppress or eliminate tumor growth by introducing genetic material into cancer cells to inactivate oncogenes or transiently change the phenotype [Molecular Cell Therapies (2014) 2:27; Journal of Vascular and Interventional Radiotherapy (2013) 24(8):1115; Journal of Immunotherapy of Cancer (2020) 8:e001]. Cytolytic gene therapy, which uses genetically engineered viruses that infect and replicate exclusively in cancer cells, has also been used. Viral therapy (OV) has shown great promise, with three OVs reaching therapeutic use worldwide in 2020. These treatments are expected to be approved as drugs [Journal of Immunotherapy of Cancer (2020) 8:e001]. These treatments have been shown to be safe, with mild side effects and minimal dropout rates.

[0003] However, there are several limitations to the use of these drugs [Journal of Gene Medicine (2005) 7:1380]: (1) resistance by patients, who eliminate the vector before it reaches its target; (2) high doses of virus particles induce a cytokine cascade (3) safety concerns due to unexpected mutations; and (4) limited delivery to tumors [Oncolytic virotherapy (2017) 6:39]. Furthermore, preclinical trials have There is a discrepancy between the results obtained in experimental studies and the antitumor effects observed in clinical trials [Oncolytic virotherapy (2017) 6:39].

[0004] Bactofection is the transfer of genetic material from bacteria (such as Salmonella) into mammalian cells. An early example of bactofection with Salmonella [Blood (1998) 92:3172] used strain SL7207 (aroA-) to transfer plasmid material to cells that spontaneously and passively transfer the spleen. The transfection efficiency was ~19% in cells, with a preference for macrophages (F4 / 80+) cells. Byrne et al. (2014) also found a ~20% bactofection efficiency when using E. coli MG1655 as a vector [Journal of Controlled Release (2014) 196:384]. They also found preferential bactofection in macrophages. This limited efficiency is mainly due to the instability of vector strains resulting from the use of plasmid systems, which are inherently variable and contribute to metabolic burden [Gene Therapy (2005) 12:364]. This is due to the need to translocate into the nucleus, possibly due to unmethylated CpG regions [Molecular Therapy (2009) 17:767]. Furthermore, translocation of unmethylated CpG islands may trigger TLR-9 responses, which are known to be intricately involved in cancer control. A relevant example demonstrating the use of bactofection in immune system control is published by Shen et al. [Microbiology and Immunology (2004) 48:329]. They used Listeria monocytogenes to translocate Mice (BALB / c) were "bactofected" to produce IL-10, IL-12, and IFNγ.

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

[0006] The present inventors have surprisingly found that RNA molecules can be delivered to target eukaryotic cells safely, efficiently, and effectively. It has been discovered that gram-negative bacteria can be modified to allow delivery of gram-negative bacteria to the host. Thus, the present invention provides a bacterial delivery system that can be used broadly across multiple disease areas.

[0007] Successful bactofection with RNA requires an RNA polymerase to synthesize RNA from a DNA template. However, the present inventors have not used a typical phage RNA polymerase. We found that enzymes have serious drawbacks that prevent successful bactofection. In the present invention, the inventors have surprisingly found that T7 RNA polymerase does not contribute to the safe, efficient, and effective delivery of RNA molecules into target eukaryotic cells and may result in undesirable toxicity in certain bacterial strains. To enable bactofection into Gram-negative bacteria, the inventors have found that modifications are necessary to improve efficiency, and that the DNA template We realized that traditional strong promoters, such as the T7 promoter, used to drive expression have been found to result in undesirable toxicity due to depletion of host bacterial cell resources. Second, we found that the presence of the T7 promoter within the ZH9 Salmonella chromosome We found that the expression of unknown genes may be promoted.

[0008] Thus, the present inventors have surprisingly found that promoter selection is critical for successful bactofection and subsequent downstream applications. Specifically, the present inventors have found two solutions to the toxicity and specificity problems described above. First, promoter selection is performed by a heterologous split RNA polymerase plasmid so that RNA production is maximized with minimal toxicity. The other is to use RNA polymerases required for endogenous gene expression. Using RNA polymerase and promoter pairs that are orthogonal to essential Gram-negative bacterial components , to avoid the specificity problem mentioned earlier.

[0009] Accordingly, in a first aspect, the present invention provides 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 an RNA polymerase binds to a promoter, wherein the RNA polymerase is encoded by a heterologous split RNA polymerase plasmid, and wherein, upon invasion of a eukaryotic cell by a Gram-negative bacterium, the RNA molecule is transcribed and can be transported into the cytoplasm of the eukaryotic cell. The present invention provides a live attenuated Gram-negative bacterium comprising a polynucleotide which is

[0010] In a second aspect, the present invention provides a heterologous polynucleotide encoding an RNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter; and ii) an RNA polynucleotide that is orthogonal to components required for endogenous gene expression in Gram-negative bacteria. When the gram-negative bacterium invades a eukaryotic cell, the RNA polymerase binds to the promoter, and the RNA molecule is transcribed and released into the cytoplasm of the eukaryotic cell. A live attenuated Gram-negative bacterium is provided, which comprises a polynucleotide capable of translocation.

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

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

[0014] In a sixth aspect, the present invention provides a method of treating, inhibiting, preventing recurrence, or controlling disease in a subject, comprising: i) a heterologous polynucleotide encoding an RNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter; and ii) an RNA polymerase that is orthogonal to components required for endogenous gene expression of said Gram-negative bacterium. a polynucleotide that encodes a gene encoding a gene encoding a gene for a nucleic acid sequence, wherein the RNA polymerase binds to the promoter and the gene encoding a gene for a nucleic acid sequence is When Rham-negative bacteria invade a eukaryotic cell, the RNA molecule is transcribed and transported into the cytoplasm of the eukaryotic cell. and administering to the subject a live attenuated Gram-negative bacterium comprising a polynucleotide capable of inhibiting the virulence of the bacterium.

[0015] In a seventh aspect, the present invention provides a method for delivering an RNA molecule to a eukaryotic cell, comprising: The method comprises the steps of: 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; and ii) contacting the Gram-negative bacterium with a eukaryotic cell, such that the Gram-negative bacterium replicates within the eukaryotic cell and the heterologous polynucleotide is transcribed and then transported from the Gram-negative bacterium into the cytoplasm of the eukaryotic cell. [Brief explanation of the drawings]

[0016] [Figure 1A]Figure 1 shows how T7 RNA polymerase (RNAP) exerts virulence on Salmonella enterica Typhi. Figure 1A shows a schematic diagram of the experimental setup. One plasmid contains T7 RNA polymerase under the control of an SPI-2-inducible promoter, and the other contains mScarlet (red fluorescent protein) under the control of the T7 promoter (T7p). Putatively active T7p was found in the Salmonella genome downstream of a prophage integrase-like gene and upstream of four unidentified open reading frames (ORFs). BLASTp searches revealed that these proteins are found across different enterobacteria, including Salmonella, Escherichia, and Shigella (data not shown). [Figure 1B] Figure 1 shows how T7 RNA polymerase (RNAP) exerts toxicity on Salmonella enterica Typhi. Figure 1B evaluates the effects of temperature and promoter strength on T7RNAP-dependent toxicity. We found that toxicity was more pronounced with strong promoters, especially at temperatures close to the growth optimum (37°C). [Figure 2] Figure 2 is a schematic diagram of a split RNAP system. In this design, the expression level of the core RNAP determines total polymerase activity and, therefore, toxicity. By carefully selecting promoter 1 (e.g., a constitutive promoter or an SPI-2-dependent promoter), strains with maximal polymerase activity at the lowest possible toxicity level can be generated. A second (or more) promoter controls expression of the "sigma" (or DNA-binding) domain of RNAP. Upon binding of sigma to the core, polymerase activity is restored and is specific to the sigma's cognate promoter. This allows for the generation of platforms in which different sigma factors control different bacteriofection circuits (e.g., sigma 1 controls therapeutic RNA production, while sigma 2 controls lysine synthesis, allowing release of therapeutic RNA into the cytoplasm of eukaryotic cells). [Figure 3]Figure 3 shows the results of expressing the split RNAP system in Escherichia coli (E. coli) and Salmonella enterica ZH9. 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 a low-copy-number plasmid containing a split RNAP carrying a specific sigma factor (depicted on the left) or a superfolder green fluorescent protein (sfGFP) (negative). Cells expressed mScarlet only in the correctly paired sigma-promoter strain. On the right, Salmonella enterica ZH9 was transformed with the above expression vectors and split RNAP plasmids carrying either the cognate sigma factor or sfGFP with a strong (H) or weak (L) ribosome binding site (RBS) to control the level of the RNAP core (center), and the PuhpT -controlled sigma factor (right) was used to evaluate the system in liquid medium. [Figure 4A] Figure 4 shows the results of an in vitro evaluation of different cytoplasmic promoters. Figure 4A shows plasmids engineered to express constitutive mScarlet, encoding red fluorescent protein, and inducible sfGFP, encoding green fluorescent protein, under the control of one of the promoters depicted. [Figure 4B] Figure 4 shows the results of in vitro evaluation of different cytoplasmic promoters. Figure 4B shows Salmonella enterica ZH9 cells harboring these plasmids were cultured in M9 medium and subjected to the addition of 1 mM Fe3+, Mg2+ (PmtgC only), or Zn2+ (PzinT only), followed by monitoring fluorescence over time. In the absence of inducers, the cells showed varying levels of expression. [Figure 5]Figure 5 shows the results of an invasion assay using Salmonella enterica ZH9 containing a reporter plasmid constitutively expressing mscarlet and sfGFP under cytoplasmic inducible promoters, visualized by microscopy 24 hours after invasion. All tested promoters (indicated above each micrograph) are in red except for PyjjZ, which indicates that sfGFP was not produced in SKOV-3 cells (an ovarian cancer cell line). [Figure 6A] Figure 6 shows the evaluation of cytoplasmic promoters in vacuolar release. Figure 6A shows the activity of the promoter puhpT, which has been reported to respond to cytoplasmic glucose-6-phosphate. puhpT was evaluated in vitro by performing an invasion assay of Salmonella Typhimurium CD12 strains harboring a reporter plasmid into SK-OV-3 cells. This reporter plasmid constitutively expresses mScarlet (red fluorescent protein) and sfGFP (green fluorescent protein) in response to glucose-6-phosphate. [Figure 6B] Figure 6 shows the evaluation of cytoplasmic promoters in vacuolar release. Figure 6B shows representative examples (two of each) of cells in vacuoles (low number, red only) and cells in the cytoplasm (high replication, yellow). [Figure 7] Figure 7 demonstrates that sigma factors are responsible for spatiotemporal regulation within host cells. Expression of sigma factors can be selectively controlled using vacuolar-inducible (ssaG, sseJ, sseA, or sifA) or cytoplasmic-inducible (fhuA, iroN, mntH, or sitA) promoters. Furthermore, the differential expression levels from each of these promoters (indicated by fluorescence from sfGFP downstream of each labeled promoter) can provide an additional layer of control over split RNAP activity. DETAILED DESCRIPTION OF THE INVENTION

[0017] Detailed explanation In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0018] As used herein, the term "attenuation," in the context of the present invention, refers to modifying a microorganism to reduce its pathogenicity and render it harmless to the host while maintaining its viability. This method is commonly used due to its ability to induce a highly specific immune response while maintaining an acceptable safety profile. Methods for obtaining attenuated microorganisms include, but are not limited to, passaging the pathogen under in vitro conditions until pathogenicity is lost, chemical mutagenesis, and genetic engineering techniques. Such attenuated microorganisms are preferably live attenuated microorganisms, although non-live attenuated microorganisms are also disclosed.

[0019] As used herein, the term "inactivating mutation" refers to a modification of the native genetic code of a particular gene or gene promoter associated with that gene, such as by altering the nucleotide code or deleting a section of nucleotides or by adding non-coding or non-naturally occurring nucleotides, such that the particular gene is not properly transcribed or translated, or the native function of that gene is abolished to an unmeasurable extent. Mutations in a gene thus inactivate the function of that gene or the protein it encodes.

[0020] As used herein, the term "non-naturally occurring bacteria or bacteria" refers to a bacterial (prokaryotic) cell that has been genetically modified or "engineered" to be altered relative to a naturally occurring cell. Such genetic modification can be, for example, the incorporation of additional genetic information into the cell, the modification of existing genetic information, or the deletion of existing genetic information. This can be accomplished, for example, by transfecting the cell with a recombinant plasmid or by making modifications directly to the bacterial genome. Additionally, bacterial cells can be genetically modified to achieve attenuation, for example, by methods of chemical mutagenesis, which will be well known to those of skill in the art. Thus, the term "non-naturally occurring bacteria or bacteria" can refer to both recombinantly and non-recombinantly modified strains.

[0021] As used herein, the term "bactofection" refers to the process of transferring 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 "bactofection" refers to the delivery of Gram-negative bacteria to a target eukaryotic cell, followed by the delivery of RNA molecules transcribed in the Gram-negative bacteria into the cytoplasm of the eukaryotic cell. Refers to the use of gram-negative bacteria for the treatment of ulcerative colitis.

[0022] As used herein, the term "split RNA polymerase" or "RNAP" refers to a system in which separate components of an RNA polymerase are encoded by separate genes and assemble, once transcribed and translated, to form a functional RNA polymerase. Specifically, RNA polymerases are divided into a "core" component and a DNA-binding component called a "sigma" or "sigma factor."

[0023] As used herein, the terms "orthogonal" or "orthogonality" are used interchangeably and refer to the biological process of orthogonalization. Orthogonalization is the ability of two or more biomolecules with similar structure and / or function to interact with each other or affect their respective substrates. Therefore, in the context of the present invention, the term "orthogonal" means that the components required for expression of a polynucleotide encoding an RNA polymerase are not genetically modified. Preferably, the term "orthogonal" refers to an RNA polymerase that is not present in a gene encoding ... This refers to the absence of components necessary for expression of the polynucleotide encoding the lysine methylase in the Salmonella enterica genome.

[0024] The term "prophylactic treatment" as used herein refers to medical intervention aimed at preventing, rather than treating or curing, an infection or disease. In the present invention, this applies in particular to vaccine compositions. The term "prevention" as used herein is not absolute and may include partial prevention of an infection or disease and / or one or more symptoms of said infection or disease. In contrast, the term "therapeutic treatment," as understood in the art, refers to a medical procedure aimed at treating or curing an infection or disease or its associated symptoms.

[0025] The present invention provides gram-negative RNA molecules that can be safely, efficiently, and effectively delivered to target eukaryotic cells. As used herein, the terms "RNA" and "ribonucleic acid" refer to the modification of ribonucleic acid. These terms and concepts are well known to those skilled in the art. Examples of such genes include messenger RNA (mRNA), small interfering RNA (siRNA), and short hairpins. Examples of the encoded RNA molecule include shRNA, microRNA (miRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), and self-amplifying (or self-replicating) RNA (saRNA). In a preferred embodiment, the encoded RNA molecule is an mRNA, siRNA, or shRNA molecule.

[0026] As used herein, the terms "mRNA" and "messenger RNA" are used interchangeably. refers to a single-stranded RNA molecule involved in protein synthesis. Eukaryotic mRNA molecules are the RNA molecules that are expressed in eukaryotic cells. It is transcribed from DNA in the nucleus and then transported from the nucleus to the cytoplasm of eukaryotic cells, where it is converted into mRNA The translation of the molecule into a protein takes place. Bacterial mRNA molecules are transcribed from non-compartmentalized DNA and undergo transcription-coupled translation in the cytoplasm. These terms and concepts will be understood by those skilled in the art. It will be well known to

[0027] As used herein, the terms "siRNA" and "short interfering RNA" are used interchangeably to refer to a specific method of RNA interference (RNAi). RNAi is a sequence-specific RNA degradation process that theoretically provides a straightforward method for knocking down, or silencing, any gene. In natural RNAi, double-stranded RNA (dsRNA) is cleaved by the RNase III / helicase protein Dicer into 19-27 nucleotide (nt) dsRNA molecules with 2-nt overhangs at the 3' end, called siRNA molecules. These siRNAs are incorporated into a multicomponent ribonuclease called the RNA-induced silencing complex (RISC). The single strand of siRNA binds to RISC and is then cleaved into siRNA molecules. The siRNA-directed endonuclease remains in place and guides the complex to a target RNA with a sequence complementary to the guide single-stranded siRNA within RISC, where it digests and inactivates the RNA.

[0028] As used herein, the terms "shRNA" and "short hairpin RNA" are used interchangeably to refer to a specific method of RNA interference (RNAi). RNAi is a sequence-specific RNA degradation process that theoretically provides a relatively simple and straightforward method for knocking down, or silencing, any gene. The stem-loop structure consists of a stem portion made of double-stranded sequence. The stem has an antisense (guide) strand on one side and a sense (passenger) strand on the other side. The stem-loop structure further contains a single-stranded loop at one end of the stem. After processing via Drosha and Dicer, shRNAs are transformed into RNA-induced silencing complexes. The sense (passenger) strand is degraded, and the antisense (guide) strand guides RISC to the mRNA with the complementary sequence. If the complementarity is perfect, RISC cleaves the mRNA. If the complementarity is imperfect, RISC inhibits the translation of the mRNA. In either case, the shRNA silences the target gene.

[0029] As used herein, the terms "saRNA," "self-amplifying RNA," and "self-replicating RNA" are used interchangeably. Once the RNA is delivered to the desired location, i.e., in the context of the present invention, the cytoplasm of the host cell, This refers to a type of mRNA that encodes a replicase that copies the original strand when it is synthesized. The use increases the efficiency of delivery and expression of RNA molecules.

[0030] As used herein, "heterologous polynucleotide" refers to a polynucleotide introduced into a Gram-negative bacterium, i.e., the introduction of a polynucleotide not previously present. A heterologous polynucleotide in the context of the present invention is an RNA molecule intended for delivery to a eukaryotic cell. The resulting RNA molecule is referred to herein as a "cargo" or "cargo molecule." In particularly preferred embodiments, the encoded RNA molecule is a mammalian RNA molecule.

[0031] The terms "vaccine composition" or "vaccine," sometimes interchangeably referred to herein as "composition," refer to a biological preparation that provides active adaptive immunity against a specific disease. Typically, a vaccine contains an agent similar to a disease-causing pathogen or a "foreign" agent, which, in the context of the present invention, is encoded by an mRNA molecule (or cargo) that is delivered to a target eukaryotic cell. Such foreign pathogens are recognized by the vaccine recipient's immune system, which destroys them and develops "memory" against the disease-causing pathogen, inducing sustained protection against future infection or disease caused by the same or similar pathogens. It is envisioned that, through a vaccination route involving the vaccine composition of the present invention, if a vaccinated subject again encounters the same pathogen or pathogen isolate against which they were vaccinated, the individual's immune system will thereby recognize the pathogen or pathogen isolate and elicit more effective protection against infection or disease. The active adaptive immunity induced in a subject as a result of a vaccine may be humoral and / or cellular. In the context of the present invention, a variety of vaccine antigens can be delivered to target eukaryotic cells of a subject using the Gram-negative bacteria disclosed herein as a delivery method, thereby priming the subject's immune system against said antigens.

[0032] The terms "tumor," "cancer," "malignancy," and "neoplasia" are used interchangeably and refer to a cell or population of cells whose growth, proliferation, or survival is greater than that of a normal counterpart, e.g., a cell proliferative or differentiative disorder. Usually, the growth is uncontrolled. The term "malignant" refers to invasion of nearby tissues. The term "metastasis" refers to the spread or dissemination of a tumor, cancer, or neoplasm to another site, location, or region within a subject, which site, location, or region is different from the primary tumor or cancer. In one embodiment, the cancer is malignant. In another embodiment, the cancer is non-malignant.

[0033] The term "effective amount" or "pharmaceutically effective amount" refers to an amount of an agent sufficient to provide a desired biological or therapeutic result, which result may include reduction, amelioration, alleviation, relief, delay, and / or amelioration of one or more signs, symptoms, or causes of a disease, or a biological effect. In some embodiments, an effective amount is an amount sufficient to delay the onset of, prolong survival of, or induce stabilization of a cancer or tumor.

[0034] 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. The amount or combination of agents may result in one or more of the following: (i) a decrease in the number of cancer cells; (ii) reduce tumor size; (iii) inhibit cancer cell invasion into peripheral organs. (iv) inhibiting (i.e., slowing to some extent, preferably stopping) tumor metastasis; (v) inhibiting tumor growth; (vi) preventing tumor onset. (vii) prevent or delay the onset and / or recurrence of the cancer; and / or (vii) alleviate to some extent one or more symptoms associated with the cancer.

[0035] For example, in the case of treating tumors, a "therapeutically effective dose" is a dose that reduces tumor size by at least about 5%, e.g., at least about 10%, or about 20%, or about 60% or more relative to baseline measurements. A reduction can be induced. Baseline measurements may be obtained from untreated subjects.

[0036] A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such an amount based on factors such as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.

[0037] The terms "treatment" or "therapy" refer to the administration of an active agent with the intent to cure, cure, alleviate, relieve, alter, relieve, ameliorate, improve, or affect a condition (e.g., a disease), the symptoms of a condition, or to prevent or delay the onset of symptoms, complications, or biochemical signs of a condition, or to arrest or inhibit further development of a disease, condition, or disorder in a statistically significant manner.

[0038] As used herein, the term "subject" is intended to include humans and non-human animals.Preferred subjects include human patients who need to enhance immune response.This method is particularly suitable for treating human patients with disorders that can be treated by enhancing immune response.In certain embodiments, this method is particularly suitable for treating neoplastic disease or infectious disease in vivo.

[0039] The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to refer to "one or more" of any described or listed components.

[0040] 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 depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" means within one standard deviation, according to the practice of one of ordinary skill in the art. or more than one standard deviation. Alternatively, "about" can mean a range of up to 20%. When a specific value is described in the specification and claims, unless otherwise specified, the meaning of "about" should be assumed to be within an acceptable error range for that particular value.

[0041] Previous strategies to increase DNA bactofection levels have involved capping the cells after entry. The aim of this study was to induce lysis of the rear vector. Microbiology (2005) 7:709; Journal of Gene Med (2002) 4:655], E. coli (90% of The transduction of lactic acid bacteria has been reported in [Journal of Controlled Release (2021) 332:233] and Salmonella typhimurium [Material Horizons (2020) 8:1454]. Another method to promote endosomal membrane lysis is to co-express hemolysin derived from Listeria monocytogenes or phospholipase C derived from Clostridium spp. [Cellular Microbiology (2005) 7(5):709]. However, this method also results in a bottleneck effect due to low transfer of the plasmid from the cytoplasm to the nucleus.

[0042] To avoid the above problems, bacteria have been used to directly deliver RNA, instead of DNA, into the cytoplasm. RNA has previously been introduced into bacteria as a delivery vector using T7 polymerase (a common method for producing large amounts of RNA) or other strong promoters (WO2020245093; Schoen et al., 2005, Cellular Microbiology, 7(5), 709-724). Translation from bacterial RNA is ensured by adding an IRES sequence to the 5' end. The main limitation of this method is that bacterial RNA expression is unstable and difficult to achieve after transfection. The presence of an IRES sequence is essential for the RNA to be degraded before it can be fully expressed in the bacterial cytoplasm. It may also enhance stability: Like DNA, the release of RNA was enhanced after lysis of intracellular bacteria.

[0043] However, the inventors have surprisingly found that the use of T7 RNA polymerase (and the corresponding T7 promoter) at high expression levels results in the synthesis of RNA molecules when targeting eukaryotic cells. It has been found that the promoter system used in the past is not conducive to safe, efficient and effective delivery, and may cause undesirable toxicity and specificity problems.Therefore, the present inventors have identified the need for modifications to the promoter system used previously in the art.As outlined above, the present inventors have resolved the problems of the promoter system used in the past by the following two solutions: First, a heterologous split RNA polymerase encoded by a plasmid was used. The second is to use an RNA polymerase that can express the gram negative genes required for endogenous gene expression. Using RNA polymerase and promoter pairs that are orthogonal to all components of bacterial genomes is.

[0044] Accordingly, in a first aspect, the present invention provides 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, the RNA polymerase is encoded by a heterologous split RNA polymerase plasmid, and the Gram-negative bacterium invades a eukaryotic cell. The RNA molecule is then transcribed and transformed into a polynucleotide that can be transported into the cytoplasm of the eukaryotic cell. The present invention provides live attenuated Gram-negative bacteria comprising:

[0045] The inventors have surprisingly found that the toxicity and specificity problems previously observed with the use of the T7 promoter can be overcome by using a split RNA polymerase system. In such systems, RNA polymerase is separated into a "core" component and a DNA-binding component, termed "sigma" or "sigma factors." These separate components of RNA polymerase are encoded by separate genes and can therefore be regulated independently.

[0046] A schematic diagram of the split RNA polymerase system is shown in Figure 2. Take T7 RNA polymerase as an example. The "core" component of RNA polymerase consists of amino acids 1 to 601 of T7 RNA polymerase. The DNA-binding component of RNA polymerase (hereafter referred to as "sigma" or "sigma factor") is composed of amino acids 600 to 883 of T7 RNA polymerase. The sigma element can contain variations that allow it to bind to different promoter sequences. To ensure binding of both partners, a synthetic coiled-coil peptide was added to induce protein association (SYNZIP). Furthermore, the core element was integrated into a low-strength promoter (proA) and a strong promoter (H). We designed two types of circuits, one expressed from a weak (L) RBS and the other from a split (L) RBS. The use of a split RNA polymerase offers the possibility of using two or more different sigma factors to control the transcription of two or more elements. Different sigma factors have different efficiencies, and these two or more outputs can be expressed in a single circuit. This opens up the possibility of expressing the RNAPs at different ratios (e.g., one cargo RNA expressed at high levels and one lysine expressed at low mRNA levels). By dividing the RNAP into two segments, we can fine-tune the expression level of the core segment to obtain maximum RNA production with minimal toxicity. Furthermore, multiple sigma elements targeted to different promoters can be expressed simultaneously, binding to an optimal pool of expressed core elements to perform independent functions (e.g., RNA production and lysine expression) at different expression levels. The inventors' surprising finding is that the use of litRNAP not only allows for greater control over the expression level of the final product, but also reduces toxicity compared to using previously described promoter systems.

[0047] In a second aspect, the present invention provides a heterologous polynucleotide encoding an RNA molecule. ii) a heterologous polynucleotide, wherein the heterologous polynucleotide is operably linked to a promoter; and ii) 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 encodes When the Gram-negative bacterium invades a eukaryotic cell, the RNA molecule is transcribed and distributed to the cytoplasm of the eukaryotic cell. The present invention provides live attenuated Gram-negative bacteria containing a polynucleotide capable of being transferred to a host.

[0048] The heterologous polynucleotide of the second aspect is operable with a promoter that can be bound by an RNA polymerase that is orthogonal to components required for endogenous gene expression in the host Gram-negative bacterium. Thus, in one embodiment, the promoter / RNA polymerase pair is operably linked to the host. It is orthogonal to the components required for endogenous gene expression in the host Gram-negative bacterium. In this context, the components required for endogenous gene expression in the host Gram-negative bacterium may include the transcription and translation machinery. Gene expression is controlled by specific systems (e.g., non-endogenous RNA polymerases). Orthogonal RNA polymerases can be controlled by effectively isolating the orthogonal RNA polymerase / promoter pairing from host components (e.g., components required for endogenous gene expression in the host Gram-negative bacterium). Orthogonal RNA polymerases are incompatible with and cannot interact with the components required for gene expression. They may be non-endogenous to the host Gram-negative bacteria and will only interact with compatible promoters and therefore initiate transcription only from these promoters. Non-endogenous (or orthogonal) RNA polymerases may interact with endogenous promoters or gene sequences of the host Gram-negative bacteria. Similarly, orthogonal promoters do not interact with endogenous RNA polymerases. does not interact with

[0049] In some embodiments, the invention provides 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 of the Gram-negative bacterium, wherein the RNA polymerase binds to the promoter, the RNA polymerase is encoded by a heterologous split RNA polymerase plasmid, and wherein the RNA molecule is transcribed upon entry of the Gram-negative bacterium into a eukaryotic cell. Disclosed is a live attenuated Gram-negative bacterium comprising a polynucleotide capable of being translocated into the cytoplasm of said eukaryotic cell.

[0050] In order for an RNA molecule to be successfully transcribed in a target eukaryotic cell by an RNA polymerase in a Gram-negative bacterium, the heterologous polynucleotide encoding the RNA molecule is linked to a promoter, which RNA polymerase binds to this and initiates transcription of the RNA molecule.

[0051] In a preferred embodiment of both the first and second aspects, the heterologous encoding RNA molecule a promoter operably linked to the polynucleotide and to which RNA polymerase binds In a more preferred embodiment, the promoter encoding the RNA molecule is a phage promoter. a promoter that is operably linked to a heterologous polynucleotide that binds to the promoter and to which RNA polymerase binds; The promoter is a strong phage promoter, and when a strong phage promoter is used, high transcription rates are initiated. Thus, a preferred embodiment of the first aspect of the invention In this manner, the phage promoter is orthogonal to the components required for endogenous gene expression in Gram-negative bacteria. It is orthogonal to components required for endogenous gene expression in Mu-negative bacteria.

[0052] As used herein, the term "phage promoter" refers to a promoter derived from a virus, particularly a bacteriophage virus (a virus that infects and replicates only in bacterial cells). Examples of phage promoters include, but are not limited to, T7p, T3p, K1Fp, and CGGp. In a preferred embodiment, the phage promoter is CGGp. In this context, virally derived polymerases (i.e., phage RNA In a preferred embodiment of the second aspect of the invention, the best performing system may consist of a CGGp phage orthogonal promoter paired with a split RNA polymerase (CGG).

[0053] Examples of possible promoter / RNA polymerase pairs according to the first aspect of the present invention are shown in Table 1. Thus, the following RNA polymerase is a split RNA polymerase. In one embodiment, the promoter operably linked to a heterologous polynucleotide encoding an RNA molecule and to which the RNA polymerase binds is a T7 promoter, and the RNA polymerase is a T7 split RNA polymerase. In one embodiment, the promoter operably linked to a heterologous polynucleotide encoding an RNA molecule and to which the RNA polymerase binds is a T7 split RNA polymerase. The promoter operably linked to the heterologous polynucleotide encoding the RNA molecule and to which the RNA polymerase binds is a T3 promoter, and the RNA polymerase is a T3 split RNA polymerase. In one embodiment, the promoter operably linked to the heterologous polynucleotide encoding the RNA molecule and to which the RNA polymerase binds is a K1F promoter, and the RNA polymerase is a K1F split RNA polymerase. In a preferred embodiment, the promoter operably linked to the heterologous polynucleotide encoding the RNA molecule and to which the RNA polymerase binds is a CGG promoter, and the RNA polymerase is a CGG split RNA polymerase. In one embodiment, the promoter / RNA polymerase combination is orthogonal to all components required for endogenous gene expression in the host Gram-negative bacterium.

[0054] The inventors of the present invention surprisingly showed that high expression levels of T7 polymerase (using a T7 promoter) are toxic. Furthermore, although the inventors showed that low expression levels of T7 polymerase allow cells to grow, even low expression levels of T7 polymerase resulted in inefficient circuit output. Therefore, in one embodiment, RNA operably linked to a heterologous polynucleotide encoding the molecule, and to which RNA polymerase binds In another embodiment, the promoter that operates on a heterologous polynucleotide encoding an RNA molecule is not a T7 promoter and the RNA polymerase is not a T7 split RNA polymerase. The promoter to which RNA polymerase binds is not the T7 promoter. , the RNA polymerase is not T7 RNA polymerase.

[0055] In one embodiment, the pairing of the T7 promoter and the RNA polymerase is not orthogonal. In another embodiment, the pairing of the T7 promoter and the split RNA polymerase is not orthogonal.

[0056] [Table 1]

[0057] In one embodiment, the sigma factor RNA polymerase is a polymerase according to SEQ ID NO: 3, 5, 7, 9. The sigma factor RNA polymerase can have a polynucleotide sequence that is 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3, 5, 7, or 9.

[0058] In embodiments, the RNA molecule is operably linked to a heterologous polynucleotide encoding the RNA molecule. and the promoter to which the RNA polymerase binds may have a polynucleotide sequence according to SEQ ID NO: 11, 12, 13, 14, or a sequence having 90% sequence identity thereto. For example, a heterologous polynucleotide encoding an RNA molecule to which the RNA polymerase binds may 91% of the promoter operably linked to SEQ ID NO: 11, 12, 13 or 14 , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0059] In one embodiment, the sigma factor RNA polymerase is a polynucleotide according to SEQ ID NO: 3. a nucleotide sequence or a sequence having 90% sequence identity to said sequence, and operably linked to a heterologous polynucleotide encoding the molecule, and an RNA polymerase The promoter to which the promoter binds is a polynucleotide sequence according to SEQ ID NO: 11, or 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 to said sequence. a heterologous polynucleotide having a sequence with 90% sequence identity to the RNA molecule and encoding the RNA molecule; In another embodiment, the promoter operably linked to 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 is a polynucleotide sequence according to SEQ ID NO: 7. a nucleotide sequence or a sequence having 90% sequence identity to said sequence, operably linked to a heterologous polynucleotide encoding the molecule, and to which RNA polymerase binds The promoter may be a polynucleotide sequence according to SEQ ID NO: 13, or a sequence corresponding to said sequence. In a preferred embodiment, the sigma factor RNase A has a sequence with 90% sequence identity to the sigma factor RNase A. The A polymerase has a polynucleotide sequence according to SEQ ID NO: 9, or a sequence with 90% sequence identity thereto, and is operably linked to a heterologous polynucleotide encoding an RNA molecule, and the promoter to which the RNA polymerase binds has a polynucleotide sequence according to SEQ ID NO: 14, or a sequence with 90% sequence identity thereto.

[0060] As used herein, the terms "sequence identity" and "sequence homology" are interchangeable and refer to the degree of homology over a defined length in a given alignment of DNA or amino acid sequences. To calculate the percent sequence identity of any of the sequences disclosed herein, sequence comparison software can be used, for example, using the default settings of the BLAST software package (V2.10.1).

[0061] In order to drive the expression of the RNA polymerases of the first and second aspects of the present invention (i.e., both split and non-split RNA polymerase systems), intracellular promoters are used. Promoters such as cytoplasmic and vacuole-dependent promoters are used.

[0062] In the context of the present invention, these promoters are designed to induce the expression of sigma factors of the above RNA polymerase systems (e.g., The promoter can be used to activate the sigma factors (as disclosed in Table 1). They are distributed throughout the genome of RAB-negative bacteria (e.g., Salmonella enterica), located upstream of related genes, and activated by numerous signals. For example, vacuolar promoters generally respond to acidification and hypoosmolarity, while cytoplasmic promoters generally respond to iron, manganese, hexose sugars, and oxidative stress. In the context of the split RNA polymerase system, In some cases, multiple sigma factors may be driven by the intracellular promoters described above. For example, expression of one sigma factor may be driven by a cytoplasmic promoter and expression of a second, different sigma factor may be driven by a vacuole-dependent promoter. Alternatively, expression of two different sigma factors may be driven by a vacuole-dependent promoter. Expression of the sigma factors may be driven by two cytoplasmic promoters or two vacuole-dependent promoters.

[0063] Thus, in a particularly preferred embodiment, the RNA polymerases of the first and second aspects of the invention The promoters used to activate the enzyme system are cytoplasmic promoters or vacuole-dependent promoters. The term "cytoplasmic promoter" as used herein refers to intracellular promoters in bacteria. In a further preferred embodiment, the cytoplasmic promoter for use in the present invention is selected from the group consisting of 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 is a vacuole-dependent promoter. The tars are 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). are selected.

[0064] The use of such promoters allows precise control of the activation of the RNA polymerase system. These enable, for example, activation to occur at specific points or times during the infection cycle. Furthermore, intracellular (SPI-2), oxidative stress response (e.g., soxP, grxA), sugar sensing (e.g., ATPase inhibitors), and other proteins are also involved. (e.g., uphT (glucose-6-phosphate), frubKA (fructose)), or metal-dependent Temporal control of expression can be achieved by using cytoplasmic promoters (e.g., iroN, fhuE, mntH, entC, fepB). These cytoplasmic promoters are not limited to RNA polymerase. The SPI-2 promoter can also be used to drive differential expression of lysine. , which promotes the export of hemolysin from Listeria and is used to activate the expression of the type-1 secretion system (T1SS), which allows the release of bacterial cells from the vacuole into the cytoplasm of eukaryotic cells, for example. It is possible.

[0065] Use in vivo or constitutive promoters to regulate the total amount of polymerase activity As used herein, the term "constitutive promoter" refers to a promoter that initiates transcription only upon receiving a specific stimulus. In the context of RNA polymerase, such promoters are responsible for the production of the RNA polymerase "core." This regulates the extent to which "sigma factors" can bind.

[0066] The invention disclosed herein involves the transfer of bacterially transcribed RNA molecules into the cytoplasm of eukaryotic host cells. The present invention relates to a method for initiating transcription of an RNA molecule, such as an mRNA, siRNA or shRNA molecule. To do this, Gram-negative bacteria must first invade the target eukaryotic cell. , for example, within the vacuole of a target eukaryotic cell, or Gram-negative bacteria that contain lysis proteins that program them to self-lyse and release the transcribed RNA molecules. Bacterial release from the phagocytic vacuole is desirable for efficient delivery of either DNA or RNA molecules, as evidenced by the high in vitro bactofection efficiency from the cytoplasmic pathogen Listeria. Thus, in one embodiment, bacteria are engineered to be able to escape from the phagocytic vacuole. For example, release from a Salmonella-containing vacuole (SCV) This can be achieved in at least two ways. First, by the addition of exogenous genes. For example, when Listeria monocytogenes-derived hly is expressed in Salmonella, it is exported from the bacterial cytoplasm to the SCV via the type 1 secretion system. Second, Salmonella SPI-1 (or SPI-2) effector proteins are secreted. It manipulates proteins (sopF and sopE) to destabilize the SCV and release bacteria into the cytoplasm. The ctor protein is injected into the cytoplasm of invaded mammalian cells and is involved in the maintenance of the initial vacuole. Literature has shown that strains lacking the sopF gene or overexpressing the sopE gene product escape from the vacuole during the early stages of invasion and either settle in the host cytoplasm or are re-encapsulated by the autophagy machinery.

[0067] As described above, the Gram-negative bacteria of the present invention express the transcribed RNA molecules in the cells of a eukaryotic host cell. They can be programmed to autolyse by incorporating lytic proteins for release into the cytoplasm. One way to achieve this is through the use of cytoplasmic promoters. Upon entering the cytoplasm of a eukaryotic cell, bacteria with cytoplasmic promoters induce their own lysis and produce RNA that enters directly into the cytoplasm, thus avoiding the transfer of genetic material into the nucleus of the target eukaryotic cell. This can be done.

[0068] In the context of Listeria, the inventors of the present invention have found that the use of a cytoplasmic promoter increases the release of RNA molecules from Listeria by approximately 10-fold. In a preferred embodiment, lysis of bacterial cells is carried out through the use of a cytoplasmic promoter. Lysis of bacterial cells can also be achieved through the use of lysin or a bacterial release protein, such as lysE, kilR, kil, or BRP.

[0069] A biotinylated enzyme comprising a promoter and an RNA polymerase that binds to the promoter. In addition to virulent Gram-negative bacteria, live attenuated Gram-negative bacteria are also used to deliver RNA molecules to target eukaryotic cells. It may further comprise a number of other accessory proteins that optimize delivery. For example, the live attenuated Gram-negative bacterium may further comprise one or more of the following accessory proteins integrated into the genome of the Gram-negative bacterium: i) a polypeptide encoding an RNA stability-enhancing component; nucleotides, preferably (when the RNA molecule is an mRNA molecule) wherein the RNA stability enhancing component is an IRES sequence; ii) polynucleotides encoding bacteriolytic proteins, preferably wherein said bacteriolytic protein is hemolysin; iii) polynucleotides encoding phospholipases; iv) a method for treating an invasion factor, preferably a method for treating an invasion factor, and / or v) a bacteriocin-releasing protein, a bacteriocin-releasing polynucleotide; Lysozyme, phage lambda lysozyme, and holin. In a preferred embodiment, lysis proteins is derived from the genus Listeria and contains polynucleotides encoding a bacteriocin release protein, bacteriophage lambda lysozyme, and a holin, allowing the bacterium to autolyse upon entry into a target eukaryotic cell.

[0070] The present invention provides methods for silencing target genes, preventing or reducing protein synthesis, or synthesizing new proteins. As will be appreciated by those skilled in the art, such methods have important and broad therapeutic benefits. The heterologous polynucleotide is functionally modified to allow transcription of the heterologous polynucleotide and production of RNA molecules in Gram-negative bacteria. The Gram-negative bacterium further comprises an appropriate RNA polymerase. The heterologous gene comprises a heterologous polynucleotide encoding an RNA molecule encoding a nucleotide sequence ... Methods for integrating proteins into bacterial genomes are well known to those skilled in the art and are described in further detail below.

[0071] The live attenuated Gram-negative bacteria of the present invention are an efficient means of delivering heterologous RNA to target eukaryotic cells. Therefore, the bacterial strains disclosed herein can be used to detect and analyze RNA molecules. a gene encoding an RNA molecule (i.e., an RNA molecule such as mRNA, siRNA, shRNA, saRNA, or miRNA) It is a recombinant strain containing ribonucleotides.

[0072] The functional RNA molecule delivered to eukaryotic cells may be messenger RNA (mRNA). Messenger RNA is transcribed from DNA and contains the genetic blueprint for making proteins. In prokaryotes, mRNA does not need to be processed and can immediately proceed to protein synthesis. In eukaryotes, the newly transcribed RNA transcript is considered pre-mRNA and is required to form mRNA. Pre-mRNA contains non-coding and coding regions called introns and exons, respectively. During pre-mRNA processing, introns are spliced ​​out and exons are joined. The 5' end of the RNA transcript contains a 7-methyl A 5' cap called guanosine is added, and the 3' end is polyadenylated. Polyadenylation is the process by which a poly(A) tail, a sequence of adenine nucleotides, is added to the transcript. The 5' cap protects mRNA from degradation, and the 3' poly(A) tail contributes to mRNA stability. The live attenuated Gram-negative bacterium can encode, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 heterologous mRNA molecules.

[0073] The mRNA of the present invention is transcribed in Gram-negative bacteria and then transported to a target eukaryotic cell for translation using the host eukaryotic cell's translation machinery. Accordingly, the invention disclosed herein provides mRNA molecules having the aforementioned modifications. 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 increase the efficiency of RNA delivery and increase expression of the RNA molecule. saRNA molecules are RNA molecules in which the polynucleotide sequence encoding the RNA molecule is transfected upon delivery to the host cell. It also encodes a replicase that allows amplification of the original RNA strand, making it "self-amplifying." In this way, the use of saRNA can provide the above advantages with a minimal amount of RNA.

[0074] The polypeptide encoded by the mRNA molecule may be any therapeutic protein used for the treatment or prevention of disease.It is easily understood that the specific therapeutic protein depends on various factors, including the specific disease to be treated.In some embodiments, the live attenuated Gram-negative bacterium does not encode a heterologous polypeptide that modifies the genome of the target eukaryotic cell.For example, the Gram-negative bacterium of the present invention does not encode an RNA that encodes a component of the CRISPR / Cas9 gene editing system, a zinc finger nuclease, or a transcription activator-like effector nuclease (TALEN).

[0075] In another embodiment, the functional RNA molecule targets a specific mRNA molecule present in a eukaryotic cell. After transcription of the siRNA or shRNA molecule in bacteria, the transcribed The resulting siRNA or shRNA molecule is transported into the cytoplasm of eukaryotic cells, where it localizes and binds to the target mRNA. Thus, the invention disclosed herein also provides a method for effectively silencing a target gene by activating the corresponding mRNA via an siRNA or shRNA molecule.

[0076] The present invention can use siRNA and / or shRNA molecules to target a single mRNA or multiple mRNAs.Therefore, the live attenuated Gram-negative bacterium of the present invention can deliver a single siRNA molecule or multiple siRNA molecules, each of which targets different target mRNAs.For example, the live attenuated Gram-negative bacterium can deliver up to 10 different heterologous siRNA or shRNA molecules, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 different heterologous siRNA or shRNA molecules. The molecule can be encoded.

[0077] siRNA or shRNA molecules can bind to any target mRNA. The target mRNA may be any mRNA encoding a protein of interest, such as an mRNA encoding a protein associated with a disease state. For example, the target mRNA may include mRNAs involved in proliferation / cell cycle, migration, angiogenesis, immune activation / inhibition, cell death, and fibrosis. Furthermore, the target mRNA may include, but is not limited to, the HIF1 protein family, the TGFβ receptor protein family, and various transcription factors that promote dysregulation of cancer cells. It will be readily understood that the specific mRNA targeted will depend on various factors, including the specific disease being treated. In some embodiments, the live attenuated Gram-negative bacterium does not encode a heterologous siRNA or shRNA that modifies the genome of the target eukaryotic cell.

[0078] The live attenuated bacteria of the present invention are Gram-negative bacteria. Examples of Gram-negative bacteria for use in the present invention include, but are not limited to, Escherichia coli, Salmonella, Shigella, Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, Legionella, Chlamydia, and Yersinia.

[0079] Preferably, the live attenuated Gram-negative bacterium is a Salmonella species. Exemplary Salmonella species for use in the present invention are Salmonella enterica and Salmonella bongori. Salmonella enterica can be further subdivided into different serotypes or serogroups. Exemplary serotypes or serostrains for use 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 Enteritidis. In a preferred embodiment, the live attenuated Gram-negative bacterium is Salmonella enterica Typhi and / or Salmonella enterica Typhimurium. In a most preferred embodiment, the live attenuated Gram-negative bacterium is Salmonella enterica Typhi.

[0080] Thus, the present invention provides attenuated bacteria that can effectively deliver RNA molecules as their cargo. The present disclosure discloses Gram-negative bacteria genetically modified to produce strains. As will be appreciated by those skilled in the art, genes can be mutated by many methods known in the art, such as homologous recombination using a recombinant plasmid targeted to the gene of interest. In this case, an engineered gene homologous to the target gene is incorporated into an appropriate nucleic acid vector (such as a plasmid or bacteriophage) and transfected into a target cell. The homologous engineered gene is then recombined with the native gene and replaced or mutated to achieve the desired inactivation mutation. Such modifications may be to the coding portion of the gene or to regulatory portions such as the promoter region. As will be appreciated by those skilled in the art, any suitable genetic modification technique can be used to mutate a gene of interest, such as a CRISPR / Cas system, e.g., CRISPR / Cas 9, to produce the bacterial strains disclosed herein.

[0081] Thus, numerous methods and techniques for genetically engineering bacterial strains are available in the art. Those skilled in the art will be familiar with techniques for introducing heterologous genes into bacteria. These techniques include those required for introducing heterologous genes into bacteria via chromosomal integration or through the introduction of stable, autosomal, self-replicating genetic elements. Exemplary methods for genetically modifying (also called "transformation" or "engineering") bacterial cells include bacteriophage infection, transduction, conjugation, lipofection, or electroporation. General discussions of these and other methods in molecular and cellular biochemistry can be found in Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harvard Laboratories Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., Protein Methods (Bollag et al., John Wiley & Sons 1996); Protein Methods (Bollag et al., John Wiley & Sons 1999).

[0082] The live attenuated Gram-negative bacteria may be selected from the group consisting of Ty21a, CVD 908-htrA, CVD 909, Ty800, M01ZH09, x9633, x639, x9640, x8444, DTY88, ZH9PA, MD58, WT05, ZH26, SL7838, SL7207, VNP20009, A1-R, or a combination thereof. In the present invention, the live attenuated Gram-negative bacterium is M01ZH09 (also called ZH9).

[0083] In one embodiment, the genetically engineered non-naturally occurring bacterium comprises Salmonella pathogenicity island 2. The live attenuated bacteria may be derived from Salmonella species, which may contain an attenuating mutation in the (SPI-2) gene and an attenuating mutation in a second gene. Suitable genes and details of such live attenuated bacteria are described in WO 2000 / 68261, which is incorporated herein by reference in its entirety. do.

[0084] In one embodiment, the SPI-2 gene is an ssa gene. For example, the present invention encompasses 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 attenuated Preferably, the attenuating mutation is in the ssaV or ssaJ gene. More preferably, the attenuating mutation is in the ssaV gene.

[0085] The genetically engineered non-naturally occurring bacterium may also contain an attenuating mutation in a second gene that may or may not be in the SPI-2 region. This mutation may be outside the SPI-2 region. For example, the present invention provides an attenuated aro gene. In a preferred embodiment, the aro gene is aroA or aroC. Preferably, the aro gene is aroC.

[0086] The genetically engineered non-naturally occurring bacterium may further comprise one or more gene cassettes that can be used to deliver additional prokaryotic molecules to support the function of the genetically engineered non-naturally occurring bacterium in modulating the immune system or to support the activity of a therapeutic protein encoded by the mRNA molecule.

[0087] In yet another embodiment, the genetically engineered non-naturally occurring bacterium may be from a Salmonella spp. and may comprise inactivating mutations in one or more genes selected from pltA, pltB, cdtB, and ttsA, and may further comprise attenuating mutations in one or more genes selected from aroA and / or aroC and / or ssaV. Details of the differences are as described in WO2019 / 110819, which is incorporated herein by reference in its entirety.

[0088] It is contemplated that inactivating mutations (e.g., deletions) in the genes pltA, pltB, and cdtB prevent Salmonella from producing typhoid toxin, and that inactivating mutations (e.g., deletions) in ttsA prevent Salmonella from secreting typhoid toxin. It is contemplated that the non-naturally occurring bacterium is particularly derived from Salmonella enterica.

[0089] The present invention provides a method for the safe, efficient, and effective delivery of heterologous RNA molecules to target eukaryotic cells. When the RNA molecule is an mRNA molecule, the resulting heterologous polypeptide can be used as a therapeutic protein. The heterologous polypeptide may be a heterologous protein and / or a heterologous antigen (depending on the indication being treated, e.g., in the context of a vaccine composition, a heterologous polypeptide may be a heterologous antigen). In a preferred embodiment, the therapeutic protein obtained is a cytokine, a chemokine, an antibody or fragment thereof, a cytotoxic agent, a cancer agent, or any combination thereof. More preferably, the therapeutic protein obtained is IL-15, IL-21, CXCL9, IL-18, IL-27 , IFNγ, IL-1, or any combination thereof. Heterologous siRNA or shRNA molecules are delivered to target eukaryotic cells by using the siRNA or shRNA delivery method disclosed herein.Using this method, any mRNA molecule can be effectively targeted, and can effectively silence related genes.Therefore, the present invention is particularly useful in the disease state where it is advantageous to block the production of specific protein.

[0090] In a preferred embodiment of the present invention, the live attenuated Gram-negative bacteria are administered intratumorally, intravenously, intraperitoneally, or orally. In a most preferred embodiment, the live attenuated Gram-negative bacteria are administered intratumorally. However, it is contemplated that other administration methods may be used in some cases. Thus, in certain embodiments, the live attenuated Gram-negative bacteria of the present invention may be administered by injection, infusion, continuous infusion, intradermal, intraarterial, intravaginal, intrarectal, intramuscular, subcutaneous, subconjunctival, intramucosal, intrapericardial, intraumbilical, intraocular, intracranial, intraarticular, intrathoracic, intratracheal, intranasal, inhalation (e.g., aerosol inhalation), via a catheter, via lavage, or by other methods known to those skilled in the art, or any combination of the above (see, for example, Remington's Pharmaceutical Sciences, 18th Ed., Mack Printing Company, 1990).

[0091] The amount of live attenuated Gram-negative bacteria administered to a subject is sufficient to deliver the heterologous RNA molecule to the target eukaryotic cell at a concentration high enough to exert the desired effect after translation within the eukaryotic cell. Those skilled in the art will appreciate that the precise amount to administer will depend on, for example, the disease to be treated, the desired protein to be translated (if the RNA molecule is an mRNA molecule), the target mRNA present in the target eukaryotic cell, and other factors. the level of translation of the RNA molecule (if the RNA molecule is an siRNA or shRNA molecule), and the medical history of the subject to be treated. It will be easy to see that this depends on many factors.

[0092] Live attenuated gram-negative bacteria are 5 From 10 12 CFU, where CFU is colony forming units. For example, a suitable dose may be between 10 5 and 10 6 Between CFU, 10 5 and 10 7 Between CFU, 10 5 and 10 8 Between CFU, 10 5 and 10 9 Between CFU, 10 5 and 10 10 Between CFU, 10 5 and 10 11 Between CFU, 10 6 and 10 7 CFU, 10 6 and 10 8 CFU, 10 6 and 10 9 CFU, 10 6 and 10 10 CFU, 10 6 and 10 11 CFU, 10 6 and 10 12 CFU, 10 7 and 10 8 CFU, 10 7 and 10 9 CFU, 10 7 and 10 10 CFU, 10 7and 10 11 CFU, 10 7 and 10 12 CFU, 10 8 and 10 9 CFU, 10 8 and 10 10 CFU, 10 8 and 10 11 CFU, 10 8 and 10 12 CFU, 10 9 and 10 10 CFU, 10 9 and 10 11 CFU, 10 9 and 10 12 CFU, 10 10 and 10 11 CFU, 10 10 and 10 12 CFU, or 10 11 and 10 12 CFU. The live attenuated Gram-negative bacteria may be administered in a single dose or multiple doses. The specific number of doses administered will depend on the particular RNA molecule being delivered or targeted, as well as the particular indication being treated. It is understood that

[0093] The object of the present invention is to deliver RNA molecules to target eukaryotic cells and have them translated into desired therapeutic proteins. The object of the present invention is to provide an effective and efficient method for delivering an iRNA molecule (when the RNA molecule is an mRNA molecule) or an iRNA molecule (when the RNA molecule is an siRNA or shRNA molecule) to a target eukaryotic cell. The target eukaryotic cell may be a mammalian cell. In a preferred embodiment, the target eukaryotic cell is a human cell. When the eukaryotic cell is a human cell, the target cell may be a cancerous human cell or a non-cancerous human cell. The cells may also be human cells.

[0094] The live attenuated Gram-negative bacterium of either the first or second aspect of the present invention disclosed herein may be for therapeutic use. For example, the live attenuated Gram-negative bacterium may be for use in treating, alleviating, inhibiting, preventing, preventing recurrence, or controlling a disease. In a preferred embodiment, the disease is a human disease. In a preferred embodiment, the disease may be a neoplastic disease, an infectious disease, a cardiovascular disease, a neurodegenerative disease, a gastrointestinal disease, a respiratory disease, a renal disease, a liver disease, an autoimmune disease, an inflammatory disease, or a genetic disorder. In a preferred embodiment, the live attenuated Gram-negative bacterium is for use in treating, alleviating, inhibiting, preventing, preventing recurrence, or controlling a neoplastic disease or an infectious disease.

[0095] When the disease being treated is a neoplastic disease, the neoplastic disease may be a solid tumor and / or a hematological tumor. The neoplastic disease may be associated with a cancer selected from the group consisting of 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, and sarcoma.

[0096] Neoplasms, tumors and cancers include benign, malignant, metastatic and non-metastatic types, any stage (I, II, III, IV or V) or grade (G1, G2, G3, etc.) of the neoplasm, tumor or cancer; or a neoplasm, tumor, cancer, or metastasis that is progressing, worsening, stabilized, or in remission. Cancers that may be treated according to the present invention include, but are not limited to, cells or neoplasms of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gingiva, head, kidney, liver, lung, nasopharynx, cervix, ovary, prostate, skin, stomach, testis, tongue, or uterus. Furthermore, the cancer may be of the following histological types, among others, but not limited to: neoplasia, malignancy; carcinoma; undifferentiated; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilonidal carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; cavernous adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; familial polyposis coli adenocarcinoma; solid tumors; malignant carcinoid tumors; bronchoalveolar adenocarcinoma; papillary adenocarcinoma; chromocell carcinoma; eosinophilic carcinoma; eosinophilic carcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-encapsulating sclerosing carcinoma; adrenocortical carcinoma; endometrial carcinoma; skin adnexal carcinoma; apocrine adenocarcinoma; sebaceous carcinoma; Ear adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary Cystadenocarcinoma; Papillary serous cystadenocarcinoma; Mucinous cystadenocarcinoma; Mucinous adenocarcinoma; Signet ring cell carcinoma; Invasive ductal carcinoma; Medullary carcinoma; Lobular carcinoma; Inflammatory carcinoma; Paget's disease of the breast; Spinach cell carcinoma; Adenosquamous carcinoma; Adenocarcinoma with squamous metaplasia; Thymoma (malignant); Ovarian stromal tumor (malignant); Sarcoma (malignant); Granulosa cell tumor (malignant); Androblastoma (malignant); Sertoli cell carcinoma; Leydig cell tumor (malignant); Lipid cell tumor (malignant); Paraganglioma (malignant); Extramammary paraganglioma, malignant; Pheochromocytoma; Angiosarcoma; 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; Stromal sarcoma; Mixed tumor; Müllerian mixed tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Mesenchymoma (malignant); Brenner tumor (malignant); Philodes tumor (malignant); Synovial sarcoma; Mesothelioma (malignant); Hemoblastoma; Embryonic carcinoma; Teratoma (malignant); Ovarian adenoma (malignant); Choriocarcinoma; Malignant mesostoma; Angiosarcoma; Malignant hemangioendothelioma; Kaposi's sarcoma; Malignant hemangiopericytoma; Lymphoid Angiosarcoma; Osteosarcoma; Soft cortical osteosarcoma; Chondrosarcoma; Malignant chondroblastoma; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Odontogenic tumor (malignant); Ablastic odontoma; Ablastic fibrosarcoma; Pinealoma (malignant); Chordoma; Glioma (malignant ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astrocytoblastoma; Glioblastoma; Oligodendroglioma; Oligodendroglioma; Primitive neuroectodermal; Small Brain sarcoma; Ganglioneuroblastoma; Neuroblastoma; Retinoblastoma; Olfactory neurogenic tumor; Malignant meningioma; Neurofibrosarcoma; Malignant neurilemmoma; Malignant granular cell tumor; Malignant lymphoma; Hodgkin's disease; Hodgkin's disease; Paraneurilemmoma; Malignant lymphoma, small lymphocytic; Malignant lymphoma, large cell, diffuse; Malignant The neoplastic disease may be a tumor associated with cancer and / or other forms of carcinoma selected from the group consisting of prostate cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colorectal cancer, pancreatic cancer, brain cancer, hepatocellular carcinoma, lymphoma, leukemia, lymphoma, follicular lymphoma, mycosis fungoides, other specified non-Hodgkin's lymphoma, malignant histiocytosis, multiple myeloma, mast cell sarcoma, immunoproliferative small intestinal disease, lymphocytic leukemia, plasma cell leukemia, erythroid leukemia, lymphosarcoma cell leukemia, myeloid leukemia, basophilic leukemia, eosinophilic leukemia, monocytic leukemia, mast cell leukemia, megakaryoblastic leukemia, myeloid sarcoma, and hairy cell leukemia. Preferably, the neoplastic disease may be a tumor associated with cancer and / or other forms of carcinoma 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. The tumor may be metastatic or malignant.

[0097] In preferred embodiments, 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 or breast cancer.

[0098] In a third aspect, the present invention provides a vaccine composition comprising a live attenuated Gram-negative bacterium, wherein the Gram-negative bacterium: i) contains a heterologous polynucleotide encoding a functional mRNA molecule; iodide, wherein the heterologous polynucleotide is operably linked to a promoter and the mRNA molecule encodes a therapeutic protein and / or therapeutic peptide; and ii) an mRNA molecule encoding an RNA polymerase that is orthogonal to components required for endogenous gene expression in the Gram-negative bacterium. a polynucleotide that encodes a gene encoding a gene encoding a gene for a nucleic acid sequence, wherein the RNA polymerase binds to the promoter and the gene encoding a gene for a nucleic acid sequence is Upon invasion of a eukaryotic cell by a Rum-negative bacterium, the mRNA molecule is transcribed and can be transported into the cytoplasm of the eukaryotic cell.

[0099] In a fourth aspect, the present invention provides a vaccine composition comprising a live attenuated Gram-negative bacterium, wherein the Gram-negative bacterium: i) contains a heterologous polynucleotide encoding a functional mRNA molecule; and ii) a polynucleotide encoding an RNA polymerase, wherein the RNA polymerase binds to the promoter, the RNA polymerase is encoded by a heterologous split RNA polymerase plasmid, and upon invasion of the Gram-negative bacterium into a eukaryotic cell, the mRNA molecule is transcribed and is capable of being transported into the cytoplasm of the eukaryotic cell.

[0100] In one embodiment, the present invention provides a method for the preparation of a heterologous polynucleotide encoding a functional mRNA molecule. ii) a heterologous polynucleotide, wherein the heterologous polynucleotide is operably linked to a promoter and the mRNA molecule encodes a therapeutic protein and / or therapeutic peptide; and ii) an RNA polymerase that is orthogonal to components required for endogenous gene expression in the Gram-negative bacterium. a polynucleotide encoding an RNA polymerase, wherein the RNA polymerase binds to the promoter and a polynucleotide, wherein the RNA polymerase is encoded by a heterologous split RNA polymerase plasmid, and wherein the mRNA molecule is transcribed and capable of being transported into the cytoplasm of the eukaryotic cell upon invasion of the Gram-negative bacterium.

[0101] In one embodiment, the vaccine compositions of the invention may be therapeutic, for example, the live attenuated Gram-negative bacteria may be for use in treating, ameliorating, inhibiting, preventing, preventing recurrence, or controlling disease.

[0102] Thus, the vaccine composition of the third or fourth aspect may comprise one or more of the embodiments described above with respect to any preceding aspect.

[0103] The phage promoter of the vaccine composition of the third or fourth aspect of the invention may be T3p, K1Fp, or CGGp.

[0104] It is particularly contemplated that the vaccine compositions disclosed herein may be used to treat, alleviate, inhibit, prevent recurrence, or control infectious diseases, such as those caused by bacteria, viruses, parasites, or fungi. In such cases, the heterologous polynucleotide transcribed into a functional mRNA molecule may be an antigen of a specific infectious disease causative agent to generate an immune response in the host. Alternatively, it is contemplated that the vaccine compositions disclosed herein may be used as cancer vaccines. In such cases, the vaccine composition comprises a Gram-negative bacterium containing a heterologous polynucleotide encoding a cancer antigen capable of generating an immune response in the host. It is therefore understood that the bacteria and methods disclosed herein can be used to prevent / treat a wide range of cancers and infectious diseases. In another example, the heterologous polynucleotide is transcribed into an siRNA or shRNA molecule designed to enhance immune anti-infective function or tissue anti-infective defense.

[0105] The vaccine compositions of the present invention may further comprise an adjuvant, a pharmaceutically acceptable carrier or excipient.

[0106] As used herein, "pharmaceutically acceptable carriers / additives / diluents / vehicles" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonicity agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and the like, and combinations thereof, as known to those skilled in the art (see, e.g., 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 (0.9% NaCl), and sterile water.

[0107] Suitable aqueous and non-aqueous carriers that can be employed in the vaccine compositions of the present invention include water, ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0108] The vaccine compositions disclosed herein can further contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of undesirable microorganisms can be ensured both by sterilization procedures, as described above, and by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, and phenol sorbic acid. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the compositions. Furthermore, absorption delaying agents, such as aluminum monostearate and gelatin, can be included to prolong absorption of injectable pharmaceutical forms. Vaccine compositions can also optionally contain additional therapeutic agents known to be effective, for example, in treating infectious or neoplastic diseases. Thus, the vaccine compositions disclosed herein can also include antiretroviral agents, antibiotics, antifungals, antiparasitics, and anticancer agents.

[0109] The vaccine composition may also contain additional components intended to enhance the immune response in a subject after administration. Examples of such additional components include aluminum hydroxide, acid aluminum salts such as aluminum chloride 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); peptidoglycan (e.g., , murein, mucopeptides, or glycoproteins such as N-opaca, muramyl dipeptide [MDP], or MDP analogs), proteoglycans (e.g., from Klebsiella pneumoniae extracts), streptococcal preparations (e.g., OK432), muramyl dipeptide, immune-stimulating complex ("Iscoms" as disclosed in EP 109 942, EP 180 564 and EP 231 039), saponin, DEAE-dextran, neutral oils (such as miglyol), vegetable oils (such as arachis oil), liposomes, polyols, the Ribi adjuvant system (see, e.g., GB-A-2 189 141), vitamin E, Carbopol, interferons (e.g., IFN-α, IFN-γ, or IFN-β), or interferons. Leukins, 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).

[0110] 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. Cut.

[0111] In a fifth aspect of the present invention, there is provided a method of treating, inhibiting, preventing recurrence or controlling disease in a subject, the method comprising: i) a heterologous polynucleotide encoding an RNA molecule, wherein said 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 said Gram-negative bacterium. a heterologous split RNA polymerase plasmid encoding the RNA polymerase, wherein the RNA polymerase is a nucleotide sequence encoding the gram-negative fragment of the gram-negative fragment of the RNA polymerase, wherein the RNA polymerase is a nucleotide sequence encoding the gram-negative fragment of the gram-negative fragment of the RNA polymerase, and ... and wherein the RNA polymerase is a nucleotide sequence encoding the gram-negative fragment of the gram-negative fragment of the RNA polymerase, and wherein the RNA polymerase is a nucleotide sequence encoding the gram-negative fragment of the gram-negative fragment of the RNA polymer Upon invasion of a eukaryotic cell by a negative bacterium, the RNA molecule is transcribed and released into the cytoplasm of the eukaryotic cell. The method comprises administering to a subject a live attenuated Gram-negative bacterium containing a polynucleotide capable of being translocated.

[0112] Thus, the method of treating, suppressing, preventing recurrence or controlling disease in a subject of the fifth aspect may include one or more of the embodiments described above with respect to any preceding aspect.

[0113] In one embodiment, the method allows for safe, efficient and effective delivery of RNA molecules to target eukaryotic cells. To achieve this, a split RNA polymerase plasmid is incorporated into the fifth aspect of the invention. This can be done.

[0114] In a sixth aspect, the present invention provides a method of treating, inhibiting, preventing recurrence or controlling disease in a subject, the method comprising administering to a subject a live attenuated Gram-negative bacterium for use in treating, alleviating, inhibiting, preventing recurrence or controlling 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 of the Gram-negative bacterium. wherein the RNA polymerase binds to the promoter, and the Gram-negative bacterium is a eukaryotic cell. Upon entry into the eukaryotic cell, the RNA molecule is transcribed and can be transported into the cytoplasm of the eukaryotic cell. including leotide.

[0115] Thus, the method of treating, suppressing, preventing or controlling disease in a subject of the sixth aspect may include one or more of the embodiments described above with respect to any preceding aspect.

[0116] In one embodiment, a method for the safe, efficient, and effective delivery of RNA molecules to target eukaryotic cells is provided. To achieve this, a cytoplasmic promoter may be incorporated into the sixth aspect of the invention.

[0117] 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 bacterium disclosed herein. do.

[0118] In a further preferred embodiment, the disease to be treated is a neoplastic disease or an infectious disease.

[0119] In a seventh aspect of the present invention, there is provided a method for delivering an RNA molecule to a eukaryotic cell, said method comprising the steps of: i) modifying a Gram-negative bacterium so that a heterologous polynucleotide encoding an RNA molecule is integrated into the bacterial genome, wherein the heterologous polynucleotide is operably linked to a promoter; and ii) contacting the Gram-negative bacterium with a eukaryotic cell, such that the Gram-negative bacterium replicates within the eukaryotic cell and the heterologous polynucleotide is transcribed and then transported from the Gram-negative bacterium into the cytoplasm of the eukaryotic cell.

[0120] Thus, the method of delivering an RNA molecule of the seventh aspect to a eukaryotic cell may include one or more of the embodiments described above with respect to any of the preceding aspects.

[0121] In a preferred embodiment, the live attenuated Gram-negative bacterium has any one or a combination of the characteristics of the live attenuated Gram-negative bacterium disclosed herein. In a preferred embodiment, the method for delivering RNA molecules to eukaryotic cells comprises in vitro It is the Bo method. [Example]

[0122] The invention will now be further described with reference to the following non-limiting examples.

[0123] Example 1: Construction and characterization of T7 RNAP expression in Salmonella A plasmid containing the T7 RNA polymerase (T7 RNAP) gene was inserted under an SPI-2 inducible promoter (i.e., a promoter that is activated inside the Salmonella-containing vacuole, the SCV, during mid-late invasion). downstream of the chloramphenicol resistance pSC101 plasmid (~5 copies / cell) , a DNA fragment synthesized by Integrated DNA Technologies (IDT) containing appropriate overhangs. The assembled plasmids contained either the pipB, ssaG, sseJ, or ssrA promoter.

[0124] In parallel, a carbenicillin-resistant p15A plasmid containing mScarlet downstream of T7p (a T7-responsive promoter) was assembled by inverse PCR and ligation of pSEVA-16 (from the Standard European Vector Architecture collection) using appropriate phosphorylated primers.

[0125] After validation, the plasmid was electroporated into Salmonella enterica Typhi ZH9. Co-transformed clones were recovered under both antibiotics. Single clones were grown in vLB liquid medium (Formedium) supplemented with 25 μg mL of each antibiotic at 37°C and 200 rpm for 16–18 h. They were then diluted 1:10 in 100 μL PCN medium in a 96-well plate and grown in a Clariostar (BMG Labtech) for 6–8 h at 37°C, recording growth and mScarlet fluorescence every 15 min. Toxicity and lack of growth (measured as OD600) were recorded. 50 μL of the final culture was then diluted to 100 μL of PCN medium containing the appropriate antibiotic. The cells were plated onto fresh vLBA medium (Formedium) containing 1000kJ / ml of lysate and cultured at 37°C for 16-18 hours. Further toxicity verification was performed (see Figure 1).

[0126] Example 2: Construction of split RNAP circuit and expression plasmid The split RNA polymerase (RNAP) entry pSC101-ori plasmid contains a weak promoter. Each gene was engineered to contain a core RNAP sequence downstream of the promoter (proA) and either a strong (H) or weak (L) ribosome binding site (RBS). The following double terminators and two placeholder sequences were placed upstream of proA: (1) a promoter cloning vector with two unique BsaI restriction sequences to allow for the cloning of any promoter using Golden Gate assembly. site; and (2) a sigma factor cloning site with a superfolder gfp sequence between two unique BbsI restriction sequences that allows for the cloning of any sigma factor with Golden Gate assembly (see Figure 2).

[0127] A p15A promoter containing a phage-dependent promoter (e.g., T7, T3, K1F, or CGG) is inserted onto the plasmid upstream of a placeholder mScarlet sequence surrounded by BbsI-sites. We constructed a transfection expression plasmid, which can be replaced with any cargo and transfected using Golden Gate Assembly.

[0128] Example 3: Verification of the split RNAP circuit Plasmids containing the sigma T7, T3, K1F, or CGG sigma factors and the split RNAP circuit were generated as described above. An initial validation test was performed by cloning the promoter zinT (responsive to Zinc) upstream of the sigma factor region.

[0129] E. coli DH5α cells were cultured with expression plasmids and combinatorial variations of sigma factors. All of the transformants (see Example 2) were co-transformed and cultured overnight in vLB (Formedium) medium. was determined and observed only in samples containing correctly paired promoter-sigma systems (see Figure 3). (see).

[0130] The plasmid with the CGG sigma factor was the most fluorescent sample, but the strong T7 sigma factor Those expressing the ma factor were toxic to ZH9 (see Figure 3).

[0131] To verify these results, the cytoplasmic promoter puhpT was expressed in BbsI-supplemented golden gel. The vectors were cloned upstream of each sigma factor element in a ribosomal reaction (using NEBridge as the buffer and standard reaction conditions). Single colonies were selected and grown in 100 μL of vLB medium supplemented with 0.4% glucose-6-phosphate (Sigma) at 37°C and 700 rpm for 48 hours, while monitoring growth and mScarlet fluorescence. Results (Figure 3) in liquid medium (vLB) after induction with glucose-6-phosphate and incubation at 37°C for 48 hours were comparable to those previously obtained in solid medium. Similar results are shown.

[0132] Example 4: Construction of promoter reporter plasmids A promoter reporter plasmid vector was constructed containing constitutively expressed mScarlet under the control of the proB promoter and an inducible sfgfp gene encoded by a divergent promoter. Any promoter can be inserted upstream of sfgfp by Golden Gate assembly. Placeholder containing two unique BsaI restriction sites for one-pot delivery Furthermore, once the promoter is characterized, sfgfp can be replaced with any gene of interest by BbsI-dependent Golden Gate assembly.

[0133] Example 5: In vitro characterization of cytoplasmic plasmids ZH9 was expressed in the cytoplasm or cytoplasm of mammalian cells using a promoter reporter plasmid constructed by BsaI-dependent Golden Gate assembly (standard conditions using NEBridge as a buffer). We introduced a series of promoters that are predicted to be active when present in the vacuole or in the nucleus (see Fig. 4). Most of these promoters are metal-dependent, and they are activated by various concentrations of Fe.3+ (cytoplasm), Zn 2+ (vacuole), Mg 2+ (vacuole), or responds to sugars (glucose-6-phosphate, cytoplasm) The resulting plasmid was transformed into ZH9, and the resulting strain was cultured in vLBA at 37°C for 16 to 18 hours. A single colony was picked and cultured in vLB at 37°C for 16–18 hours, then diluted 1:100 in 100 μL of M9 minimal medium and the corresponding inducible metabolites (uhpT (Glucose-6-Phosphate), zinT (Zn 2+ ), mtgC (Mg 2+ ), soxS (unknown), and sfbA (unknown) were added or not. Samples were grown in Clariostar at 37°C for 16-18 hours, and growth (OD600) and sfGFP fluorescence were recorded. .

[0134] All metal-dependent promoters showed inhibition of sfGFP upon addition of the corresponding metabolite (see Figure 4). In unknown cases, no significant variation was observed.

[0135] Example 6: Verification of repression of cytoplasmic promoters in vacuoles Salmonella ZH9 transformed with the promoter reporter plasmid (see above) was SKOV-3 cells were prepared for invasion assay using standard protocols.

[0136] Briefly, a single colony was grown in vLB at 37°C for 16-18 hours, then diluted in fresh vLB and grown at 37°C for approximately 5 hours to an OD of 1.5. Then, gently centrifuge at 2000 x g for 15 minutes. The cells were pelleted by centrifugation at RT and washed twice with ice-cold PBS. After the final wash, the cells were resuspended in 10% glycerol in PBS at 1:10 of the original culture volume, aliquoted into 200 μL incubation stocks, and stored at -80°C until needed. One aliquot was thawed and the stock was serially diluted with PBS. The cells were diluted to 100 ml, plated on vLB plates, and incubated at 37°C for 16-18 hours. The resulting cultures were used to calculate the cell concentration as CFU. CFU was calculated by counting the number of surviving colonies in the dilution series. and is usually determined by 10% of the stock. 10 ~10 12 CFU / mL.

[0137] On the day of the assay, circulating SKOV-3 cells were infected with 50 bacteria per mammalian cell. The invader strain was thawed in advance to a multiplicity of infection (MOI) adjusted to After incubation of both cells for 1 hour, non-invaded bacteria were washed off and The cells were killed with mycin. Samples were visualized after 24 hours and showed bacteria in the vacuole in red, except in the case of the yjjZ promoter (i.e., constitutive mScarlet expression only) (see Figure 5).

[0138] Example 7: Verification of cytoplasmic expression of the cytoplasmic promoter puhpT A promoter reporter plasmid containing puhpT (see above) was transfected into Salmonella enterica Typhimurium CD12 strain (Salmonella Typhimurium TML aroCΔΔ sifA). This strain is defective in the SifA SPI-2 effector, which may result in vacuolar disruption during the late stages of invasion, resulting in escape. Bacterial invasion strains were prepared as previously described and co-cultured with SKOV-3 cells at an MOI of 50. Bacteria were observed under a microscope 3, 6, and 24 hours after invasion. At 6 hours, bacteria were They showed signs of overgrowth (growth outpacing host cells) and green fluorescence, confirming signs of escape (see Figure 6).

[0139] An array that forms part of the description SEQ ID NO: 1-T7 core (DNA sequence) ATGAACACGATTAACATCGCTAAGAACGACTTCTCTGACATCGAACTGGCTGCTATCCCGTTCAACACTCTGGCTGACCATTACGGTGAGCGTTTAGCTCGCGAACAGTTGGCCCTTGAGCATGAGTCTTACGAGATGGGTGAAGCACGCTTCCGCAAGATGTTTGAGCGTCAACTTAAAGCTGGTGAGGTTGCGGATAACGCTGCCGCCAAGCCTCTCATCACTACCCTACTCCCTAAGATGATTGCACGCATCAACGACTGGTTTGAGGAAGTGAAAGCTAAGCGCGGCAAGCGCCCGACAGCCTTCCAGTTCCTGCAAGAAATCAAGCCGGAAGCCGTAGCGTACATCACCATTAAGACCACTCTGGCTTGCCTAACCAGTGCTGACAATACAACCGTTCAGGCTGTAGCAAGCGCAATCGGTCGGGCCATTGAGGACGAGGCTCGCTTCGGTCGTATCCGTGACCTTGAAGCTAAGCACTTCAAGAAAAACGTTGAGGAACAACTCAACAAGCGCGTAGGGCACGTCTACAAGAAAGCATTTATGCAAGTTGTCGAGGCTGACATGCTCTCTAAGGGTCTACTCGGTGGCGAGGCGTGGTCCTCGTGGCATAAGGAAGATTCTATTCATGTAGGAGTACGCTGCATCGAGATGCTCATTGAGTCAACCGGAATGGTTAGCTTACACCGCCAAAATGCTGGCGTAGTAGGTCAAGACTCTGAGACTATCGAACTCGCACCTGAATACGCTGAGGCTATCGCAACCCGTGCAGGTGCGCTGGCTGGCATCTCTCCGAT SEQ ID NO:2-T7 core (amino acid sequence) MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEAR FGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTH SKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGK PIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETTVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDEN SEQ ID NO:3-T7 Sigma (DNA sequence) AAGAACACTGGTGAAATCCTGAGAAAGTCAAGCTGGGCACTAAGGCACTGGCTGGTCCAATGGCTGGCTTACGGTGTTACTCGCAGTGTGCTAAGAGTTCAGTCATGACGCTGGCTTACGGGTCCAAAGAGTTCGGCTTCCGTCAACAAGTGCTGGAAGATACCATTCAGCCAGCTATTGATTCCGGCAAGGGTCTGATGTTCACTCAGCCG AATCAGGCTGCTGGATACATGGCTAAGCTGATTTGGGAATCTGTGAGCGTGACGGTGGTAGCTGCGGTTGAAGCAATGAACTGGCTTAAGTCTGCTGCTAAGCTGCTGGCTGCTGAGGTCAAAGATAAAAGACTGGAGAGATTCTTCGCAAGCGTTGCGCTGTGCATTGGGTAACTCCTGATGGTTTCCCTGTGTGGCAGGAATACAAGAAGC CTATTCAGACGCGCTTGAACCTGATGTTCCTCGGTCAGTTCCGCTTACAGCCTACCATTAACACCAACAAAGATAGCGAGATTGATGCACAAACAGGAGTCTGGTATCGCTCCTAACTTTGTACACAGCCAAGACGGTAGCCACCTTCGTAAGACTGTAGTGTGGGCACACGAGAAGTACGGAATCGAATCTTTTGCACTGATTCACGACT CCTTCGGTACGATTCCGGCTGACGCTGCGAACCTGTTCAAAGCAGTCGCGAAACTATGGTTGACACATATGAGTCTTGTGATGTACTGGCTGATTTCTACGACCAGTTCGCTGACCAGTTGCACGAGTTCCAATTGGACAAAATGCCAGCACTTCCGGCTAAAGGTAACTTGAACCTCCGTGACATCTTAGAGTCGGACTTCGCGTTCGCGTA SEQ ID NO:4-T7 シグマ(amino acid sequence) KNTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAAKLLAAEVKDKKTGEIRKRCAVHWVTPDGFPVWQEYKK PIQKRLDMIFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA SEQ ID NO:5-T3 シグマ(DNA sequence) AAGAACACTGGTGAAATCCTGAGAAAGTCAAGCTGGGCACTAAGGCACTGGCTGGTCCAATGGCTGGCTTACGGTGTTACTCGCAGTGTAGCTAAGCGTTCAGTCATGACGCTGGCTTACGGGTCCAAAGAGTTCGGCTTCCGTCAACAAGTGCTGGAAGATACCATTCAGCCAGCTATTGATTCCGGCAAGGGTCTGAT GTTCACTCAGCCGAATCAGGCTGCTGGATACATGGCTAAGCTGATTTGGGAATCTGTGAGCGTGACGGTGGTAGCTGCGGTTGAAGCAATGAACTGGCTTAAGTCTGCTGCTAAGCTGCTGGCTGAGGTCAAAGATAAAAAAGACTGGAGAGATTCTTCGCAAGCGTTGCGCTGTGCATTGGGTAACTCCTGATGGTT TCCCTGTGTGGCAGGAATACAAGAAGCCTATTCAGAAGCGCCTGGACATGATTTTCTTGGGTCAATTTCGCTTGCAACCTACCATTAACACCAACAAAGATAGCGAGATTGATGCACACAAACAGGAGTCTGGTATCGCTCCTAACTTTGTACACAGCCAAGACGGTAGCCACCTTCGTAAGACTGTAGTGTGGGCACAC GAGAAGTACGGAATCGAATCTTTTGCACTGATTCACGACTCCTTCGGTACGATTCCGGCTGACGCTGCGAACCTGTTCAAAGCAGTCGCGAAACTATGGTTGACACATATGAGTCTTGTGATGTACTGGCTGATTTCTACGACCAGTTCGCTGACCAGTTGCACGAGTTCCAATTGGACAAAATGCCAGCACTTCCGGC TAAAGGTAACTTGAACCTCCGTGACATCTTAGAGTCGGACTTCGCGTTCGCGTA

[0140] SEQ ID NO:6-T3 シグマ(amino acid sequence) KNTGEISKVKLGTKALAGQWLYGVTRSVTKRRSVMTLAYGSKEFGFRQVLEDTIQPAIDSGKGLMFTQPNQAAAGIMAKLIWESVSVTVVAAVEAMNWLKSAAAKLAEVKKTGKTGEILKRCAVHWVTPDGFPVWQEYKKPIQKRLDMIFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLKTVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNRDILESDFAFA SEQ ID NO:7-K1F シグマ(DNA sequence) AAGAACACTGGTGAAATCCTGAGAAAGTCAAGCTGGGCACTAAGGCACTGGCTGGTCCAATGGCTGGCTTACGGTGTTACTCGCAGTGTAGCTAAGCGTTCAGTCATGACGCTGGCTTACGGGTCCAAAGAGTTCGGCTTCCGTCAACAAGTGCTGGAAGATACCATTCAGCCAGCTATTGATTCCGGCAAGGGTCTGATGTTCACTCAGCCG AATCAGGCTGCTGGATACATGGCTAAGCTGATTTGGGAATCTGTGAGCGTGACGGTGGTAGCTGCGGTTGAAGCAATGAACTGGCTTAAGTCTGCTGCTAAGCTGCTGGCTGCTGAGGTCAAAGATAAAAGACTGGAGAGATTCTTCGCAAGCGTTGCGCTGTGCATTGGGTAACTCCTGATGGTTTCCCTGTGTGGCAGGAATACAAGAAGC CTATTCAGACGCGCTTGAACCTGAGGTTCCTCGGTTCGTTCAACCTCCAGCGACCGTCAACACCAACAAAGATAGCGAGATTGATGCACAAACAGGAGTCTGGTATCGCTCCTAACTTTGTACACAGCCAAGACGGTAGCCACCTTCGTAAGACTGTAGTGTGGGCACACGAGAAGTACGGAATCGAATCTTTTGCACTGATTCACGACT CCTTCGGTACGATTCCGGCTGACGCTGCGAACCTGTTCAAAGCAGTCGCGAAACTATGGTTGACACATATGAGTCTTGTGATGTACTGGCTGATTTCTACGACCAGTTCGCTGACCAGTTGCACGAGTTCCAATTGGACAAAATGCCAGCACTTCCGGCTAAAGGTAACTTGAACCTCCGTGACATCTTAGAGTCGGACTTCGCGTTCGCGTA SEQ ID NO:8-K1F シグマ(amino acid sequence) KNTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAAKLLAAEVKDKKTGEIRKRCAVHWVTPDGFPVWQEYKK PIQTRNLRFLGSFNLQPTVNTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA SEQ ID NO:9-CGGシグマ(DNA sequence) AAGAACACTGGTGAAATCCTGAGAAAGTCAAGCTGGGCACTAAGGCACTGGCTGGTCCAATGGCTGGCTTACGGTGTTACTCGCAGTGTAGCTAAGCGTTCAGTCATGACGCTGGCTTACGGGTCCAAAGAGTTCGGCTTCCGTCAACAAGTGCTGGAAGATACCATTCAGCCAGCTATTGATTCCGGCAAGGGTCTGATGTTCACTCAGCCG AATCAGGCTGCTGGATACATGGCTAAGCTGATTTGGGAATCTGTGAGCGTGACGGTGGTAGCTGCGGTTGAAGCAATGAACTGGCTTAAGTCTGCTGCTAAGCTGCTGGCTGCTGAGGTCAAAGATAAGAAAACTGGAGAGATTCTTCGCAAGCGTTGCGCTGTGCATTGGGTAACTCCTGATGGTTTCCCTGTGTGGCAGGAATACAAGAAGC CTATTAAAACGCGCGTGCATATTATGTTCCTCGGTCAGTTCGAAATGCAGCCTACCATTAACACCAACAAAGATAGCGAGATTGATGCACGCAAACAGGAGTCTGGTATCGCTCCTAACTTTGTACACAGCCAAGACGGTAGCCACCTTCGTAAGACTGTAGTGTGGGCACACGAGAAGTACGGAATCGAATCTTTGCACTGATTCACGACT CCTTCGGTACGATTCCGGCTGACGCTGCGAACCTGTTCAAAGCAGTCGCGAAACTATGGTTGACACATATGAGTCTTGTGATGTACTGGCTGATTTCTACGACCAGTTCGCTGACCAGTTGCACGAGTTCCAATTGGACAAAATGCCAGCACTTCCGGCTAAAGGTAACTTGAACCTCCGTGACATCTTAGAGTCGGACTTCGCGTTCGCGTA SEQ ID NO:10-CGGシグマ(amino acid sequence) KNTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKK PIKTRVHIMFLGQFEMQPTINTNKDSEIDARKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA

[0141] SEQ ID NO: 11-T7p (DNA sequence) TAATACGACTCACTATAGG SEQ ID NO: 12-T3p (DNA sequence) TAATAACCCTCACTATAGG SEQ ID NO: 13-K1Fp (DNA sequence) TAATAACTATCACTATAGG SEQ ID NO:14-CGGp (DNA sequence) TAATACCGGTCACTATAGG SEQ ID NO: 15-SYNZIP 18 (DNA sequence) GGAGGTTCAGGTGGTGGATCCAACGAAAAAGAACTGAAATCCAAAAAAGCGGAACTGCGCAACCGTATCGAACAGCTGAACAGAAACGTGAACAACTGAAGCAGAAAAATCGCGAACCTGCGCGTAAAGAAATCGAAGCTTACAAATAAT

[0142] SEQ ID NO:16-SYNZIP18 (amino acid sequence, C-terminus) GGSGGSNEKEELKSKKAELRNRIEQLKQREQLKQKIANLRKEIEAYK SEQ ID NO: 17-SYNZIP 17 (DNA sequence) ATGAGCATCGCGGCGACCCTGGAGAACGATCTGGCGCGTCTGGAAAACGAAAACGCTCGTCTCGAAAAAGACATCGCGAACCTGGAACGTGACCTGGCGAAACTGGAGCGTGAAGAAGCGTACTTC SEQ ID NO:18-SYNZIP 17 (amino acid sequence, N-terminus) MSIAATLENDLARLENENARLEKDIANLERDLAKLEREEAYF SEQ ID NO: 19-proA (DNA sequence) TTTACGGGCATGCATAAGGCTCGtAGGCATTC SEQ ID NO:20-Strong RBS (H) (DNA sequence) TACTAGAGTCATTTATGAAAGTACTAG

[0143] SEQ ID NO:21 - Weak RBS (L) (DNA sequence) TACTAGAGTCAGCCAAGAAAGTACTAG SEQ ID NO:22-PzinT (DNA sequence) AAGCGAGTAGTCACAAAAATTATGCCGCCTGTGCGCGGATATCTGCAAAGCCTGTGCCGAAGAGTGTGCAAGGCACGATCACGACCATTGCCAGAATTGCGCGCGGGCATGCAGCCAATGCGCAGACGCCTGCCTTAAAATGGCCGCGTAATTTTTCTTCCGCCATTAGCTCAACCGGATAGAGCATAGAGCTTCTACCT CTAAGGTTCGGGGTTCAATTCCTCGATGGCGGACCAGTTGATATCAAAAAAGGCCACCTGCGCGGTGGCCGCTGAGTTTCTGTTGAAATAAATGCAATGTTATAATAACAATCATCTTTTAAGAAAGATGAGGGTAACGTTTTGGTGATTCATTTAAAAAAACTGACAATGCTTCTGGGAATGCTGTTGGTAAATAGT SEQ ID NO:23-PuhpT (DNA sequence) GCACTGGACCGGTTTTTGCGGTCATCGCCATCGCGGCGGCGGATCTGCGCTGCTGGTTGCCATTTCTGAACGCTCAGGCCACGCGAAACCCACGAAGCGTGATACCTCACCTGTTGCGCTGAATGGCAAAACTAAGAAATTTTCGGTTGCCTGACGCTGTCGCAGGCCACTACTTCGTGATTTACAATGCTGCCATTCGATTCGATTCGCATTCGCAGCAGGTATAAAAATTAGCTCAGGAGTAATCC SEQ ID NO:24 - PfhuA (DNA sequence) TACGCTGTGCCAGCAGGGCGAGATGATGCAGCAGCAACAGCAGCCGTCAGGCAATCCGTTCGATCAGTCGTCTCAGCCGCAGCAGCCTGCGCAGCAACAGCCGCCGAAAGAAGAGAAGAGCGACGGCGTTGCCGGCTGGATTAAGGAGATGTTTGGCGGCAATTAATCACGGTAATAGTGCCGGGTGGCGCTGTGCTTACTCGGTCCACACCGTTACGACCCCATTATGTGCGACGTAGGCCGAATAAGACGCTTACATCGCCATCCGGCAAATCCTCCATAAATAACATTTCAGTCTAATTTATTAACCCTTCCTTTTCATCTGGTTGTTTCTTAACCCCTTAGTTTTCGTAGGGCCGCGTATCGCTTGCCATTGCGACGATATTTCGCCTATCATGCTGCGGTTATAATAATAATTATCGTTTACGTTATCATTCACTTTCATCAGAGATATACCAATGGCGCGTCTTAAAATTGCTCAGCCAAACTCCTCACTGCGTAAAATCGCAGTTGTAGTAGCCAC SEQ ID NO:25 - PentC (DNA sequence) TTTGCCGGGGCCAACCGGCGTCCTGGGCTAAGGATATTCCTGAAATTGATAAACCAACCACTAAAAAGCAGCCAACGATAAAAGGCGGAGGTCTCACAATAGCGTCCTGTTATTAATAAAGTTAATGCTTCTCATTTTCATGTCAGCGGCAGCGAGATGCAAGCCTTAGTGCCATTTAACTCATGACCAGAGTTGACAGAGCGACGTTTTACTCTTAGGTTAGCGCA CTAAAAATAGAAATAATAATCATTATTATACACAAAATCATTCAAGAAGCATCGCGACGGCAAGGGAAGAATCCCCGCGGGACATAGATAACTGTGTGACCGGGGTTTCTGATCGCAGCCAACAAGAGGCAGCTTGAAAGATGAAGTGTATATAAGCCTTTATCATTGGAGGATGATATGTGCACTGGCCGAGGACGCTCAGGAGACAATGGCAACGCTTGCT

[0144] SEQ ID NO:26-PfhuE (DNA sequence) CATTGACGGTTGGGATCAGGATATTGGGAATAATCAAGATATGCGTCGGCGCCTGCGGTGAAATATCACGAAAGGCGGTGACCAGCTCATCCTGATAAACGATATCCGAAGGGATTTCACGACGAATAATTTTGCTGAATATAGTTTCTTCTGCCACGACGTTTTCCTTTTTCATAATAGCCCTTGCAGCGCACATGCTACGCCGAGCCATACTGCGAGTATAGAGTATGAGCGAGTTA CGACGCTCTTTCAACTTTAACCCACGATTTATTAAGCGAAAAAATGACTACATGCTGGACTTACCGCCATATCCCCCTGCCATGACGCCTTTGTACGCTTAAAAAATTTCTAGTTTCCTGGTTATACCGTTTTAACACATTTAATACAAATGCGTATATTTCTCATTTGCATTTTTATACGCATTAACTAGCAAAGAATGAAAGGTTCAACGTCATACGTCCTGAACTTACCCCAATAACAAGCAAGGATTTCAGATGTCTTTCATTCAATA SEQ ID NO:27-PsfbA (DNA sequence) GGATGTTGGCGTTAAGACGTCTTATCCGGCATTTCCTACGCCATTCCCGCCTGACACGCCTGAGCCTGCTCGTACATTTAACGACACAGGAACATCATGAAATAATTTCAAGGACAGCCTGTGATCTGTGTCATGTAGAAATAGCCTCCTGTTGGTGGCCAAAACAGACGATGCCGCATGAACGGCAGCATCACTTAAAATGGAGAAATTATGGGGATTCGTCAGAGTTACGCATT SEQ ID NO:28-PyjjZ (DNA sequence) GCCGACACGGCGTTGAGAAACAAGAAAGACGTAAAGAAAACTGATACTTCTTAATACGAAGCGACCGCCAGGATGGGGTTGTCATGGGTATTGTCGTTATTTATCGGTGATATACACGGAATCGGGCGCCAACATGAAAATAACGTATGAGAAAAGGTCGCCTAAAGCGAGGTGTTGTT GTTTTTACGTTAACAGTCGGACATTTATCACCTTACTGAATACGTGTCATCAACCGTTAAGTAAAACTCATCTCTTTAGCTTTCTCCCTGGCTGACAAATGAGAAAATATCATATGATATTGGTTATCATTATCAATTCCAGAGGTGAAACCATGTTGCAGCGGACGTTAGGCAGCGG SEQ ID NO:29 - Pstnc3080 (DNA sequence) CGCCGCCATCACCGGTAATTGACGAAACCCTTGCGCCGGCAGCGTATCCGCTGCATCCGGCACAGATCGACTTCACCATCTCAAATTCAAATCCACGGCTCATCGCCGCCGCTGTGTGGCGTGGCACTGAACAGTTGCGAGTGTAAATAGTAATGCTGCATGATTCGTTCCATTAAAAATGAGAAAA SEQ ID NO:30 - PsitA (DNA sequence) CGGCTGATAGCAGTGCACCGGGCACTGCTTACAGGCGGGTTTTTCCTCGCCAAATACGCATTTATCAAGACGTTTTTGCGCGTAGGCGAACAGCGCGTCGTAATGCCCCTGCACCGCTGACGCCTGTGGACACTGGCTTTCATACAGCGCGATCATTTTTTTGATCGTCAGTTTTTCACGAGCGATACGTTTACCAGGCATCGTGCTCTCTCCGAACATTAAGATGCATTTATTTTACACCTTATCCCTCTTTAGCACTATCACTGCATATCGTCGCCATTACGCAAATAAGAATTATTTTCATTTATTCATGCCTTGTGCTATATAACATAGCAAAGGCTATATTCGATGATTAATTAACCACATTGTTGCGAGGGATACTATGACGAATCTACATCGTCTAAAAACACTCCTGATTGCCGGTATTGTCGCGATACT

[0145] SEQ ID NO:31 - PmntH (DNA sequence) TTTGCTCCAAATATGAGGCAGGTTTAATTTTCGTGCACATTCTATGCAACAGCTGTAAAGAAAACGAGATCCAACACACACTATAATAAGGACCTGTGACGAGATTCAAAATTAGTGATCTGTAATACACTTTTACTGTACTGAATATGAAATGAAAAGTTATATCAGTGTGCTAATCTTGT AATGTTAAGCCAAACTGTTCTGATACAGGTCGCCATCGTATCGGTCTATCGTTTCACACTATCAAAGTAATCACCCGTACCCATTGAATGCACTTGATAATCATTATCAATGAACATAGCATGAAACATAGCAAAGGCTATGTTTTTGAGGCAAAAGATGACTGACAATCGCGTAGAGAATAG SEQ ID NO:32-PfepA (DNA sequence) AATAAAACAGTAGCTGCCGGCCAGTTAGCGCTAAGCGCCGTGCTCCAAGCGCAGACATCCGTTCCGGCAATACGCGCCATCGTCTGAGGCTGCGCGTTTTGATGATGATCGGTTACGCCAGTGATGTAGACCCAGACACGCCGTATCGGGGAGTGTGTTTCGTTTCCCTGCGGGTCGGCCCACCAAAAGTGACCCGATAATTTCCGTCTTTTTCCCGTATCCATTCCGGCCGGTTTTCGTCCT CCACCAGGCCTCACTTCCCGTTGCCAGCGCCTCTTTCATTATAACCCTGTGTTTTATTAGATTTTGTATATAAAAGGTGAAATATATTGATAATATTATTGATAACTATTTGCATTTGCAATAGCGTATTGTAGCGCTATGGGACGCGCGAACACAATTTCACCACCCGGCCAATGCCTTTGACGGGCGCTTTGGCTTATGTGGCTAAAAAAAGCAGGATATACAATGAACAAGAAGATTCATTC SEQ ID NO:33-PsufA (DNA sequence) CTCATTCAGCACCTGAAATGCCGGAAAGAGTTTATAACTAACCTATAGCTCAAACTGAGTTATAGAACCGCAGCGGATTATAAAGAGCGCAACGCCAGGTATCCATACAAAAATGGGGTTCTGACCTCGCCCGCCGCAATGTCGACAGCCTATTAATTAAATAGTCATTTTTCTATACATCTTTTCGTTTTTGACCTGCCAGAACGGTTAATGTCTTATAAATC ATTACTTATCAAAAAGTTAAGTGGTTTTTGTCTGTCGTATGACCTGGCGGACAGGGTCTATGCTTAATAAAAGGCGCTCAATATGACCATTTGTTGGAAAGCCCCTGCGGTTAAGGGGTTGAAGTGATAATCATTATCACTAACATGCTGTTATATCCTGGTGATTTAGAACGCGAGGTAACTCTATGGAATTGCATTCAGGCACGTTTAACCCGGAGGAACTTC SEQ ID NO:34-PsoxS (DNA sequence) TTTCGCAGCGGACAGTCGCTACGCGATAAACAGCCGCAGCCGATACAACCGTCCAGCTCATCGCGCAACGCCACCAGCGTATGAATACGTCGGTCTAACTCTTCGCGGCCACTGCGAGGAGAGCTGCTTCCACTCTTTCGGCCTTAACGTATGCCCTTCCG GCAAGATACCAAACGCGTCGCCGATAGTTGCCAGCGGGATGCCGATACGCTGGGCAATCTTGATAATCGCGACATAACGCAAACACGTCACGCTTGTATCGCCGTTGGTTACCGCTATTACGGATACTGGTAATTAGCCCTTTGCTTTCATAGAAGTGCAGGGCGGACACAGCAACACCG CTACGTTTCGCAACTTCCCCCGGCGTCAGTAAGGCTTTTAAACGGGGAGATTTTTTTTCCATAAATCGCTTTACCTCCAAGTTAACTTGAGGAATTATACTCGCCCGCAGACAAAACGACGAATCGAATACTGTTTAAGAGGCAACAATATGTCGCATCAGCAGATAATTCAGACCCTTAT SEQ ID NO:35-PiroN (DNA sequence) AAGCGCCTGATAAATATTACCAGGCGCTTTGTATGTTGGTGCCAACATCACTTTCATCATCAAATATCGAATGGCTACAATCGTATCCGATCCCGCCATACCAGACGGAAAGTCGCTGGCAAACTGTAAGAATGGTTCGCCGTCGGCAGGGAAGCGGCGTGAACCCTGAACCGTGCTATACCATCTTACCTGGGTGTTTCTTGATTAACGATCTGAAAAATAGTTTTATTTTATCTATTCTTGTTTGTAAAACCTCCGTT CAGTAGGCGCATTCTGCCCCCCTTCCCGGATTTACTGGCAAAGCGGAGCCCGGACAGAGAGTCATATTGCAAAATCCCGTTTCCGTTTTTTTATTACCAGATTTTGTGGTCGAAAGATTGCCTTTTCCTTAATTGAATGATAATTATTATCATTAGCATATGATAATAATTACTATATAGACGTAACCTGGCAAGGATGTGAGCTTGAGGGCAACAGCGCTACTTTTAGATATTTAGGGAATGGGTATGAGAGTTAAAAAGTTC

[0146] SEQ ID NO:36-PfepB (DNA sequence) CAAACTGCTGGCGCAATTTCTGCTGGAAAGGGCTATCCGGGCGAATCTCCGGCAACCGCAGGCTCGGTATAACGGGCGAAGCACCCGGAAGTGGTAAAACTGCGATACGGCGACATGAAAAAAAGCGATCGGGAGCAAGCGTTGCCATTGTCTCCTGAGGCTTCCTCGGCCAGTGACATATCCATATCATCCTCCAATGATAAAGGCTTATATACACTTCATCTTTCAAGCTGCCTCTTTGTTGGCTGCG ATCAGAAACCCCGGTCACACAGTTATCTATGCCCGCGGGGATTCTTCCCTTGCCGTCGCGATGCTTCTTGAATGATTTTGTGTATAATAATGATTATTATTCTATTTTTAGTGCGCTAACCTAAGAGTAAAACGTCGCTCTGTCAACTCTGGTCATGAGTTAAATGGCACTAAGGCTTGCATCTCGCTGCCGCTGACATGAAAATGAGAAGCATTAACTTTATTAATAACAGGACGCTATTGTGAGACT SEQ ID NO:37-LysE (amino acid sequence) MVRWTLWDTLAFLLLLSLLLPSLLIMFIPSTFKRVPSSWKALNLRKTLLMASSVRLKPLNCSRLPVCYAQETLTFLLTQKKTCVKNYVRKE SEQ ID NO:38-LysE (DNA sequence) ATGGTACGCTGGACTTTGTGGGATACCCTGCTCCTGCTCCTGGTTGAGTTATTGCTGCCGTATTGCTTATGTTCATCCCGTCAACATTCAAACGGCCTGTCTCATCATGAAGGCGCTGAATTTAACGGAAAACATTATTAATGGCGTCGAGCGTGGTACGGTAAAGCCGCTGAATTGTTCGCGTTACCTGGTCGTCGCGCAGGAAACACTGACCTGCTTACTGACGCAGAAAACGTGGTCGTCAAAAAAATTACGTGCGGAAGGAGAG SEQ ID NO:39-kilR (amino acid sequence) MIAHFGTDEIPRQCVTPGDYVLHEGRTYIASANNIKRKLYIRNLLTCTFITDRMIKVF SEQ ID NO:40-kilR (DNA sequence) ATGATAGCACATCATTCGGCACCGAATGAATCCCCCGCCAGTGCGTTACCCCTGGTGATTACGTTCTGCATGAAGGTCGACGTATCGCGTCCGCCAACAACATCAAAAAACGTAAACTTTACATCCGTAATCTTACAACAAAGACGTTCATCACTGATCGTGATTGATTAAAGTATTATT

[0147] SEQ ID NO:41-Kil (amino acid sequence) MRKRFFVGIFAINLVGCQANYIRDVQGGTIAPSSSKLTGIAVQ* SEQ ID NO:42-Kil(DNA sequence) ATGCGCAAAAGGTTCTTCGTAGGTATTTTCGCCATCAACCTGTTGGTGGGATGTCAAGCAAATTATATCCGTGACGTACAAGGTGGCACAATCGCGCCATCGAGTTCGAGTAAATTAACCGGAATCGCTGTTCAATAG SEQ ID NO:43-BRP (amino acid sequence) MKATKLVLGAVILGSTLLAGCQANYIRDVQGTVAPSSSELTGIAVN SEQ ID NO:44-BRP(DNA sequence) ATGAAAGCGCCAAAACTGTACTGGCGCGGTAATCGGGTTCTCTGCTGCAGGCTGCCAGGCAACTATCCGATGTTCAGGGTGAACGGTGCAGCATCGCTCGCTCGCTCTGAACTGACGGGATCGCGGTTAAC SEQ ID NO: 45-(stn3250) (DNA sequence) CAGAAGTCATAGGTATTGGAAGCGCCGCGACTGCTTACAGTTACGCCCGGCGTGTAACCCAACGCTTCTTTTACTGACTGGAATTGATGCATCTGCATCTCTTCGTTAGTGACCACCGAACCGACTGTGGCGTTTTTTCGATAGATGTATCAGTTTTGG TGGTAGTGGCGGAACGCTTCGCGGCGATGGTCGGAGCCGGTCCCCAGGCACTTTCCTGCGGCGGCAGGCGCTGCGGTTACGGTAATGGTTTCTTCTTCGGTTGAACCGCCGCCTGTGCATAGACAGACATGCCGCTAACCGCTGTGGCTACTACAACTGCGATTTTACGCAGGAGGATTTGGCTGAGCAATTTTAAGACGCGCCATTGGTATATCTCTGATGAAAGTGAATGATAACGTAAACGATAATTATTATAACCGCAGCATGATAGGCACAGACAACGG

[0148] SEQ ID NO: 46-(mtgC)(DNA sequence) CGTTTAGCATCCCTTTTCTGGTGGAACCCATTTTTTCCTCGTCATGTTGTTTTATTTTTTTACGTGCAGGCATCATAACAGAGCTATCGCCGGCATTAAGCAGGAATTTATTGTTTAATGATTTCAGACGAGCCTGTTATTGACATAATATTGTCATTTTTTTGTCACGGGAAATATCAAACAAACTTAAACAAATCGTCACTATCCCCGCCTTTGCACTTTACAGAACATATTGACTGACTATAATAAGCGCAAATTCATGCAGGAGTAATATGTTGGACAGTCACTTTTACGTAAATCATCTGGCAAGTTAACGCACGCTATTCCTGCGCTGCTTGCCGAACCGGTGGGCAGCAATCTCCCCTTGTGACGATTGTCATCCCAATAATGTTACAACACGCGCATTGTCGCGAGGTAATCGTCATGTTCATGTTTAAACACGCTTTATTTCCTCCGCCGTTAACACGACGCTAATTGCCTCAGGGCAGAAATTTGTCGTGTGCTAAATATAGCACGTACTTATTCTTCCAGAAAAAATGGAGGAACGTATGTTAATGTTTCCTTATATTTTAAATTTACTGGCCGCTATGCT The original text seems to be a DNA sequence in a rather raw form. Let's try to make it more legible by adding some spaces and line breaks for better readability: CGTTTAGCATCCCTTTTCTGGTGGAACCCATTTTTTCCTCGTCATGTTGTTTTATTTTTTTACGTGCAGGCATCATAACAGAGCTATCGCCGGCATTAAGCAGGAATTTATTGTTTAATGATTTCAGACGAGCCTGTTATTGACATAATATTGTCATTTTTTTGTCACGGGAAATATCAAACAAACTTAAACAAATCGTCACTATCCCCGCCTTTGCACTTTACAGAACATATTGACTGACTATAATAAGCGCAAATTCATGCAGGAGTAATATGTTGGACAGTCACTTTTACGTAAATCATCTGGCAAGTTAACGCACGCTATTCCTGCGCTGCTTGCCGAACCGGTGGGCAGCAATCTCCCCTTGTGACGATTGTCATCCCAATAATGTTACAACACGCGCATTGTCGCGAGGTAATCGTCATGTTCATGTTTAAACACGCTTTATTTCCTCCGCCGTTAACACGACGCTAATTGCCTCAGGGCAGAAATTTGTCGTGTGCTAAATATAGCACGTACTTATTCTTCCAGAAAAAATGGAGGAACGTATGTTAATGTTTCCTTATATTTTAAATTTACTGGCCGCTATGCT SEQ ID NO: 47 - (sseJ)(DNA sequence) It should be noted that this is a very long DNA sequence, and its analysis and understanding often require specific biological and genetic context knowledge. If you have more specific requirements or need further processing related to this sequence, it would be beneficial to provide additional information. If you want to keep it in the original format without the added readability enhancements, the translation remains as follows: CGTTTAGCATCCCTTTTCTGGTGGAACCCATTTTTTCCTCGTCATGTTGTTTTATTTTTTTACGTGCAGGCATCATAACAGAGCTATCGCCGGCATTAAGCAGGAATTTATTGTTTAATGATTTCAGACGAGCCTGTTATTGACATAATATTGTCATTTTTTTGTCACGGGAAATATCAAACAAACTTAAACAAATCGTCACTATCCCCGCCTTTGCACTTTACAGAACATATTGACTGACTATAATAAGCGCAAATTCATGCAGGAGTAATATGTTGGACAGTCACTTTTACGTAAATCATCTGGCAAGTTAACGCACGCTATTCCTGCGCTGCTTGCCGAACCGGTGGGCAGCAATCTCCCCTTGTGACGATTGTCATCCCAATAATGTTACAACACGCGCATTGTCGCGAGGTAATCGTCATGTTCATGTTTAAACACGCTTTATTTCCTCCGCCGTTAACACGACGCTAATTGCCTCAGGGCAGAAATTTGTCGTGTGCTAAATATAGCACGTACTTATTCTTCCAGAAAAAATGGAGGAACGTATGTTAATGTTTCCTTATATTTTAAATTTACTGGCCGCTATGCT SEQ ID NO: 47 - (sseJ)(DNA sequence) CACATAAAACACTAGCACTTTAGCAATAATAGTCGGATGATAAGTTTGTCTGTTTTTCCTGAGTATCAAGCCAGCTCATACTCACGCCAGCACACTAAAATCAGGAGTGGCTTCTTTTTAGATCTTTGCCTTAGCCAGGCGCACACTCAATAATGATAGCAGTCAGATAATATGTACCAGGCATTAACCTC ACGTTGTTGATGATATATTTACTTCGTTGAAAAACAATAAACATTGTATGTATTTTATTGGCGACGAAAAACTGTTAAAGAAGCGTAATTCCATATACACCATTTACCTGATTACTTTTCTTGCTAATATTTGCTAATTAATTATTTGCTAAAGCGTGTTTAATAAAGTAAGGAGGAGGCACAGCACAACGG SEQ ID NO: 48-(ssaG) (DNA sequence) TATTGCCATCGCGGATGTCGCCTGGTCTTATCTACCATCATAAACATCATTTGCCTATGGCTCACGACAGTATAGGCAATGCCGTTTTTATATTGCTAATTGTTTGCCAATCAACGCAAAGTATGGCGATTGCTAAAGCCGTCTCCCTGGGCGGTAGATTAGCCTTAACCGCGACGGTAATGACTCATTCATACTGGAGTGGGTAGTTTGGGACTACAGCCTCATTATTAG AGCGTCTTAATGATATTACCTATGGACTAATGAGTTTTACTCGCTTCGGTATGGATGGGATGGCAATGACCGGTATGCAGGTCAGCAGCCCATTATATCGTTTGCTGGCTCAGGTAACGCCAGAAACAACGTGCGCCGGAGTAATCGTTTTCAGGTATATACCGGATGTTCATTGCTTTCTAAATTTTGCTATGTTGCCAGTATCCTTACGATGTATTTTTAAGGAAAAGC SEQ ID NO: 49-(sseA)(DNA sequence) SEQ ID NO: 50 - (sifA) (DNA sequence) ATAAGCGATTAATTGCGCAACGCTAACAAATCCACACGCATCCAGGCATGAAGTTTATTCAAGGGTAAACTTCATGCCTTCGGCATAAAAAACGCATGAAAGAAGTTGCCGCCAGTATTGCAAATCTACAACATCATCCGCGGTAGTCCTTCTTTTATTTTTACCTGTAGCGACGCTATCACAGACAGTAATGCGTTTATACGCGAAGCTCTCAGGTTTTATACTGATTGCCAGTCTCTTTTAAAAATTATATTACATCCGATGCGCCCGCAGTTGAGATAAAAAGGGTCGATTTAATCAATTATGTAGTCATTTTTACTCCAGTATAAGTGAGATTAAG

Claims

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

2. 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. nucleotides, wherein the RNA polymerase binds to the promoter, and the Gram-negative bacterium When the enters a eukaryotic cell, the RNA molecule is transcribed and transported into the cytoplasm of the eukaryotic cell. A live attenuated gram-negative bacterium containing a polynucleotide capable of

3. The RNA polymerase is orthogonal to components required for endogenous gene expression in Gram-negative bacteria. Item 1. A live attenuated Gram-negative bacterium according to Item 1.

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

5. The polynucleotide encoding the RNA polymerase is either a cytoplasmic promoter or a vacuole-dependent promoter. and a cytoplasmic promoter selected from the group consisting of uhpT, mntH, entC, fhuE, iroN, fepB, fepA, fhuA, sitA, stn3250, sufA, yjjZ, soxS, sfbA, or any combination thereof, and a vacuole-dependent promoter selected from the group consisting of uhpT, mntH, entC, fhuE, iroN, fepB, fepA, fhuA, sitA, stn3250, sufA, yjjZ, soxS, sfbA, or any combination thereof.

5. The live attenuated Gram-negative bacterium of claim 1, wherein the motor is selected from the group consisting of zinT, mtgC, ssaG, sseJ, or any combination thereof.

6. The sigma factor of RNA polymerase is operably linked to a heterologous polynucleotide.

10. A live attenuated Gram-negative bacterium according to any one of the preceding claims, which binds to a motor.

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

8. The sigma factor of RNA polymerase is CGG sigma factor RNA polymerase, and heterologous polynucleotide 7. The method of claim 6, wherein the promoter operably linked to the polypeptide is a CGG promoter. Live attenuated gram-negative bacteria.

9. The heterologous polynucleotide encoding the RNA molecule is operably linked to a phage promoter 10. The live attenuated Gram-negative bacterium of any one of the preceding claims, wherein the bacterium is

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

11. 10. Any of the preceding claims, wherein the RNA molecule is an mRNA molecule, an siRNA molecule, or an shRNA molecule.

2. A live attenuated Gram-negative bacterium according to claim 1.

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

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

14. A heterologous polynucleotide encoding an RNA molecule does not modify the genome of a eukaryotic cell.

10. The live attenuated Gram-negative bacterium according to claim 1 .

15. The live attenuated gram-negative bacteria are Salmonella spp. A live attenuated Gram-negative bacterium according to any one of claims 1 to 4.

16. The live attenuated gram-negative bacterium is Salmonella Enterica. Preferably, the live attenuated gram-negative bacteria is Salmonella enterica serovar Typhi and / or Salmonella enterica - Salmonella enterica serovar Typhimurium 10. A live attenuated Gram-negative bacterium according to claim 1, which is used to treat a bacterial infection.

17. 10. The live attenuated Gram-negative bacterium of any one of the preceding claims, wherein the live attenuated Gram-negative bacterium is a genetically modified, non-naturally occurring bacterium.

18. The live attenuated gram-negative bacteria is selected from the group consisting of 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, and preferably is a live attenuated gram-negative bacterium.

10. The live attenuated bacterium according to any one of the preceding claims, wherein the toxified gram-negative bacterium is M01ZH09. Toxigenic gram-negative bacteria.

19. 10. The live attenuated virus of any one of the preceding claims, wherein the RNA molecule is mammalian. gram-negative bacteria.

20. The live attenuated Gram-negative cell of claim 5, wherein the cytoplasmic promoter is derived from SPI-2. Bacteria.

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

22. 10. The live attenuated Gram-negative bacterium of any one of the preceding claims, wherein the live attenuated Gram-negative bacterium further comprises one or more of the following accessory proteins: i) RNA stability enhancement A polynucleotide encoding a strong component, preferably wherein said RNA stability enhancing component comprises an IRES sequence. ii) a polynucleotide encoding a lytic protein, preferably iii) a polynucleotide encoding a phospholipase; iv) a gene encoding an invasion factor, preferably one in which the phospholipase is phospholipase C; and / or v) a bacteriocin-releasing protein, a bacteriocin-releasing polynucleotide; phage λ lysozyme, and holin.

23. 10. The method of claim 9, wherein the live attenuated gram-negative bacteria is administered intratumorally, intravenously, intraperitoneally, or orally, preferably the gram-negative bacteria is administered intratumorally. A live attenuated gram-negative bacterium as described.

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

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

26. 24. A live attenuated gram-negative bacterium for use according to claim 23, wherein the live attenuated gram-negative bacterium is for use in the treatment, alleviation, inhibition, prevention or control of a neoplastic disease, an infectious disease, a cardiovascular disease, a neurodegenerative disease, a gastrointestinal disease, a respiratory disease, a renal disease, a liver disease, an autoimmune disease, an inflammatory disease or a genetic disease, preferably the live attenuated gram-negative bacterium is used for the treatment, alleviation, inhibition, prevention or control of a neoplastic or infectious disease.

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

28. Solid tumors and / or hematological malignancies include prostate cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, brain tumor, mesothelioma, hepatocellular carcinoma, lymphoma, leukemia, gastric cancer, prostate cancer, endometrial cancer, uterine cancer, vulvar / vaginal cancer, cervical cancer, and ovarian cancer.

26. The live attenuated Gram-negative bacterium for use according to claim 25, wherein the cancer is selected from thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer or sarcoma, preferably 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 a live attenuated gram-negative bacterium, the gram-negative bacterium being: 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 peptide; and ii) a polynucleotide encoding an RNA polymerase that is orthogonal to components required for endogenous gene expression in the Gram-negative bacterium. a nucleotide sequence of the gram-negative bacterium, wherein the RNA polymerase binds to the promoter, and A vaccine composition comprising a polynucleotide that, upon entry into a cell, is capable of transcribing said mRNA molecule and transporting it into the cytoplasm of said eukaryotic cell.

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

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

32. A vaccine composition according to claims 29 to 31, which comprises a live attenuated Gram-negative bacterium according to any one of claims 1 to 24.

33. 1. A method of treating, preventing, inhibiting, preventing recurrence, or controlling a disease in a subject, comprising: i) a heterologous polynucleotide encoding an RNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter; and ii) a heterologous polynucleotide encoding an RNA polymerase. a polynucleotide encoding an RNA polymerase that binds to the promoter, the RNA polymerase being encoded by a heterologous split RNA polymerase plasmid; and When the Gram-negative bacterium invades a eukaryotic cell, the RNA molecule is transcribed and expressed in the cytoplasm of the eukaryotic cell. administering to said subject a live attenuated Gram-negative bacterium comprising a polynucleotide capable of being transferred to said subject.

34. 1. A method of treating, preventing, inhibiting, preventing recurrence, or controlling a disease in a subject, 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 the Gram-negative bacterium is a eukaryotic cell. Once inside the cytoplasm, the RNA molecule can be transcribed and transported into the cytoplasm of the eukaryotic cell. administering to a subject a live attenuated Gram-negative bacterium comprising a polynucleotide,

35. 35. A method for treating, preventing, suppressing, preventing recurrence or controlling a disease in a subject according to claim 33 or 34, comprising a live attenuated Gram-negative bacterium for use according to any one of claims 1 to 24.

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

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

38. A method for delivering an RNA molecule to a eukaryotic cell according to claim 36, comprising the step of: A method comprising the live attenuated Gram-negative bacterium for use according to any one of claims 1 to 4.

Images