WO2024079358A1

WO2024079358A1 - Bactofection

Info

Publication number: WO2024079358A1
Application number: PCT/EP2023/078576
Authority: WO
Inventors: Marc Biarnes CARRERA; Georgia PETSIOU
Original assignee: Prokarium Limited
Priority date: 2022-10-13
Filing date: 2023-10-13
Publication date: 2024-04-18
Also published as: GB202215134D0

Abstract

The present invention relates to a modified live attenuated Gram-negative bacteria, wherein said bacteria has been modified in such a way that RNA molecules can be safely and efficiently delivered to target eukaryotic cells. As such, the present invention relates a to a bacterial delivery system and various uses and methods thereof.

Description

BACTOFECTION

FIELD OF INVENTION

The present invention relates to a modified live attenuated Gram-negative bacteria and uses thereof.

BACKGROUND OF INVENTION

Gene therapy has been used to transfer genetic material into cancerous cells to deactivate oncogenes or transiently change their phenotype to reduce or abolish tumor growth [Molecular Cell Therapies (2014) 2:27; Journal of Vascular and Interventional Radiotherapy (2013) 24(8):1115; Journal of Immunotherapy of Cancer (2020) 8:e001], Oncolytic virotherapy (OV), which employs genetically modified viruses to infect and replicate exclusively in cancerous cells, has shown great promise, with 3 OVs being approved globally for treatment in 2020 [Journal of Immunotherapy of Cancer (2020) 8:e001], These therapies have been shown to be safe, with mild adverse effects and minimal shedding.

However, several limitations exist when using these agents [Journal of Gene Medicine (2005) 7:1380]: (1) generation of anti-viral antibodies by the patient that clear the vector before reaching its target; (2) high doses of viral particles can result in cytokine cascade, affecting patient’s health, (3) safety concerns due to unforeseen mutations, and (4) limited tumoral delivery [Oncolytic virotherapy (2017) 6:39], Furthermore, there is a discrepancy between the results obtained in pre-clinical studies and antitumor effects observed in clinical trials [Oncolytic virotherapy (2017) 6:39],

Bactofection is the process of transduction of genetic material from a bacterium (e.g., Salmonella) into a mammalian cell. Early examples of bactofection using Salmonella [Blood (1998) 92:3172] employed the strain SL7207 aroA~), which spontaneously and passively transferred plasmid material into splenocytes - preferentially transducing macrophages (F4/80⁺) with efficiencies of -19%. Byrne et al. 2014 also identified a bactofection efficiency of -20 % [Journal of Controlled Release (2014) 196:384] when using E. coli MG 1655 as a vector. They too identified a preferential bactofection in macrophages. This limited efficiency is attributed not only to the instability of the vector strains, mainly due to the use of plasmid systems, which are inherently variable and cause metabolic burden [Gene Therapy (2005) 12:364] but due to the need for the plasmid to travel into the nuclei, possibly due to unmethylated CpG areas [Molecular Therapy (2009) 17:767], Additionally the transfer of unmethylated-CpG islands may help trigger TLR-9 responses (known to participate in a complex way in cancer regulation). A relevant example demonstrating the use of bactofection in the regulation of the immune system is the publication by Shen et al. [Microbiology and Immunology (2004) 48:329], who used Listeria monocytogenes to “bactofect” mice (BALB/c) to produce IL-10, IL-12, or lFNgamma.

There remains a need for new delivery systems and methods of delivery of heterologous polypeptides to target eukaryotic cells.

SUMMARY OF INVENTION

The inventors of the present invention have surprisingly found that Gram-negative bacteria can be modified in such a way that RNA molecules can be safely, efficiently, and effectively delivered to target eukaryotic cells. Accordingly, the present invention provides a bacterial delivery system with broad usability across numerous disease areas.

In order to achieve successful bactofection with RNA, RNA polymerase is required to synthesise the RNA from a DNA template. However, the inventors of the present invention have identified that typical phage RNA polymerases have significant disadvantages that prevent successful utilization of bactofection. Firstly, the inventors of the present invention have surprisingly found that T7 RNA polymerase does not contribute to the safe, efficient, and effective delivery of RNA molecules to target eukaryotic cells, and can result in unwanted toxicity in certain bacterial strains. In enabling Gram-negative bacteria for bactofection, the inventors of the present invention have realised that modifications are required for improved efficiency, and that conventional strong promoters, such as T7 promoters, used to drive expression of the DNA template, were found to give rise to unwanted toxicity, due to depletion of the host bacterial cell resources. Secondly, the inventors have identified that the presence of a T7 promoter inside the ZH9 Salmonella chromosome may drive the expression of unknown genes, some of which could be toxic, thus leading to specificity problems.

The present inventors have therefore surprisingly found that the choice of promoter is important for successful bactofection and any subsequent downstream application. Specifically, the inventors have identified two solutions to the problem of toxicity and specificity outlined above. The first is to use a RNA polymerase that is encoded by a heterologous split RNA polymerase plasmid such that RNA production can be maximized with minimal toxicity. The second is to use a RNA polymerase and promoter pairing that is orthogonal to any components of the Gram-negative bacterium that is required for endogenous gene expression such that the specificity issues previously described are circumvented.

Accordingly, in a first aspect, the present invention provides a live attenuated Gram-negative bacterium comprising i) a heterologous polynucleotide encoding an RNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter; and ii) a polynucleotide encoding an RNA polymerase, wherein the RNA polymerase binds to the promoter, wherein the RNA polymerase is encoded by a heterologous split RNA polymerase plasmid, and wherein upon invasion of the Gram-negative bacteria to a eukaryotic cell, the RNA molecule is transcribed and is capable of being transferred to the cytoplasm of the eukaryotic cell.

In a second aspect, the invention provides a live attenuated Gram-negative 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 which is orthogonal to components required for endogenous gene expression in the Gram-negative bacterium, wherein the RNA polymerase binds to the promoter, and wherein upon invasion of the Gram-negative bacteria to a eukaryotic cell, the RNA molecule is transcribed and is capable of being transferred to the cytoplasm of the eukaryotic cell.

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

In a fourth aspect, the present invention provides a vaccine composition comprising a live attenuated Gram-negative bacterium, wherein the Gramnegative bacterium comprises i) a heterologous polynucleotide encoding a functional mRNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter and the mRNA molecule encodes a therapeutic protein and/or therapeutic peptide; and ii) a polynucleotide encoding an RNA polymerase, wherein the RNA polymerase binds to the promoter, wherein the RNA polymerase is encoded by a heterologous split RNA polymerase plasmid, and wherein upon invasion of the Gram-negative bacteria to a eukaryotic cell, the mRNA molecule is transcribed and is capable of being transferred to the cytoplasm of the eukaryotic cell.

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

In a sixth aspect, the present invention provides a method of treating, inhibiting, preventing recurrence or controlling a disease in a subject, wherein the method comprises administering to the subject a live attenuated Gram-negative bacterium comprising i) a heterologous polynucleotide encoding an RNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter; and ii) a polynucleotide encoding an RNA polymerase which is orthogonal to components required for endogenous gene expression in the Gram-negative bacterium, wherein the RNA polymerase binds to the promoter, and wherein upon invasion of the Gram-negative bacteria to a eukaryotic cell, the RNA molecule is transcribed and is capable of being transferred to the cytoplasm of the eukaryotic cell.

In a seventh aspect, the present invention provides a method for delivering an RNA molecule into a eukaryotic cell, said method comprising 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; ii) contacting the Gram-negative bacterium with the eukaryotic cell such that the Gram-negative bacterium replicates within said eukaryotic cell, such that the heterologous polynucleotide is transcribed and subsequently transferred from the Gramnegative bacterium to the cytoplasm of the eukaryotic cell.

DESCRIPTION OF FIGURES Figure 1 shows how T7 RNA polymerase (RNAP) can be toxic to Salmonella enterica Typhi. Figure 1 A shows a schematic representation of the experimental setup. Salmonella enterica ZH9 was transformed with two plasmids, one containing the T7 RNA polymerase under control of SPI-2 inducible promoters and the other containing mScarlet (red fluorescent protein) under control of the T7 promoter (T7p). A putative active T7p was found inside the Salmonella genome, downstream of a prophage integrase-like gene and upstream of four uncharacterized open reading frames (ORF), one having homology to phage regulatory proteins. A BLASTp search showed these proteins can be found across different enteric bacteria, such as Salmonella, Escherichia, or Shigella (data not shown). Figure 1 B shows the evaluation of the effect of temperature and promoter strength on T7RNAP-dependent toxicity. It was found that toxicity was more marked under strong promoters, especially at temperatures close to optimal of growth (37°C).

Figure 2 shows a schematic representation of the split RNAP system. In this design, the expression levels of the core RNAP determine the total polymerase activity, and thus toxicity. Careful selection of promoter 1 (e.g., constitutive promoter or SPI-2 -dependent promoter) allows for the generation of a strain with maximum polymerase activity at the lowest possible toxicity levels. A second (or more) promoter controls expression of the “sigma” (or DNA-binding) region of the RNAP. Upon binding of sigma to the core, the polymerase activity is restored and is specific for the sigma’s cognate promoter. This allows for the generation of a platform where different sigma factors control different bactofection circuits (e.g., sigma 1 controls therapeutic RNA production and sigma 2 controls lysin synthesis, thus enabling the release of the therapeutic RNA into the cytosol of the eukaryotic cell).

Figure 3 shows the results from expressing the split RNAP system in Escherichia coli (E.coli) and Salmonella enterica ZH9. On the left, E. coli DH5a cells were transformed with a medium-copy number expression plasmid, containing a polymerase phage promoter (e.g., T7, T3, K1 F, or CGG) upstream of mScarlet reporter gene, and a low-copy number plasmid, containing split RNAP with specific sigma factors (depicted on the left) or superfolder green fluorescent protein (sfGFP) (negative). Cells only expressed mScarlet in correctly paired sigma-promoter strains. On the right, the Salmonella enterica ZH9 was transformed with the above-described expression vectors and either the split RNAP plasmid with the cognate sigma factor or sfGFP, either with a strong (H) or a weak (L) ribosome binding site (RBS) to control the levels of RNAP core (middle) and with PuhpT -controlled sigma factors (right), which was used to assess the system in liquid media.

Figure 4 shows the results of assessing different cytosolic promoters in vitro. Figure 4A shows plasmids generated to express constitutive mScarlet, encoding a red fluorescent protein, and inducible sfGFP, encoding for a green fluorescent protein, controlled by any of the depicted promoters. Figure 4B shows Salmonella enterica ZH9 cells harbouring these plasmids were grown in M9 media and either supplemented with 1 mM Fe³⁺, Mg²⁺ (only PmtgC ), Zn²⁺ (only PzinT) and fluorescence was monitored over time. Cells showed various levels of expression in the absence of an inducer.

Figure 5 shows the results of visualizing invasion assays with Salmonella enterica ZH9, containing reporter plasmids that constitutively express mscarlet and sfGFP under cytosol-inducible promoters in the microscope 24 hours post-invasion. All tested promoters (specified above each micrograph) with the exception of PyjjZ are red, indicating a lack of sfGFP production on SKOV-3 cells (an ovarian cancer cell line).

Figure 6 shows the evaluation of cytosolic promoters in vacuole release. Figure 6A shows the activity of the promoter puhpT, which has been reported to be responsive to cytosolic glucose-6-phosphate. puhpT was evaluated in cellulo by performing an invasion assay of Salmonella Typhimurium CD12 harbouring a reporter plasmid onto SK-OV-3 cells. The reporter plasmid constitutively expresses a mScarlet (red fluorescent protein) and sfGFP (green fluorescent protein) as a response to glucose-6-phosphate. Figure 6B shows representative examples (two each) of cells in vacuole (few, red only) and in the cytosol (hyper- replicative, yellow).

Figure 7 shows that the sigma factor is responsible for spatiotemporal control inside host cells. Sigma factor expression can be selectively controlled using vacuole-inducible (ssaG, sseJ, sseA, or sifA) or cytosol-inducible (fhuA, iroN, mntH, or sitA) promoters. Additionally, the different levels of expression from each of these promoters (shown as fluorescence from a sfGFP downstream of each of the labelled promoters) can offer an additional layer of control over the split RNAP activity.

DETAILED DESCRIPTION

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.

As used herein, the term “attenuated” in the context of the present invention, refers to the alteration of a microorganism to reduce its pathogenicity, rendering it harmless to the host, whilst maintaining its viability. This method is commonly used due to its ability to elicit a highly specific immune response whilst maintaining an acceptable safety profile. Methods of obtaining attenuated microorganisms may include, but are not limited to, passing the pathogens under in vitro conditions until virulence is lost, chemical mutagenesis and genetic engineering techniques. Such an attenuated microorganism is preferably a live attenuated microorganism, although non-live attenuated microorganisms are also disclosed.

As used herein, the term “inactivating mutations” refers to modifications of the natural genetic code of a particular gene or gene promoter associated with that gene, such as modification by changing the nucleotide code or deleting sections of nucleotide or adding non-coding nucleotides or non-natural nucleotides, such that the particular gene is either not transcribed or translated appropriately or is expressed into a non-active protein such that the gene’s natural function is abolished or reduced to such an extent that it is not measurable. Thus, the mutation of the gene inactivates that gene’s function or the function of the protein which that gene encodes.

As used herein, the term “non-natural bacterium or bacteria” refers to bacterial (prokaryotic) cells that have been genetically modified or “engineered” such that it is altered with respect to the naturally occurring cell. Such genetic modification may for example be the incorporation of additional genetic information into the cell, modification of existing genetic information or indeed deletion of existing genetic information. This may be achieved, for example, by way of transfection of a recombinant plasmid into the cell or modifications made directly to the bacterial genome. Additionally, a bacterial cell may be genetically modified by way of chemical mutagenesis, for example, to achieve attenuation, the methods of which will be well known to those skilled in the art. As such, the term “non-natural bacterium or bacteria” may refer to both recombinantly modified and non- recombinantly modified strains of bacteria.

As used herein, the term “bactofection” refers to the process of transduction of genetic material from a bacterium (e.g., Salmonella) into a eukaryotic cell. Preferably, the eukaryotic cell is a mammalian cell. More preferably, the eukaryotic cell is a human cell. Specifically, in the context of the present invention, the term “bactofection” refers to the use of Gram-negative bacteria to deliver RNA molecules transcribed in the Gram-negative bacteria to the cytosol of a eukaryotic cell following delivery of the Gram-negative bacteria to the target eukaryotic cell.

The term “split RNA polymerase” or “RNAP”, as used herein, refers to a system in which separate components of an RNA polymerase are encoded on separate genes, and when transcribed and translated, assemble to form the functional RNA polymerase. Specifically, the RNA polymerase may be separated into a “core” component, and a DNA-binding component, referred to as “sigma” or “sigma factor”. As used herein, the terms “orthogonal” or “orthogonality” are used interchangeably, and refer to the biological process of orthogonalization. Orthogonalization is the purposeful inability of two or more biomolecules, which have similar structure and/or function, to interact with one another, or affect their respective substrates. Accordingly, in the context of the present invention, the term “orthogonal” refers to the components required for expression of the polynucleotide encoding the RNA polymerase not being present in the Gramnegative bacteria. Preferably, the term “orthogonal” refers to the components required for expression of the polynucleotide encoding the RNA polymerase not being present in the Salmonella enterica genome.

The term “prophylactic treatment”, as used herein, refers to a medical procedure whose purpose is to prevent, rather than treat or cure, an infection or disease. In the present invention, this applies particularly to the vaccine composition. The term “prevent” as used herein is not intended to be absolute and may also include the partial prevention of the infection or disease and/or one or more symptoms of said infection or disease. In contrast, the term “therapeutic treatment” refers to a medical procedure with the purpose of treating or curing an infection or disease or the associated symptoms thereof, as would be appreciated within the art.

The present invention relates to modifications of Gram-negative bacteria such that RNA molecules can be safely, efficiently, and effectively delivered to target eukaryotic cells. As used herein, the terms “RNA” and “ribonucleic acid” are used interchangeably, and refer to nucleic acids composed of uracil, adenine, guanine, and cytosine ribonucleic acid bases. These terms and concepts will be well known to those in the art. Types of RNA molecules include, for example, messenger RNA (mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), transfer RNA (tRNA), ribosomal RNA (rRNA) and self-amplifying (or self-replicating) RNA (saRNA). In a preferred embodiment, the RNA molecule to be encoded is a mRNA, siRNA or shRNA molecule. As used herein, the terms “mRNA” and “messenger RNA” are used interchangeably and refer to a single-stranded RNA molecule involved in protein synthesis. Eukaryotic mRNA molecules are transcribed from DNA in the nucleus of a eukaryotic cell, and subsequently exported from the nucleus into the cytoplasm of the eukaryotic cell, where translation of the mRNA molecule into proteins takes place. Bacterial mRNA molecules are transcribed from DNA that is non-compartmentalised and translated in the cytosol coupled to transcription. These terms and concepts will be well known to those in the art.

As used herein, the terms “siRNA” and “short interfering RNA” are used interchangeably and refer to a particular method of RNA interference (RNAi). RNAi is a sequence-specific RNA degradation process that provides a direct way to knock down, or silence, theoretically any gene. In naturally occurring RNAi, a double-stranded RNA (dsRNA) is cleaved by an RNase lll/helicase protein, Dicer, into siRNA molecules, a dsRNA of 19-27 nucleotides (nt) with 2-nt overhangs at the 3' ends. These siRNAs are incorporated into a multicomponent-ribonuclease called RNA-induced silencing complex (RISC). One strand of siRNA remains associated with RISC and guides the complex toward a target RNA that has sequence complementary to the guider single-stranded siRNA in RISC. This siRNA-directed endonuclease digests the RNA, thereby inactivating it.

As used herein, the terms “shRNA” and “short hairpin RNA” are used interchangeably and refer to a particular method of RNA interference (RNAi). RNAi is a sequence-specific RNA degradation process that provides a relatively easy and direct way to knock down, or silence, theoretically any gene. shRNAs consist of a stem-loop structure. The stem-loop structure consists of a stem portion that comprises a double-stranded sequence. The double-stranded stem portion comprises an antisense (guide) strand on one side of the stem, and a sense (passenger) strand on the other side of the stem. The stem-loop structure further comprises a single-stranded loop portion at one end of the stem. Following processing via Drosha and Dicer, the shRNA is loaded into the RNA-induced silencing complex (RISC). The sense (passenger) strand is degraded, and the antisense (guide) strand directs RISC to mRNA that has a complementary sequence. In the case of perfect complementarity, RISC cleaves the mRNA. In the case of imperfect complementarity, RISC represses the translation of the mRNA. In both of these cases, the shRNA leads to target gene silencing.

As used herein, the term “saRNA”, “self-amplifying” and “self-replicating RNA” are used interchangeably and refer to a type of mRNA that encodes a replicase that copies the original strand of RNA once it has been delivered to the desired location, i.e. in the context of the present invention, to the cytosol of a host cell. The use of saRNA enhances efficiency of RNA delivery and expression of the RNA molecule.

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

The term “vaccine composition”, or “vaccine”, which from herein may be referred to interchangeably as the “composition”, relates to a biological preparation that provides active acquired immunity to a particular disease. Typically, the vaccine contains an agent, or “foreign” agent, that resembles the disease-causing pathogen, and in the context of the present invention will be encoded by an mRNA molecule (or cargo) to be delivered to the target eukaryotic cell. Such a foreign agent would be recognised by a vaccine-receiver’s immune system, which in turn would destroy said agent and develop “memory” against the disease-causing pathogen, inducing a level of lasting protection against future infection or disease from the same or similar pathogens. Through the route of vaccination, including those vaccine compositions of the present invention, it is envisaged that once the vaccinated subject again encounters the same pathogen or pathogen isolate of which said subject was vaccinated against, the individual’s immune system may thereby recognise said pathogen or pathogen isolate and elicit a more effective defence against infection or disease. The active acquired immunity that is induced in the subject as a result of the vaccine may be humoral and/or cellular in nature. In the context of the present invention, various vaccine antigens can be delivered to a target eukaryotic cell of a subject using the Gram-negative bacteria herein disclosed as a method of delivery, thus priming the subject’s immune system against said antigen.

The terms "tumour," "cancer", “malignancy” and "neoplasia" are used interchangeably and refer to a cell or population of cells whose growth, proliferation or survival is greater than growth, proliferation or survival of a normal counterpart cell, e.g., a cell proliferative or differentiative disorder. Typically, the growth is uncontrolled. The term "malignancy" refers to invasion of nearby tissue. The term "metastasis" refers to spread or dissemination of a tumour, cancer or neoplasia to other sites, locations, or regions within the subject, in which the sites, locations or regions are distinct from the primary tumour or cancer. In one embodiment, the cancer is malignant. In an alternative embodiment, the cancer is non-malignant.

The terms "effective amount" or "pharmaceutically effective amount" refer to a sufficient amount of an agent to provide the desired biological or therapeutic result. That result can be reduction, amelioration, palliation, lessening, delaying, and/or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. In reference to cancer, an effective amount may comprise an amount sufficient to cause a tumour to shrink and/or to decrease the growth rate of the tumour (such as to suppress tumour growth) or to prevent or delay other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay development or prolong survival or induce stabilisation of the cancer or tumour. 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 therapeutically effective amount of the agent or combination may result in one or more of the following: (i) reduce the number of cancer cells; (ii) reduce tumour size; (iii) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumour metastasis; (v) inhibit tumour growth; (vi) prevent or delay occurrence and/or recurrence of tumour; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer.

For example, for the treatment of tumours, a "therapeutically effective dosage" may induce tumour shrinkage by at least about 5 % relative to baseline measurement, such as at least about 10 %, or about 20 %, or about 60 % or more. The baseline measurement may be derived from untreated subjects.

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

The term "treatment" or "therapy" refers to administering an active agent with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect a condition (e.g., a disease), the symptoms of the condition, or to prevent or delay the onset of the symptoms, complications, biochemical indicia of a disease, or otherwise arrest or inhibit further development of the disease, condition, or disorder in a statistically significant manner.

As used herein, the term "subject" is intended to include human and non-human animals. Preferred subjects include human patients in need of enhancement of an immune response. The methods are particularly suitable for treating human patients having a disorder that can be treated by augmenting the immune response. In a particular embodiment, the methods are particularly suitable for treatment of neoplastic disease or infectious disease in vivo.

The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the indefinite articles "a" or "an" should be understood to refer to "one or more" of any recited or enumerated component.

As used herein, "about" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e. , the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, "about" can mean a range of up to 20%. When particular values are provided in the application and claims, unless otherwise stated, the meaning of "about" should be assumed to be within an acceptable error range for that particular value.

Previous strategies to increase the levels of bactofection of DNA have been to induce lysis of the carrier vector after cell invasion. This has been shown for L monocytogenes [Cellular Microbiology (2005) 7:709; Journal of Gene Med (2002) 4:655], E. coli (90% reported transduction) [Journal of Controlled Release (2021) 332:233], and Salmonella Typhimurium [Material Horizons (2020) 8:1454], Others [Cellular Microbiology (2005) 7(5):709], co-expressed hemolysin from Listeria or phospholipase C from Clostridium spp. to increase lysis of endosomal membranes. However, the above mentioned method still results in a bottleneck effect due to the low transference of plasmid from the cytosol into the nuclei.

To circumvent the above issue, bacteria have been used to release RNA directly into the cytosol, instead of DNA. RNA has previously been transferred using bacteria as a delivery vector using T7 polymerase (which is a common method to produce large amounts of RNA) or other strong promoters (W02020245093; Schoen et aL, 2005, Cellular Microbiology, 7(5), 709-724). Translation from bacterial RNA is ensured by adding an IRES sequence on the 5’. The major limitation of this method comes from the instability of bacterial RNA expression, which can be degraded before it can be transfected. The presence of the IRES sequence may also enhance the stability of RNA in the bacterial cytosol. As with DNA, the release of RNA was enhanced after lysis of the bacteria inside the cell.

However, the inventors of the present invention have surprisingly found that the use of T7 RNA polymerase (and corresponding T7 promoter) at high expression levels is not conducive to the safe, efficient, and effective delivery of RNA molecules when targeting eukaryotic cells, and can result in unwanted toxicity and specificity issues. Thus, the present inventors have identified that modifications to previously used promoter systems in the art is required. As outlined above, the inventors have overcome the problems with previously used promoter systems via the following two solutions. The first is to use a RNA polymerase that is encoded by a heterologous split RNA polymerase plasmid and the second is to use a RNA polymerase and promoter pairing that is orthogonal to any components of the Gram-negative bacterium that is required for endogenous gene expression.

The inventors of the present invention have surprisingly found the toxicity and specificity issues previously seen with the use of the T7 promoter can be overcome by using a split RNA polymerase system. In such a system, the RNA polymerase is separated into a “core” component and a DNA-binding component, referred to as “sigma” or “sigma factor”. These separate components of the RNA polymerase are encoded on separate genes and can therefore be controlled individually.

A schematic representation of the split RNA polymerase system is shown in Figure 2. Using the T7 RNA polymerase as an example, the “core” component of the RNA polymerase is composed of amino acids 1 to 601 of the T7 RNA polymerase. The DNA-binding component of the RNA polymerase, hereafter referred to as “sigma” or “sigma factor”, is composed of amino acids 600 to 883 of the T7 RNA polymerase. The sigma element can contain variations that allow it to bind to different promoter sequences. In order to ensure the binding of both partners, synthetic coil-coil peptides that direct protein association were added (SYNZIP). Additionally, two variants of the circuit were designed, where the core element was expressed from a low-strength promoter (proA) and either a strong (H) or weak (L) RBS. The use of split RNA polymerases offers the possibility of using two or more different sigma factors to control transcription of two or more elements. Different sigma factors have different efficiencies, opening the possibility of having these two or more outputs expressed at different ratios (e.g., one cargo RNA expressed at high levels and one lysin at low mRNA levels). Splitting the RNAP into two segments allows for fine-tuning the levels of expression of the core segment to obtain maximal RNA production with minimal toxicity. Moreover, multiple sigma elements, targeting different promoters, can be simultaneously expressed, which will bind to the optimal pool of expressed core elements to perform independent functions (e.g., RNA production and lysin expression) at different expression levels. It is a surprising finding of the inventors that the use of a split RNAP as described above not only allows for more control over the expression levels of the end product but results in reduced toxicity compared to using the previously described promoter systems.

In a second aspect, the invention provides a live attenuated Gram-negative bacterium comprising i) a heterologous polynucleotide encoding an RNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter; and ii) a polynucleotide encoding an RNA polymerase which is orthogonal to components required for endogenous gene expression in the Gramnegative bacterium, wherein the RNA polymerase binds to the promoter, and wherein upon invasion of the Gram-negative bacteria to a eukaryotic cell, the RNA molecule is transcribed and is capable of being transferred to the cytoplasm of the eukaryotic cell.

The heterologous polynucleotide of the second aspect is operably linked to a promoter that can be bound by an RNA polymerase which is orthogonal to the components required for endogenous gene expression in a host Gram-negative bacterium. Accordingly, in one embodiment, the promoter/RNA polymerase pairing is orthogonal to the components required for endogenous gene expression in a host Gram-negative bacterium. In this context, the components required for endogenous gene expression in the host Gram-negative bacterium may include transcriptional and translational machinery. Gene expression can be orthogonally regulated by effectively insulating certain systems (for example, non-endogenous RNA polymerase/promoter pairings) from host components (for example, components required for endogenous gene expression in a host Gram-negative bacterium). In a host Gram-negative bacterium, the orthogonal RNA polymerase would be incompatible with and unable to interact with the components required for endogenous gene expression of the host Gram-negative bacterium. The orthogonal RNA polymerase may be non-endogenous to the host Gram-negative bacterium, and would interact only with promoters with which it is compatible, therefore exclusively initiating transcription from these promoters. A non- endogenous (or orthogonal) RNA polymerase would not interact with endogenous components, such as endogenous promoters and gene sequences, of the host Gram-negative bacterium. Likewise, an orthogonal promoter would not interact with endogenous RNA polymerases.

In some embodiments, the present invention discloses a live attenuated Gramnegative bacterium comprising i) a heterologous polynucleotide encoding an RNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter; and ii) a polynucleotide encoding an RNA polymerase which is orthogonal to components required for endogenous gene expression in the Gramnegative bacterium, wherein the RNA polymerase binds to the promoter, wherein the RNA polymerase is encoded by a heterologous split RNA polymerase plasmid, and wherein upon invasion of the Gram-negative bacteria to a eukaryotic cell, the RNA molecule is transcribed and is capable of being transferred to the cytoplasm of the eukaryotic cell.

In order for the RNA molecule to be successfully transcribed by RNA polymerase within the Gram-negative bacteria whilst inside the target eukaryotic cell, the heterologous polynucleotide that encodes for the RNA molecule is linked to a promoter, to which the RNA polymerase binds to initiate transcription of said RNA molecule.

In a preferred embodiment of both the first and second aspect, the promoter operably linked to the heterologous polynucleotide encoding the RNA molecule, and to which the RNA polymerase binds, is a phage promoter. In a more preferred embodiment, the promoter operably linked to the heterologous polynucleotide encoding the RNA molecule, and to which the RNA polymerase binds, is a strong phage promoter, such that when the strong phage promoter is used, a high rate of transcription is initiated. Accordingly, in a preferred embodiment of the first aspect of the present invention, said phage promoter is orthogonal to components required for endogenous gene expression in the Gram-negative bacterium. With respect to the second aspect of the present invention, said phage promoter is orthogonal to components required for endogenous gene expression in the Gramnegative bacterium.

As used herein, the term “phage promoter” refers to promoters which are derived from viruses, in particular, bacteriophage viruses, which are viruses that infect and replicate only in bacterial cells. Examples of phage promoters include, but are not limited to, T7p, T3p, K1 Fp, and CGGp. In a preferred embodiment, the phage promoter is CGGp. In this context, a polymerase which is derived from a virus (i.e. , a phage RNA polymerase) is paired with a phage promoter. In a preferred embodiment of the second aspect of the present invention, the bestperforming system may comprise a CGGp phage orthogonal promoter paired with a split RNA polymerase (CGG).

Examples of possible promoter/RNA polymerase pairings of the first aspect of the present invention are shown in Table 1. Accordingly, the following RNA polymerases are split RNA polymerases. In one embodiment, the promoter operably linked to the heterologous polynucleotide encoding the 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 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 K1 F promoter and the RNA polymerase is a K1 F 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 above promoter/RNA polymerase pairings are all orthogonal to the components required for endogenous gene expression in a host Gram-negative bacterium.

The inventors of the present invention have surprisingly shown that high expression levels of T7 polymerase (using a T7 promoter) are toxic. Additionally, despite the inventors showing that low expression levels of T7 polymerase allow for growth of the cells, even low expression levels of T7 polymerase resulted in inefficient circuit output. Therefore, in one embodiment, the promoter operably linked to the heterologous polynucleotide encoding the RNA molecule, and to which the RNA polymerase binds, is not a T7 promoter and the RNA polymerase is not a T7 split RNA polymerase. In another embodiment, the promoter operably linked to the heterologous polynucleotide encoding the RNA molecule, and to which the RNA polymerase binds, is not a T7 promoter and the RNA polymerase is not a T7 RNA polymerase.

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

Table 1 : Examples of possible promoter/RNA polymerase pairings. Tick symbols (^) indicate pairings between promoters and sigma factors of the RNA polymerase.

In an embodiment, the sigma factor RNA polymerase may have a polynucleotide sequence according to SEQ ID NO: 3, 5, 7, 9 ora sequence having 90% sequence identity to said sequence. For example, the sigma factor RNA polymerase may have a polynucleotide sequence having 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 3, 5, 7 or 9.

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

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

As used herein, the term “sequence identity” and “sequence homology” are interchangeable and refers to the number of identical residues over a defined length in a given alignment of a DNA sequence or an amino acid sequence. To calculate % sequence identity of any of the sequences herein disclosed, sequence comparison software may be used, for example, using the default settings on the BLAST software package (V2.10.1).

In order to drive expression of the RNA polymerase of the first and second aspect of the invention (i.e. both split RNA polymerase systems and non-split RNA polymerase systems), intracellular promoters, for example, cytosolic and vacuoledependent promoters, are used.

In the context of the present invention, these promoters may be used to activate the sigma factor of the RNA polymerase systems described above (for example, the sigma factors disclosed in Table 1) under specific conditions, for example, at a specific time or at a specific time of the infection cycle. Said promoters are dispersed throughout the genome of Gram-negative bacteria (e.g. Salmonella enterica), positioned upstream of relevant genes and are activated through a myriad of signals. For example, vacuole promoters commonly respond to acidification and low osmolarity, whilst the cytosolic promoters commonly respond to iron, manganese, hexose sugars and oxidative stress. In the context of a split RNA polymerase system, multiple sigma factors may be driven by the intracellular promoters described above. For example, the expression of one sigma factor may be driven by a cytosolic promoter and the expression of a second, different, sigma factor may be driven by a vacuole-dependent promoter. Alternatively, the expression of two different sigma factors may be driven by two cytosolic promoters or two vacuole-dependent promoters.

Accordingly, in a particularly preferred embodiment, the promoter used to activate the RNA polymerase systems of the first and second aspect of the present invention are cytosolic promoters or vacuole dependent promoters. The term “cytosolic promoter”, as used herein, refers to intracellular promoters in bacteria. In an even more preferred embodiment, the cytosolic promoters for use in the present invention may include, but are not limited to, uhpT (SEQ ID NO:23 ), mntH (SEQ ID NO: 31), entC (SEQ ID NO: 25), fhuE (SEQ ID NO: 26), iroN (SEQ ID NO: 35), fepB (SEQ ID NO: 36), fepA (SEQ ID NO: 32), fhuA (SEQ ID NO:29), sitA (SEQ ID NO: 30), stn3250 (SEQ ID NO: 45 ), sufA (SEQ ID NO: 33), yjjZ (SEQ ID NO: 28), soxS (SEQ ID NO: 34), sfbA (SEQ ID NO: 27) and the vacuole dependent promoter is selected from the group comprising 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).

The use of such promoters allows for the precise control of the activation of the RNA polymerase system, for example, activation can be initiated at specific locations or times of the infection cycle. Temporal control of expression may additionally be achieved by using intracellular (SPI-2), oxidative stress-responsive (e.g., soxP, grxA), sugar-sensing (e.g., uphT (glucose-6-phosphate), frubKA (fructose)), or metal-dependent promoters (e.g., iroN, fhuE, mntH, entC, fepB). These cytosolic promoters can be used to drive differential expression of lysins as well as RNA polymerases. SPI-2 promoters can also be used to activate the expression of type-1 secretion systems (T1SS) which facilitate the export of hemolysins from Listeria and enable, for example, the release of bacterial cells from a vacuole into the cytoplasm of a eukaryotic cell, thus granting bacteria access to the cytoplasm of a eukaryotic cell.

In order to regulate the total amount of polymerase activity, in vivo or constitutive promoters may be utilised. The term “constitutive promoter” herein used refers to a promoter that initiates transcription only when it receives a specific stimuli. In the context of the split RNA polymerase of the present invention, such promoters are responsible for the production of the “core” of the RNA polymerase and therefore regulate the extent to which the “sigma factor” can bind.

The invention herein disclosed relates to a method by which RNA molecules transcribed within a bacterium can be transferred to the cytosol of a eukaryotic host cell. In order for the transcription of the RNA molecules, such as mRNA, siRNA or shRNA molecules to be initiated, the Gram-negative bacteria must first invade within the target eukaryotic cell. The RNA molecule may be transferred to the cytoplasm in multiple ways, for example, within the vacuoles of the target eukaryotic cell, or the Gram-negative bacterium may be programmed to self-lyse via inclusion of a lysis protein to release the transcribed RNA molecule. Release from a phagocytic vacuole of the bacteria is desirable for efficient delivery of either DNAor RNA molecules, as evidenced by the higher in vitro bactofection efficiency from the cytosolic pathogen Listeria. Accordingly, in one embodiment, the bacterium is designed to allow escape from a phagocytic vacuole. For example, escape from Sa/mone//a-containing vacuoles (SCVs) can be achieved in at least two ways. Firstly, by the addition of exogenous genes. For example, expression of hly from Listeria in Salmonella, results in export from the cytosol of the bacterium into the SCV using a type 1 secretion system. Secondly, by manipulation of Salmonella’s SPI-1 (or SPI-2) effector proteins (sopF and sopE) to destabilise the SCV and release the bacteria into the cytosol. The effector proteins are injected into the cytosol of the invaded mammalian cell and are involved in the maintenance of the early vacuole. It has been shown in the literature that invasion with strains having a deletion of the sopF or overexpression of the sopE gene products undergo early vacuole escape and either establish in the host cytosol or are encapsulated again via autophagy mechanisms.

As described above, the Gram-negative bacteria of the present invention may be programmed to self-lyse via inclusion of a lysis protein to release the transcribed RNA molecule into the cytoplasm of the eukaryotic host cell. One method by which this is achieved is the use of a cytosolic promoter. When entering the cytoplasm of a eukaryotic cell, bacteria with cytosolic promoters induce their own lysis, and produce RNA that enters directly into the cytosol, thus avoiding the transfer of genetic material into the nucleus of the target eukaryotic cell.

In the context of Listeria, the inventors of the present invention have found that the use of a cytosolic promoter enabled around a ten times increase in the release of the RNA molecule from the Listeria bacteria. Therefore, in a preferred embodiment, lysis of the bacterial cell is to be carried out via the use of a cytosolic promoter. Lysis of the bacterial cell can also be achieved by using a lysin or bacterial release protein. For example, lysE, kiIR, kil, or BRP. In addition to the live attenuated Gram-negative bacterium comprising a promoter, and an RNA polymerase that binds to said promoter, the live attenuated Gramnegative bacterium may further comprise numerous other auxiliary proteins which optimise the delivery of the RNA molecule to the target eukaryotic cell. For example, the live attenuated Gram-negative bacterium may further comprise one or more of the following auxiliary proteins integrated into the genome of the Gramnegative bacterium: i) a polynucleotide encoding an RNA stability enhancing component, preferably (in the case when the RNA molecule is an mRNA molecule) wherein the RNA stability enhancing component is a IRES sequence; ii) a polynucleotide encoding a lysis protein, preferably wherein said lysis protein is hemolysin; iii) a polynucleotide encoding a phospholipase, preferably wherein the phospholipase is phospholipase C; iv) a polynucleotide encoding an invasion factor; and/or v) a bacteriocin release protein, a bacteriophage lambda lysozyme, and holin.ln a preferred embodiment the lysis protein is derived from a Listeria spp., the phospholipase is derived from Clostridium spp. or Listeria spp., and the invasion factor is derived from Yersina spp. The inclusion of a polynucleotide encoding a bacteriocin release protein, a bacteriophage lambda lysozyme, and holin enables the bacterium to self lyse upon entering the target eukaryotic cell.

The present invention provides a way in which target genes can be silenced, protein synthesis prevented or reduced, or new proteins synthesised. As the skilled person will appreciate, such a method has a significant and wide-reaching therapeutic benefit. In order to allow for transcription of the heterologous polynucleotide and production of an RNA molecule in the Gram-negative bacterium, the heterologous polynucleotide is operably linked to a functional promoter, with the Gram-negative bacterium further comprising a heterologous polynucleotide encoding a suitable RNA polymerase. These components work together to enable the synthesis of the RNA molecule within the Gram-negative bacteria intended for delivery to a eukaryotic cell. The methods by which heterologous genetic material can be incorporated into the bacterial genome are well known to the skilled person and are further detailed below. The live attenuated Gram-negative bacterium of the present invention can act as an efficient and reliable method of delivering heterologous RNA to a target eukaryotic cell. Accordingly, the bacterial strains herein disclosed are recombinant strains comprising a polynucleotide that encodes for an RNA molecule (i.e., an RNA molecule such as mRNA, siRNA, shRNA, saRNA, or miRNA).

The functional RNA molecule to be delivered to the eukaryotic cell may be a messenger RNA (mRNA). Messenger RNA is transcribed from DNA and contains the genetic blueprint to make proteins. Prokaryotic mRNA does not need to be processed and can proceed to synthesize proteins immediately. In eukaryotes, a freshly transcribed RNA transcript is considered a pre-mRNA and needs to undergo maturation to form mRNA. A pre-mRNA contains non-coding and coding regions known as introns and exons, respectively. During pre-mRNA processing, the introns are spliced, and the exons are joined together. A 5’ cap known as 7- methylguanosine is added to the 5’ end of the RNA transcript and the 3’ end is polyadenylated. Polyadenylation refers to the process where a poly(A) tail, which is a sequence of adenine nucleotides, is added to the transcript. The 5’ cap protects the mRNA from degradation, and the 3’ poly(A) tail contributes to the stability of mRNA and aids it in transport. The live attenuated Gram-negative bacterium may encode up to 10 different heterologous mRNA molecules, for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 different heterologous mRNA molecules.

The mRNA of the present invention is transcribed within the Gram-negative bacteria and subsequently transferred to the target eukaryotic cell for translation using the translational machinery of the host eukaryotic cell. Accordingly, the invention herein disclosed provides a mRNA molecule 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 lead to enhanced efficiency of RNA delivery and increased expression of the RNA molecule. The saRNA molecule is able to “self-amplify” due to the polynucleotide sequence encoding the RNA molecule also encoding a replicase that enables the amplification of the original strand of RNA upon delivery to the host cell. Using saRNA thus enables the advantages described above whilst only requiring a minimal dose of RNA.

The polypeptide that is encoded by the mRNA molecule may be any therapeutic protein for use in the treatment or prevention of disease. It will be readily understood that the specific therapeutic protein will depend on a variety of factors, not least 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 Gramnegative bacterium of the present invention does not encode components of the CRISPR/Cas9 gene editing system, zinc finger nucleases or RNA encoding transcription activator-like effector nucleases (TALENs).

In another embodiment, the functional RNA molecule is a siRNA or shRNA molecule, which targets a specific mRNA molecule present within the eukaryotic cell. Following transcription of an siRNA or shRNA molecule within the bacteria, the transcribed siRNA or shRNA molecule is transferred to the cytoplasm of the eukaryotic cell where it locates and binds to the target mRNA. The invention herein disclosed therefore also provides a method in which a target gene can effectively be silenced by the degradation of the corresponding mRNA via an siRNA or shRNA molecule.

The present invention may target a single mRNA or multiple mRNAs via the use of siRNA and/or shRNA molecules. Accordingly, the live attenuated Gramnegative bacterium of the present invention may deliver a single siRNA molecule or multiple siRNA molecules, each one targeting a different target mRNA. For example, the live attenuated Gram-negative bacterium may encode 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 siRNA or shRNA molecule may bind to any target mRNA. The target mRNA may be any mRNA encoding a protein of interest, for example, a mRNA encoding a protein associated with a disease state. For example, target mRNAs may include mRNAs involved in proliferation/cell cycle, migration, angiogenesis, immune activation/inhibition, cell death and fibrosis. Additionally, target mRNAs may include, but are not limited to the HIF1 protein family, the TGFb receptors protein family and various transcriptional factors that drive dysregulation of cancer cells. It will be readily understood that the specific mRNA to be targeted will depend on a variety of factors, not least the specific disease to be 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.

The live attenuated bacterium of the present invention is a Gram-negative bacterium. 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.

Preferably, the live attenuated Gram-negative bacterium is a Salmonella species. Examples of Salmonella species for use in the present invention are Salmonella enterica and Salmonella bongori. Salmonella enterica can be further sub-divided into different serotypes or serovars. Examples of said serotypes or serovars 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 enterica 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

Salmohnella enterica Typhi. In another embodiment of the present invention, the live attenuated Gramnegative bacterium is a genetically engineered non-natural bacterium.

Accordingly, the present invention discloses Gram-negative bacteria that have been genetically altered to produce attenuated bacterial strains that can effectively deliver RNA molecules as their cargo. As would be understood by a person of skill in the art, genes may be mutated by a number of well-known methods in the art, such as homologous recombination with recombinant plasmids targeted to the gene of interest, in which case an engineered gene with homology to the target gene is incorporated into an appropriate nucleic acid vector (such as a plasmid or a bacteriophage), which is transfected into the target cell. The homologous engineered gene is then recombined with the natural gene to either replace or mutate it to achieve the desired inactivating mutation. Such modification may be in the coding part of the gene or any regulatory portions, such as the promoter region. As would be understood by a person of skill in the art, any appropriate genetic modification technique may be used to mutate the genes of interest, such as the CRISPR/Cas system, e.g., CRISPR/Cas 9, to produce the bacterial strains herein disclosed.

Thus, numerous methods and techniques for genetically engineering bacterial strains will be well known to the person skilled in the art. These techniques include those required for introducing heterologous genes into the bacteria either via chromosomal integration or via the introduction of a stable autosomal selfreplicating genetic element. Exemplary methods for genetically modifying (also referred to as "transforming" or “engineering”) bacterial cells include bacteriophage infection, transduction, conjugation, lipofection or electroporation. A general discussion on these and other methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); which are hereby incorporated by reference. The live attenuated Gram-negative bacterium may be selected from the group comprising Ty21a, CVD 908-htrA, CVD 909, Ty800, M01ZH09, x9633, x639, X9640, X8444, DTY88, ZH9PA, MD58, WT05, ZH26, SL7838, SL7207, VNP20009, A1-R, or any combinations thereof. In a preferred embodiment, the live attenuated Gram-negative bacterium is M01ZH09 (also referred to as ZH9).

In one embodiment, the genetically engineered non-natural bacterium may be derived from a Salmonella species that may comprise an attenuating mutation in a Salmonella Pathogenicity Island 2 (SPI-2) gene and an attenuating mutation in a second gene. Suitable genes and details of such a live attenuated bacterium is as described in WO 2000/68261 , which is hereby incorporated by reference in its entirety.

In one embodiment, the SPI-2 gene is an ssa gene. For example, the invention includes an attenuating mutation in one or more of ssa ssaJ, ssaU, ssaK, ssaL, ssaM, ssaO, ssaP, ssaQ, ssaR, ssaS, ssaT, ssaD, ssaE, ssaG, ssa/, ssaC and ssa/-/. Preferably, the attenuating mutation is in the ssa\/ or ssa J gene. Even more preferably, the attenuating mutation is in the ssa\/ gene.

The genetically engineered non-natural bacterium may also comprise an attenuating mutation in a second gene, which may or may not be in the SPI-2 region. The mutation may be outside of the SPI-2 region and involved in the biosynthesis of aromatic compound. For examples, the invention includes an attenuating mutation in an aro gene. In a preferred emnodiment, the aro gene is aroA or aroC. Even more preferably, the aro gene is aroC.

The genetically engineered non-natural bacterium may further comprise one or more gene cassettes. Such gene cassettes may be used to deliver additional prokaryotic molecules to support the function of the genetically engineered non- natural bacterium to condition the immune system, or to support the activity of the therapeutic protein encoded by an mRNA molecule. In yet another embodiment, the genetically engineered non-natural bacterium may be derived from a Salmonella species and may comprise inactivating mutations in one or more genes selected from pltA, pltB, cdtB and ttsA and further comprises attenuating mutations in one or more genes selected from aroA and/or aroC and/or ssaV Details of said genes and mutations are as described in WO 2019/110819, which is hereby incorporated by reference in its entirety.

It is envisaged that inactivating mutations (e.g., deletions) in the genes pltA, pltB and cdtB will prevent the Salmonella species from producing the typhoid toxin and that inactivating mutations (e.g. deletions) in ttsA will prevent the Salmonella species from secreting the typhoid toxin. It is envisaged that the non-natural bacterium may be derived from Salmonella enterica, in particular.

The present invention allows for the safe, efficient, and effective delivery of a heterologous RNA molecule to a target eukaryotic cell. . In the case where the RNA molecule is an mRNA molecule, the resulting heterologous polypeptide may be a therapeutic protein and/or a heterologous antigen (dependent on the indication to be treated, for example, in the context of a vaccine composition, the heterologous polypeptide may be a heterologous antigen). In a preferred embodiment, the resulting therapeutic protein is a cytokine, a chemokine, an antibody or a fragment thereof, a cytotoxic agent, a cancer agent or any combination thereof. Even more preferably, the resulting therapeutic protein may be IL-15, IL-21 , CXCL9, IL-18, IL-27, IFNy, IL-1 , or any combination thereof. In another embodiment, a heterologous siRNA or shRNA molecule is delivered to a target eukaryotic cell by using the siRNA or shRNA delivery method herein disclosed. Using this method, any mRNA molecule can be effectively target and in effect, the associated gene silenced. Accordingly, the present invention has particular use in disease states in which preventing the production of a particular protein would be advantageous. In a preferred embodiment of the present invention, the live attenuated Gramnegative bacteria is administered intratumourally, intravenously, intraperitoneally, or orally administered. In a most preferred embodiment, the live attenuated Gramnegative bacteria is administered intratumourally. However, it is also contemplated that other methods of administration may be used in some cases. Therefore, in certain instances the live attenuated Gram-negative bacterium of the present invention may be administered by injection, infusion, continuous infusion, intradermally, intraarterially, intralesionally, intravaginally, intrarectally, intramuscularly, subcutaneously, subconjunctival, , mucosally, intrapericardially, intraumbilically, intraocularally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, inhalation (e.g. aerosol inhalation), via a catheter, via a lavage, or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990).

The amount of the live attenuated Gram-negative bacterium administered to the subject is sufficient to deliver the heterologous RNA molecule to the target eukaryotic cell in high enough concentrations for it to have the desired effect once translated in the eukaryotic cell. The skilled person will readily understand that the precise amount to be administered will be dependent on a number of factors, for example, the disease to be treated, the desired protein to be translated (in the case when the RNA molecule is an mRNA molecule), the translated level of the target mRNA present (in the case when the RNA molecule is an siRNA or shRNA molecule) in the target eukaryotic cell and the medical history of the subject to be treated.

The live attenuated Gram-negative bacterium may be administered at a dose of between 10⁵ and 10¹² CFU, where CFU is a colony-forming unit. For example, suitable doses may be between 10⁵ and 10⁶ CFU, 10⁵ and 10⁷ CFU, 10⁵ and 10⁸ CFU, 10⁵ and 10⁹ CFU, 10⁵ and 10¹° CFU, 10⁵ and 10¹¹ CFU, 10⁶ and 10⁷ CFU, 10⁶ and 10⁸ CFU, 10⁶ and 10⁹ CFU, 10⁶, and 10¹° CFU, 10⁶ and 10¹¹ CFU, 10⁶ and 10¹² CFU, 10⁷ and 10⁸ CFU, 10⁷ and 10⁹ CFU, 10⁷ and 10¹° CFU, 10⁷ and 10¹¹ CFU, 10⁷ and 10¹² CFU, 10⁸ and 10⁹ CFU, 10⁸ and 10¹° CFU, 10⁸ and 10¹¹ CFU, 10⁸ and 10¹² CFU, 10⁹ and 10¹° CFU, 10⁹ and 10¹¹ CFU, 10⁹ and 10¹² CFU, 10¹° and 10¹¹ CFU, 10¹° and 10¹² CFU, or 10¹¹ and 10¹² CFU. The live attenuated Gram-negative bacterium may be administered in a single dose or in multiple doses. The specific number of doses to be administered are understood to be dependent on the specific RNA molecule to be delivered or targeted, as well as the specific indication to be treated.

The purpose of the present invention is to provide an effective and efficient way of delivering RNA molecules to a target eukaryotic cell to be translated into the desired therapeutic protein (in the case when the RNA molecule is an mRNA molecule), or of delivering iRNA molecules to a target eukaryotic cell (in the case when the RNA molecule is an siRNA or shRNA molecule). The target eukaryotic cell may be a mammalian cell. In a preferred embodiment, the target eukaryotic cell is a human cell. Where the eukaryotic cell is a human cell, the target cell may be a cancerous human cell or a non-cancerous human cell.

The live attenuated Gram-negative bacterium of either the first aspect or the second aspect of the present invention herein disclosed may be for therapeutic use. For example, the live attenuated Gram-negative bacterium may be for use in the treatment, reduction, inhibition, prevention, prevention of recurrence, or control of 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 the treatment, reduction, inhibition, prevention, prevention of recurrence, or control of a neoplastic disease or an infectious disease.

Where the disease to be treated is a neoplastic disease, the neoplastic disease may be associated with a solid tumour and/or a haematological malignancy. Such diseases include a sarcoma, carcinoma, adenocarcinoma, melanoma, myeloma, blastoma, glioma, lymphoma or leukaemia. In a preferred embodiment, the neoplastic disease is associated with a solid tumour. In particular aspects, the neoplastic disease is associated with a cancer selected from prostate cancer, oesophageal cancer, liver cancer, renal cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, brain cancer, mesothelioma, hepatocellular cancer, lymphoma, leukaemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer or sarcoma.

Neoplasia, tumours and cancers include benign, malignant, metastatic and non- metastatic types, and include any stage (I, II, III, IV or V) or grade (G1 , G2, G3, etc.) of neoplasia, tumour, or cancer, or a neoplasia, tumour, cancer or metastasis that is progressing, worsening, stabilized or in remission. Cancers that may be treated according to the invention include but are not limited to cells or neoplasms of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestines, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to the following: neoplasm, malignant; carcinoma; undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumour, malignant; bronchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumour, malignant; thecoma, malignant; granulosa cell tumour, malignant; androblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumour, malignant; lipid cell tumour, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumour; Mullerian mixed tumour; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; Brenner tumour, malignant; phyllodes tumour, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumour of bone; Ewing's sarcoma; odontogenic tumour, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumour; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumour, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; 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 tumours associated with a cancer selected from prostate cancer, liver cancer, renal cancer, lung cancer, breast cancer, colorectal cancer, pancreatic cancer, brain cancer, hepatocellular cancer, lymphoma, leukaemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, head and neck cancer, skin cancer and soft tissue sarcoma and/or other forms of carcinoma. The tumour may be metastatic or a malignant tumour.

In a preferred embodiment, the neoplastic disease is associated with a cancer selected from bladder cancer, lung cancer, mesothelioma, hepatocellular cancer, melanoma, oesophageal cancer, gastric cancer, ovarian cancer, colorectal cancer, head and neck cancer or breast cancer.

In a third aspect, the present invention provides a vaccine composition comprising a live attenuated Gram-negative bacterium, wherein the Gram-negative bacterium comprises i) a heterologous polynucleotide encoding a functional mRNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter and the mRNA molecule encodes a therapeutic protein and/or therapeutic peptide; and ii) a polynucleotide encoding an RNA polymerase which is orthogonal to components required for endogenous gene expression in the Gram-negative bacterium, wherein the RNA polymerase binds to the promoter, and wherein upon invasion of the Gram-negative bacteria to a eukaryotic cell, the mRNA molecule is transcribed and is capable of being transferred to the cytoplasm of the eukaryotic cell. In a fourth aspect, the present invention provides a vaccine composition comprising a live attenuated Gram-negative bacterium, wherein the Gramnegative bacterium comprises i) a heterologous polynucleotide encoding a functional mRNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter and the mRNA molecule encodes a therapeutic protein and/or therapeutic peptide; and ii) a polynucleotide encoding an RNA polymerase, wherein the RNA polymerase binds to the promoter, wherein the RNA polymerase is encoded by a heterologous split RNA polymerase plasmid, and wherein upon invasion of the Gram-negative bacteria to a eukaryotic cell, the mRNA molecule is transcribed and is capable of being transferred to the cytoplasm of the eukaryotic cell.

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

In an embodiment, the vaccine composition of the present invention may be for therapeutic use. For example, the live attenuated Gram-negative bacterium may be for use in the treatment, reduction, inhibition, prevention, prevention of recurrence, or control of a disease.

Therefore, the vaccine composition of the third or fourth aspect may comprise one or more of the aforementioned embodiments in respect to any preceding aspect. The phage promoter of the vaccine composition of the third or fourth aspect of the present invention may be T3p, K1 Fp, or CGGp.

It is particularly envisaged that the vaccine composition herein disclosed may be used in the treatment, reduction, inhibition, prevention of recurrence or control of an infectious disease, for example, a disease caused by a bacteria, a virus, a parasite or a fungi. In such instances, the heterologous polynucleotide to be transcribed into a functional mRNA molecule may be an antigen of the causative agent of the specific infectious disease in order to produce an immune response in the host. Alternatively, it is envisaged that the vaccine composition herein disclosed may be used as a cancer vaccine. In such instances the vaccine composition comprises Gram-negative bacteria comprising a heterologous polynucleotide encoding a cancer antigen that is capable of producing an immune response in the host. It is therefore appreciated that a wide-range of cancers and infectious diseases can be prevented/treated using the bacterium and methods herein disclosed. In other instances, the heterologous polynucleotide is to be transcribed into an siRNA or shRNA molecule which is designed to enhance immune anti-infectious function or tissue anti-infectious defences.

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

As used herein, "pharmaceutically acceptable camer/adjuvant/diluent/excipient" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavouring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289- 1329). Examples include, but are not limited to disodium hydrogen phosphate, soya peptone, potassium dihydrogen phosphate, ammonium chloride, sodium chloride, magnesium sulphate, calcium chloride, sucrose, borate buffer, sterile saline solution (0.9 % NaCI) and sterile water.

Suitable aqueous and non-aqueous carriers that may be employed in the vaccine compositions of the invention include water, ethanol, polyols (such as 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.

The vaccine compositions herein disclosed may further contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of unwanted microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminium monostearate and gelatin. The vaccine composition may also optionally include additional therapeutic agents, known to be efficacious in, for example, infectious disease or neoplastic disease. Accordingly, the vaccine composition herein disclosed may also comprise antiretroviral drugs, antibiotics, antifungals, antiparasitics and anticancer agents.

The vaccine composition may also comprise additional components intended for enhancing an immune response in a subject following administration. Examples of such additional components include but are not limited to; aluminium salts such as aluminium hydroxide, aluminium oxide and aluminium phosphate, oil-based adjuvants such as Freund's Complete Adjuvant and Freund's Incomplete Adjuvant, mycolate-based adjuvants (e.g., trehalose dimycolate), bacterial lipopolysaccharide (LPS), peptidoglycans (e.g., mureins, mucopeptides, or glycoproteins such as N-Opaca, muramyl dipeptide [MDP], or MDP analogs), proteoglycans (e.g., extracted from Klebsiella pneumoniae), streptococcal preparations (e.g., OK432), muramyldipeptides, Immune Stimulating Complexes (the "Iscoms" as disclosed in EP 109942, EP 180564 and EP 231 039), saponins, DEAE-dextran, neutral oils (such as miglyol), vegetable oils (such as arachis oil), liposomes, polyols, the Ribi adjuvant system (see, for instance, GB-A-2 189 141 ), vitamin E, Carbopol, interferons (e.g., IFN-alpha, IFN-gamma, or IFN-beta) or interleukins, particularly those that stimulate cell mediated immunity (e.g., IL-2, IL- 3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1 , IL-12, IL-13, IL-14, IL-15, IL-16 and IL-17).

The live attenuated Gram-negative bacterium of the vaccine composition herein disclosed may include any one of, or any combination of the features of the live attenuated Gram-negative bacterium herein disclosed.

In a fifth aspect of the present invention provides a method of treating, inhibiting, preventing recurrence or controlling a disease in a subject, wherein the method comprises administering to the subject a live attenuated Gram-negative bacterium comprising i) a heterologous polynucleotide encoding an RNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter; and ii) a polynucleotide encoding an RNA polymerase which is orthogonal to components required for endogenous gene expression in the Gram-negative bacterium, wherein the RNA polymerase binds to the promoter, wherein the RNA polymerase is encoded by a heterologous split RNA polymerase plasmid, and wherein upon invasion of the Gram-negative bacteria to a eukaryotic cell, the RNA molecule is transcribed and is capable of being transferred to the cytoplasm of the eukaryotic cell. Therefore, the method of treating, inhibiting, preventing recurrence or controlling a disease in a subject of the fifth aspect may comprise one or more of the aforementioned embodiments in respect to any preceding aspect.

In one embodiment, a split RNA polymerase plasmid may be incorporated into the fifth aspect of the present invention in order to enable the safe, efficient, and effective delivery of RNA molecules to target eukaryotic cells.

In a sixth aspect, the present invention, a method of treating, inhibiting, preventing recurrence or controlling a disease in a subject is provided, wherein the method comprises administering to the subject a live attenuated Gram-negative bacterium for use in the treatment, reduction, inhibition, prevention of recurrence, or control of a 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 which is orthogonal to components required for endogenous gene expression in the Gram-negative bacterium, wherein the RNA polymerase binds to the promoter, and wherein upon invasion of the Gram-negative bacteria to a eukaryotic cell, the RNA molecule is transcribed and is capable of being transferred to the cytoplasm of the eukaryotic cell.

Therefore, the method of treating, inhibiting, preventing recurrence or controlling a disease in a subject of the sixth aspect may comprise one or more of the aforementioned embodiments in respect to any preceding aspect.

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

In a preferred embodiment, the live attenuated Gram-negative bacterium has any one of, or any combination of the features of the live attenuated Gram-negative bacterium herein disclosed. In a further preferred embodiment, the disease to be treated is a neoplastic disease or an infectious disease.

In a seventh aspect of the present invention, a method for delivering an RNA molecule into a eukaryotic cell is provided, said method comprising 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; ii) contacting the Gram-negative bacterium with the eukaryotic cell such that the Gram-negative bacterium replicates within said eukaryotic cell, such that the heterologous polynucleotide is transcribed and subsequently transferred from the Gramnegative bacterium to the cytoplasm of the eukaryotic cell.

Therefore, the method for delivering an RNA molecule into a eukaryotic cell of the seventh aspect may comprise one or more of the aforementioned embodiments in respect to any preceding aspect.

In a preferred embodiment, the live attenuated Gram-negative bacterium has any one of, or any combination of the features of the live attenuated Gram-negative bacterium herein disclosed. In a further preferred embodiment, the method for delivering an RNA molecule into a eukaryotic cell is an in vivo method.

EXAMPLES

The invention is further described with reference to the following non-limiting examples:

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

A plasmid containing the T7 RNA polymerase (T7 RNAP) gene downstream of SPI-2-inducible promoters (/.e., promoters that activate inside the Salmonellacontaining vacuole, SCV, during mid-late stages of invasion) were assembled onto Chloramphenicol-resistant pSC101 plasmids (~5 copies/cell) via Gibson assembly of DNA fragments synthesized by integrated DNA technologies (IDT) and that contained appropriate overhangs. Plasmids assembled contained either pipB, ssaG, sseJ, or ssrA promoters.

In parallel, carbenicillin-resistant p15A plasmids containing mScarlet downstream of T7p (T7-responsive promoter) were assembled by inverse-PCR and relegation of pSEVA-16 (from Standard European Vector Architecture collection) using appropriate phosphorylated primers.

After validation, plasmids were co-transformed into Salmonella enterica Typhi ZH9 via electroporation and recovered under both antibiotics. Single clones were grown at 37°C and 200 rpm for 16-18 h in vLB liquid media (Formedium) supplemented with 25 pg mL'¹ of each antibiotic, after which these were diluted 1 : 10 in 100 pL PCN media on a 96-well plate in and grown at 37°C for 6-8 h in a Clariostar (BMG Labtech) whilst recording growth and mScarlet fluorescence every 15 min. Toxicity and lack of growth (measured as OD₆oo) was recorded. Additional validation of toxicity was performed by plating 50 pL of final cultures in fresh vLBA media (Formedium) containing appropriate antibiotics and grown for 16-18 h at 37°C (See Figure 1).

Example 2: Construction of split RNAP circuits and expression plasmids

A split RNA polymerase (RNAP) entry pSC101 -o plasmid was generated to contain the core RNAP sequence downstream a weak promoter (proA), either a strong (H) or weak (L) Ribosome Binding Site (RBS). Upstream of proA, a double terminator and two placeholder sequences were placed: (1) a promoter cloning site, with two unique Bsal restriction sequences that allow cloning of any promoter desired via Golden Gate Assembly; and (2) a sigma factor cloning site, with superfolder gfp sequence between two unique Bbsl restriction sequences that allow cloning of any sigma factor desired via Golden Gate Assembly (see Figure 2). Bactofection expression plasmids were built on plasmids with a p15A ori containing a phage-dependent promoter (e.g., T7, T3, K1 F, or CGG) upstream of a placeholder mScarlet sequence surrounded by Bbsl-sites. This sequence can be replaced by any desired cargo to transfect via Golden Gate Assembly.

Example 3: Validation of split RNAP circuits

Plasmids containing the split RNAP circuits with sigma T7, T3, K1 F, or CGG sigma factors were generated as described above. A first validation test was done by cloning promoter zinT (responsive for Zinc) upstream of the sigma factor region.

E. coli DH5a cells were co-transformed with all the combinatorial variations of expression plasmids and sigma factors (see Example 2) and grown in vLB (Formedium) media overnight. Fluorescence was measured and only observed on samples containing the correctly paired promoter-sigma systems (See Figure 3).

Correctly paired promoter-sigma plasmids were then co-transformed into Salmonella enterica ZH9 (no sigma used as negative control) and grown in vLB media at 37°C for 16-18 h. Plasmid with CGG sigma factor was the most fluorescent sample, whereas those expressing strong T7 sigma factor were toxic to ZH9 (See Figure 3).

To validate these results, the cytosolic promoter puhpT was cloned upstream of each sigma factor element via Bbsl-depenendent Golden Gate reaction (using NEBridge as buffer and standard reaction conditions). These variants were cotransformed into Salmonella enterica ZH9 as previously described and grown in vLBA at 37°C for 16-18 h. Single colonies were selected and grown for 48 h at 37°C and 700 rpm in 100 pL of vLB media supplemented with 0.4 % glucose-6- phosphate (Sigma), whilst recording growth and mScarlet fluorescence. Results (Figure 3) depict similar results in liquid media (vLB) after induction with Glucose- 6-Phosphate and incubation for 48 h at 37 °C than those obtained previously in solid media. Example 4: Construction of promoter reporter plasmids

A promoter reporter plasmid vector was built to contain mScarlet constitutively expressed under control of promoter proB and inducible sfgfp encoded divergently one from the other. Upstream sfgfp a placeholder sequence was placed to contain two unique Bsal restriction sites that allow one-pot introduction of any desired promoter via Golden Gate Assembly. Additionally, once the promoter has been characterised, the sfgfp can be replaced for any gene of interest via Bbsl- dependent Golden Gate Assembly.

Example 5: In vitro characterisation of cytosolic plasmids

Promoter reporter plasmids, built via Bsal-dependent Golden Gate Assembly (NEBridge as buffer, standard conditions), were used as template to introduce a series of promoters putatively active when ZH9 is present in the cytosol or vacuole of mammalian cells (see Figure 4). Most of these promoters are metal-dependent and respond to varying concentrations of Fe³⁺ (cytosol), Zn²⁺ (vacuole), Mg²⁺ (vacuole), or sugars (glucose-6-phosphate, cytosol). The resulting plasmids were transformed into ZH9 and the resulting strains were grown in vLBA at 37°C for 16-18 h. Single colonies were picked and grown in vLB at 37°C for 16-18 h before being diluted 1 :100 in 100 pL of M9 minimal media, either with or without the corresponding inducing metabolite (Fe³⁺ for all but uhpT (Glucose-6-Phosphate), zinT (Zn²⁺), mtgC (Mg²⁺), soxS (unknown), sfbA (unknown). Samples were then grown in a Clariostar at 37°C for 16-18 h whilst recording growth (OD₆oo) and sfGFP fluorescence.

All metal-dependent promoters showed inhibition of sfGFP upon addition of the corresponding metabolite (see Figure 4). Where unknown, no significant variability was observed.

Example 6: Validation of repression of cytosolic promoters in vacuole

Salmonella ZH9 previously transformed with promoter reporter plasmids (see above) was prepared for invasion assays onto SKOV-3 cells using standard protocol. Briefly, single colonies were grown at 37°C for 16-18 h in vLB before being diluted in fresh vLB and grown at 37°C for approximately 5 h, until OD₆oo ~ 1 .5. Cells were then pelleted by gentle centrifugation at 2000xg for 15 min and washed with ice- cold PBS twice. After the last wash, cells were re-suspended in 1 :10 of the initial culture volume of 10% glycerol PBS and aliquoted in 200 pL invasion stocks and stored at -80°C until required. One aliquot was thawed and used to calculate the cell concentration as CFU by serially diluting the stock in PBS, plating those in vLB and growing them at 37°C for 16-18 h. CFU was determined by counting the number of viable colonies in the dilution series and typically ranged between 1O^1o-1O¹² CFU/mL of stock.

On the assay day, circulating SKOV-3 cells were incubated together with previously thawed invasion stocks to an adjusted multiplicity of infection (MOI) of 50 bacterial cells per 1 mammalian cell. Both cell types were incubated together for 1 h, after which non-invaded bacteria were washed away and killed with gentamycin. Samples were visualised 24 h after, showing bacteria in vacuole as red (/.e., only constitutive mScarlet expression) except in the case of yjjZ promoter (See Figure 5).

Example 7: Validation of expression of cytosolic promoter puhpT in cytosol The promoter reporter plasmid containing puhpT (see above) was transformed into Salmonella enterica Typhimurium CD12 strain (Salmonella Typhimurium TML AaroC Asif A), which is defective in SifASPI-2 effector, leading to potential vacuole disruption, and thus escape, during late stages of invasion. Bacterial invasion stocks were prepared as previously described and grown in co-culture with SKOV- 3 cells in MOI 50, as previously done. Bacteria were imaged by microscopy 3 h, 6 h, and 24 h after invasion. At 6 h pi, accounts of escape were identified (see Figure 6) with bacteria showing signs of hyper-replication (growth overtaking the host cell) and displaying green fluorescence. SEQUENCES FORMING PART OF THE DESCRIPTION

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

ATGAACACGATTAACATCGCTAAGAACGACTTCTCTGACATCGAACTGGCTGCTA

TCCCGTTCAACACTCTGGCTGACCATTACGGTGAGCGTTTAGCTCGCGAACAGT

TGGCCCTTGAGCATGAGTCTTACGAGATGGGTGAAGCACGCTTCCGCAAGATG

TTTGAGCGTCAACTTAAAGCTGGTGAGGTTGCGGATAACGCTGCCGCCAAGCC

TCTCATCACTACCCTACTCCCTAAGATGATTGCACGCATCAACGACTGGTTTGAG

GAAGTGAAAGCTAAGCGCGGCAAGCGCCCGACAGCCTTCCAGTTCCTGCAAG

AAATCAAGCCGGAAGCCGTAGCGTACATCACCATTAAGACCACTCTGGCTTGCC

TAACCAGTGCTGACAATACAACCGTTCAGGCTGTAGCAAGCGCAATCGGTCGG

GCCATTGAGGACGAGGCTCGCTTCGGTCGTATCCGTGACCTTGAAGCTAAGCA

CTTCAAGAAAAACGTTGAGGAACAACTCAACAAGCGCGTAGGGCACGTCTACA

AGAAAGCATTTATGCAAGTTGTCGAGGCTGACATGCTCTCTAAGGGTCTACTCG

GTGGCGAGGCGTGGTCCTCGTGGCATAAGGAAGATTCTATTCATGTAGGAGTAC

GCTGCATCGAGATGCTCATTGAGTCAACCGGAATGGTTAGCTTACACCGCCAAA

ATGCTGGCGTAGTAGGTCAAGACTCTGAGACTATCGAACTCGCACCTGAATACG

CTGAGGCTATCGCAACCCGTGCAGGTGCGCTGGCTGGCATCTCTCCGATGTTC

CAACCTTGCGTAGTTCCTCCTAAGCCGTGGACTGGCATTACTGGTGGTGGCTAT

TGGGCTAACGGTCGTCGTCCTCTGGCGCTGGTGCGTACTCACAGTAAGAAAGC

ACTGATGCGCTACGAAGATGTTTACATGCCTGAGGTGTACAAAGCGATTAACATT

GCGCAAAACACCGCATGGAAAATCAACAAGAAAGTCCTAGCGGTCGCCAACGT

AATCACCAAGTGGAAGCATTGTCCGGTCGAGGACATCCCTGCGATTGAGCGTG

AAGAACTCCCGATGAAACCGGAAGATATCGACATGAATCCTGAGGCTCTCACCG

CGTGGAAACGTGCTGCCGCTGCTGTGTACCGCAAGGACAAGGCTCGCAAGTC

TCGCCGTATCAGCCTTGAGTTCATGCTTGAGCAAGCCAATAAGTTTGCTAACCAT

AAGGCCATCTGGTTCCCTTACAACATGGACTGGCGCGGTCGTGTTTACGCTGT

GTCAATGTTCAACCCGCAAGGTAACGATATGACCAAAGGACTGCTTACGCTGGC

GAAAGGTAAACCAATCGGTAAGGAAGGTTACTACTGGCTGAAAATCCACGGTGC

AAACTGTGCGGGTGTCGATAAGGTTCCGTTCCCTGAGCGCATCAAGTTCATTGA

GGAAAACCACGAGAACATCATGGCTTGCGCTAAGTCTCCACTGGAGAACACTT

GGTGGGCTGAGCAAGATTCTCCGTTCTGCTTCCTTGCGTTCTGCTTTGAGTACG

CTGGGGTACAGCACCACGGCCTGAGCTATAACTGCTCCCTTCCGCTGGCGTTT

GACGGGTCTTGCTCTGGCATCCAGCACTTCTCCGCGATGCTCCGAGATGAGGT

AGGTGGTCGCGCGGTTAACTTGCTTCCTAGTGAAACCGTTCAGGACATCTACG

GGATTGTTGCTAAGAAAGTCAACGAGATTCTACAAGCAGACGCAATCAATGGGA

CCGATAAcgaagtagttaccgtgaccgatgagaac SEQ ID NO:2-T7 core (amino acid sequence)

MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFER

QLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVA

YITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKR

VGHVYKKAFMQWEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVS

LHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCWPPKPWTGITGG

GYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVI

TKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISL

EFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIG

KEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPF

CFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPS

ETVQDIYGIVAKKVNEILQADAINGTDNEWTVTDEN

SEQ ID NO:3-T7 sigma (DNA sequence)

AAGAACACTGGTGAAATCTCTGAGAAAGTCAAGCTGGGCACTAAGGCACTGGC

TGGTCAATGGCTGGCTTACGGTGTTACTCGCAGTGTGACTAAGAGTTCAGTCAT

GACGCTGGCTTACGGGTCCAAAGAGTTCGGCTTCCGTCAACAAGTGCTGGAAG

ATACCATTCAGCCAGCTATTGATTCCGGCAAGGGTCTGATGTTCACTCAGCCGA

ATCAGGCTGCTGGATACATGGCTAAGCTGATTTGGGAATCTGTGAGCGTGACGG

TGGTAGCTGCGGTTGAAGCAATGAACTGGCTTAAGTCTGCTGCTAAGCTGCTG

GCTGCTGAGGTCAAAGATAAAAAGACTGGAGAGATTCTTCGCAAGCGTTGCGC

TGTGCATTGGGTAACTCCTGATGGTTTCCCTGTGTGGCAGGAATACAAGAAGCC

TATTCAGACGCGCTTGAACCTGATGTTCCTCGGTCAGTTCCGCTTACAGCCTAC

CATTAACACCAACAAAGATAGCGAGATTGATGCACACAAACAGGAGTCTGGTAT

CGCTCCTAACTTTGTACACAGCCAAGACGGTAGCCACCTTCGTAAGACTGTAGT

GTGGGCACACGAGAAGTACGGAATCGAATCTTTTGCACTGATTCACGACTCCTT

CGGTACGATTCCGGCTGACGCTGCGAACCTGTTCAAAGCAGTGCGCGAAACTA

TGGTTGACACATATGAGTCTTGTGATGTACTGGCTGATTTCTACGACCAGTTCGC

TGACCAGTTGCACGAGTCTCAATTGGACAAAATGCCAGCACTTCCGGCTAAAG

GTAACTTGAACCTCCGTGACATCTTAGAGTCGGACTTCGCGTTCGCGTA

SEQ ID NO:4-T7 sigma (amino acid sequence)

KNTGEISEKVKLGTKALAGQWLAYGVTRSVTKSSVMTLAYGSKEFGFRQQVLEDTI

QPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTWAAVEAMNWLKSAAKLLAAEV

KDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKD

SEIDAHKQESGIAPNFVHSQDGSHLRKTWWAHEKYGIESFALIHDSFGTIPADAAN

LFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESD

FAFA SEQ ID NO:5-T3 sigma (DNA sequence)

AAGAACACTGGTGAAATCTCTGAGAAAGTCAAGCTGGGCACTAAGGCACTGGC

TGGTCAATGGCTGGCTTACGGTGTTACTCGCAGTGTGACTAAGCGTTCAGTCAT

GACGCTGGCTTACGGGTCCAAAGAGTTCGGCTTCCGTCAACAAGTGCTGGAAG

ATACCATTCAGCCAGCTATTGATTCCGGCAAGGGTCTGATGTTCACTCAGCCGA

ATCAGGCTGCTGGATACATGGCTAAGCTGATTTGGGAATCTGTGAGCGTGACGG

TGGTAGCTGCGGTTGAAGCAATGAACTGGCTTAAGTCTGCTGCTAAGCTGCTG

GCTGCTGAGGTCAAAGATAAAAAGACTGGAGAGATTCTTCGCAAGCGTTGCGC

TGTGCATTGGGTAACTCCTGATGGTTTCCCTGTGTGGCAGGAATACAAGAAGCC

TATTCAGAAGCGCCTGGACATGATTTTCTTGGGTCAATTTCGCTTGCAACCTACC

ATTAACACCAACAAAGATAGCGAGATTGATGCACACAAACAGGAGTCTGGTATC

GCTCCTAACTTTGTACACAGCCAAGACGGTAGCCACCTTCGTAAGACTGTAGTG

TGGGCACACGAGAAGTACGGAATCGAATCTTTTGCACTGATTCACGACTCCTTC

GGTACGATTCCGGCTGACGCTGCGAACCTGTTCAAAGCAGTGCGCGAAACTAT

GGTTGACACATATGAGTCTTGTGATGTACTGGCTGATTTCTACGACCAGTTCGCT

GACCAGTTGCACGAGTCTCAATTGGACAAAATGCCAGCACTTCCGGCTAAAGG

TAACTTGAACCTCCGTGACATCTTAGAGTCGGACTTCGCGTTCGCGTA

SEQ ID NO:6-T3 sigma (amino acid sequence)

KNTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTI

QPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTWAAVEAMNWLKSAAKLLAAEV

KDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQKRLDMIFLGQFRLQPTINTNKD

SEIDAHKQESGIAPNFVHSQDGSHLRKTWWAHEKYGIESFALIHDSFGTIPADAAN

LFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESD

FAFA

SEQ ID NO:7-K1F sigma (DNA sequence)

AAGAACACTGGTGAAATCTCTGAGAAAGTCAAGCTGGGCACTAAGGCACTGGC

TGGTCAATGGCTGGCTTACGGTGTTACTCGCAGTGTGACTAAGCGTTCAGTCAT

GACGCTGGCTTACGGGTCCAAAGAGTTCGGCTTCCGTCAACAAGTGCTGGAAG

ATACCATTCAGCCAGCTATTGATTCCGGCAAGGGTCTGATGTTCACTCAGCCGA

ATCAGGCTGCTGGATACATGGCTAAGCTGATTTGGGAATCTGTGAGCGTGACGG

TGGTAGCTGCGGTTGAAGCAATGAACTGGCTTAAGTCTGCTGCTAAGCTGCTG

GCTGCTGAGGTCAAAGATAAAAAGACTGGAGAGATTCTTCGCAAGCGTTGCGC

TGTGCATTGGGTAACTCCTGATGGTTTCCCTGTGTGGCAGGAATACAAGAAGCC

TATTCAGACGCGCTTGAACCTGAGGTTCCTCGGTTCGTTCAACCTCCAGCCGA

CCGTCAACACCAACAAAGATAGCGAGATTGATGCACACAAACAGGAGTCTGGTA

TCGCTCCTAACTTTGTACACAGCCAAGACGGTAGCCACCTTCGTAAGACTGTAG

TGTGGGCACACGAGAAGTACGGAATCGAATCTTTTGCACTGATTCACGACTCCT TCGGTACGATTCCGGCTGACGCTGCGAACCTGTTCAAAGCAGTGCGCGAAACT

ATGGTTGACACATATGAGTCTTGTGATGTACTGGCTGATTTCTACGACCAGTTCG

CTGACCAGTTGCACGAGTCTCAATTGGACAAAATGCCAGCACTTCCGGCTAAAG

GTAACTTGAACCTCCGTGACATCTTAGAGTCGGACTTCGCGTTCGCGTA

SEQ ID NO:8-K1 F sigma (amino acid sequence)

KNTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTI

QPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTWAAVEAMNWLKSAAKLLAAEV

KDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLRFLGSFNLQPTVNTNK

DSEIDAHKQESGIAPNFVHSQDGSHLRKTWWAHEKYGIESFALIHDSFGTIPADAA

NLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILES

DFAFA

SEQ ID NO:9-CGG sigma (DNA sequence)

AAGAACACTGGTGAAATCTCTGAGAAAGTCAAGCTGGGCACTAAGGCACTGGC

TGGTCAATGGCTGGCTTACGGTGTTACTCGCAGTGTGACTAAGCGTTCAGTCAT

GACGCTGGCTTACGGGTCCAAAGAGTTCGGCTTCCGTCAACAAGTGCTGGAAG

ATACCATTCAGCCAGCTATTGATTCCGGCAAGGGTCTGATGTTCACTCAGCCGA

ATCAGGCTGCTGGATACATGGCTAAGCTGATTTGGGAATCTGTGAGCGTGACGG

TGGTAGCTGCGGTTGAAGCAATGAACTGGCTTAAGTCTGCTGCTAAGCTGCTG

GCTGCTGAGGTCAAAGATAAGAAAACTGGAGAGATTCTTCGCAAGCGTTGCGC

TGTGCATTGGGTAACTCCTGATGGTTTCCCTGTGTGGCAGGAATACAAGAAGCC

TATTAAAACGCGCGTGCATATTATGTTCCTCGGTCAGTTCGAAATGCAGCCTACC

ATTAACACCAACAAAGATAGCGAGATTGATGCACGCAAACAGGAGTCTGGTATC

GCTCCTAACTTTGTACACAGCCAAGACGGTAGCCACCTTCGTAAGACTGTAGTG

TGGGCACACGAGAAGTACGGAATCGAATCTTTTGCACTGATTCACGACTCCTTC

GGTACGATTCCGGCTGACGCTGCGAACCTGTTCAAAGCAGTGCGCGAAACTAT

GGTTGACACATATGAGTCTTGTGATGTACTGGCTGATTTCTACGACCAGTTCGCT

GACCAGTTGCACGAGTCTCAATTGGACAAAATGCCAGCACTTCCGGCTAAAGG

TAACTTGAACCTCCGTGACATCTTAGAGTCGGACTTCGCGTTCGCGTA

SEQ ID NO:10-CGG sigma (amino acid sequence)

KNTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTI

QPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTWAAVEAMNWLKSAAKLLAAEV

KDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIKTRVHIMFLGQFEMQPTINTNKD

SEIDARKQESGIAPNFVHSQDGSHLRKTWWAHEKYGIESFALIHDSFGTIPADAAN

LFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESD

FAFA 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)

GGAGGTTCAGGTGGTGGATCCAACGAAAAAGAAGAACTGAAATCCAAAAAAGC

GGAACTGCGCAACCGTATCGAACAGCTGAAACAGAAACGTGAACAACTGAAGC

AGAAAATCGCGAACCTGCGTAAAGAAATCGAAGCTTACAAATAAT

SEQ ID NO:16-SYNZIP 18 (amino acid sequence, C-terminus)

GGSGGGSNEKEELKSKKAELRNRIEQLKQKREQLKQKIANLRKEIEAYK

SEQ ID NO:17-SYNZIP 17 (DNA sequence)

ATGAGCATCGCGGCGACCCTGGAGAACGATCTGGCGCGTCTGGAAAACGAAAA

CGCTCGTCTCGAAAAAGACATCGCGAACCTGGAACGTGACCTGGCGAAACTGG

AGCGTGAAGAAGCGTACTTC

SEQ ID NO:18-SYNZIP 17 (amino acid sequence, N- terminus)

MSIAATLENDLARLENENARLEKDIANLERDLAKLEREEAYF

SEQ ID NO:19-proA (DNA sequence)

TTTACGGGCATGCATAAGGCTCGTAGGCTATATTC

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

TACTAGAGTCATTTATGAAAGTACTAG

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

TACTAGAGTCAGCCAAGAAAGTACTAG SEQ ID NO:22-PzinT (DNA sequence)

AAGCGAGTAGTCACAAAAATTATGCCGCCTGTGCGCGGATATCTGCAAAGCCTG

TGCCGAAGAGTGTGCAAGGCACGATCACGACCATTGCCAGAATTGCGCGCGG

GCATGCAGCCAATGCGCAGACGCCTGCCTTAAAATGGCCGCGTAATTTTTCTTC

CGCCATTAGCTCAACCGGATAGAGCATAGAGCTTCTACCTCTAAGGTTCGGGGT

TCAATTCCTCGATGGCGGACCAGTTGATATCAAAAAAGGCCACCTGCGCGGTG

GCCGCTGAGTTTCTGTTGAAATAAATGCAATGTTATAATATAACAATCATCTTTCTA

AGAAAGATGAGGGTAACGTTTTGGTGATTCATTTAAAAAAACTGACAATGCTTCT

GGGAATGCTGTTGGTAAATAGT

SEQ ID NO:23-PuhpT (DNA sequence)

GCACTGGACCGGTTTTTTTGCGGTCATCGCCATCGCGGCGGGGATCTCCGCGC

TATTGCTGTTGCCATTTCTGAACGCTCAGGCCCCACGCGAAACCCACGAAGCG

TGATACACCTCACCTTTTTGCGCTGAATGGGGCAAAACTAAGAAATTTTCCCGGT

TTTGCCTGGACGCTGTCGCAGGCCACTTTTCCTGTGGATTTTTACAATGCCTGC

CATTCGCAGGTATAAAAATTAGCTCAGGAGTAATCC

SEQ ID NO:24-PfhuA (DNA sequence)

TACGCTGTGCCAGCAGGGCGAGATGATGCAGCAGCAACAGCAGCCGTCAGGC

AATCCGTTCGATCAGTCGTCTCAGCCGCAGCAGCCTGCGCAGCAACAGCCGCC

GAAAGAAGAGAAGAGCGACGGCGTTGCCGGCTGGATTAAGGAGATGTTTGGC

GGCAATTAATCACGGTAATAGTGCCGGGTGGCGCTGTGCTTACTCGGTCCACAC

CGTTACGACCCCATTATGTGCGACGTAGGCCGAATAAGACGCTTACATCGCCAT

CCGGCAAATCCTCCATAAATAACATTTCAGTCTAATTTATTAACCCTTCCTTTTCAT

CTGGTTGTTTCTTAACCCCTTAGTTTTCGTAGGGCCGCGTATCGCTTGCCATTGC

GACGATATTTCGCCTATCATGCTGCGGTTATAATAATAATTATCGTTTACGTTATCA

TTCACTTTCATCAGAGATATACCAATGGCGCGTCTTAAAATTGCTCAGCCAAACT

CCTCACTGCGTAAAATCGCAGTTGTAGTAGCCAC

SEQ ID NO:25-PentC (DNA sequence)

TTTGCCGGGGCCAACCGGCGTCCTGGGCTAAGGATATTCCTGAAATTGATAAAC

CAACCACTAAAAGCAGCCAACGATAAAAGGCGGAGAGTCTCACAATAGCGTCCT

GTTATTAATAAAGTTAATGCTTCTCATTTTCATGTCAGCGGCAGCGAGATGCAAG

CCTTAGTGCCATTTAACTCATGACCAGAGTTGACAGAGCGACGTTTTACTCTTAG

GTTAGCGCACTAAAAATAGAAATAATAATCATTATTATACACAAAATCATTCAAGAA

GCATCGCGACGGCAAGGGAAGAATCCCCGCGGGCATAGATAACTGTGTGACCG

GGGTTTCTGATCGCAGCCAACAAAGAGGCAGCTTGAAAGATGAAGTGTATATAA

GCCTTTATCATTGGAGGATGATATGGATATGTCACTGGCCGAGGACGCTCAGGA

GACAATGGCAACGCTTGCT SEQ ID NO:26-PfhuE (DNA sequence)

CATTGACGGTTGGGATCAGGATATTGGGAATAATCAAGATATGCGTCGGCGCCT

GCGGTGAAATATCACGAAAGGCGGTGACCAGCTCATCCTGATAAACGATATCCG

AAGGGATTTCACGACGAATAATTTTGCTGAATATAGTTTCTTCTGCCACGACGTT

TTCCTTTTTCATAATAGCCCTTGCAGCGCACATGCTACGCCGAGCCATACTGCGA

GTATAGAGTATGAGCGAGTTACCGACGCTCTTTCAACTTTAACCCACGATTTATTA

AGCGAAAAATGACTACATGCTGGACTTACCGCCATATCCCCCTGCCATGACGCC

TTTGTACGCTTAAAAAATATTTCTAGTTTCCCTGGTTATACCGTTTTACACATTTAA

TACAAATGCGTATATTTCTCATTTGCATTTTTATACGCATTAACTAGCAAAGAATGA

AAAGGTTCAACGTCATACGTCCTGAACTTACCCCAATAACAAGCAAGGATTTTCA

GATGTCTTTCATTCAATA

SEQ ID NO:27-PsfbA (DNA sequence)

GGATGTTGGCGTTAAGACGTCTTATCCGGCATTTCCCTTTACGCCATTCCCGCC

TGACACACGCCTGAGCGCCTGCCTCGTACATTTAACGACACACCAGGAAACATC

ATGAAATAATTTCAAGGACAGCAGGCTGTGATCTGTGTCATGTTAAGAAATAGCC

TTTCGTTTGGGCCAAAACAGACGATGCCGCATGAACGGCATCCGGCACAGCAT

CACACTATTTAAAATGGAGAAATTATGGGATTGCGTCAGAGTTTACGCATT

SEQ ID NO:28-PyjjZ (DNA sequence)

GCCGACACGGCGTTGAGAAACAAGAAAAGACGTAAAGAAAACTGATACTTCTTA

ATACGAAGCGACCGCCAGGATGGGGTTGTCATGGGTAATTGTCGTTATTTATCG

GTGATATACACGGAATCGGGCGCCAACATGAAAATAACGTATGAGAAAAGGTCG

CCTAAAGCGAGGTGTTGTTGTTTTTACGTTAACAGTCGGACAATTTATCACCTTA

CTGAATACGTGTCATCAACCGTTAAGTAAAACTCATCTCTTTAGCTTTCTCCCTG

GCTGACAAATGAGAAAATATATCATATGATATTGGTTATCATTATCAATTCCAGAGG

TGAAACCATGTTGCAGCGGACGTTAGGCAGCGG

SEQ ID N0:29-Pstnc3080 (DNA sequence)

CGCCCGCCATCACCACCGGTAATTGACGAAACCCTTGCGCCCGGAGCGTATCC

GCTGCATCCGGCACCAGATCGACGTTCACCATCTCAAATTCAAATCCACGGCTT

TCCATCGCCCGCTTTGTGGCGTGGCACTGAACACAGTTATTGCGAGTGTAAATA

GTAATGCTCATGATTCGTATTTCCATTTAAAATGAGAAA

SEQ ID NO:30-PsitA (DNA sequence)

CGGCTGATAGCAGTGCACCGGGCACTGCTTACAGGCGGGTTTTTCCTCGCCAA

ATACGCATTTATCAAGACGTTTTTGCGCGTAGGCGAACAGCGCGTCGTAATGCC

CCTGCACCGCTGACGCCTGTGGACACTGGCTTTCATACAGCGCGATCATTTTTT TGATCGTCAGTTTTTCACGAGCGATACGTTTACCAGGCATCGTGCTCTCTCCGA

ACATTAAGATGCATTTATTTTACACCTTATCCCTCTTTAGCACTATCACTGCATATC

GTCGCCATTACGCAAATAAGAATTATTTTCATTTATTCATGCCTTGTGCTATATAAC

ATAGCAAAGGCTATATTCGATGATTAATTAACCACATTGTTGCGAGGGATACTATG

ACGAATCTACATCGTCTAAAAACACTCCTGATTGCCGGTATTGTCGCGATACT

SEQ ID NO:31-PmntH (DNA sequence)

>

TTTGCTCCAAATATGAGGCAGGTTTAATTTTCGTGCACATTCTATGCAACAGCTG

TAAAGAAAACGAGATCCAACACACACTATAATAAGGACCTGTGACGAGATTCAAA

ATTAGTGATCTGTAATACACTTTTACTGTACTGAATATGAAAATGAAAAGTTATATC

AGTGTGCTAATCTTGTAATGTTAAGCCAAACTGTTCTGATACAGGTCGCCATCGT

ATCGGTCTATCGTTTCACACTATCAAAGTAATCACCCGTACCCATTGAAATGCACT

TGATAATCATTATCAATGAACATAGCATGAAACATAGCAAAGGCTATGTTTTTGAG

GCAAAAGATGACTGACAATCGCGTAGAGAATAG

SEQ ID NO:32-PfepA (DNA sequence)

AATAAAACAGTAGCTGCCGCGCCAGTTAGCGCTAAGCGCCGTGCTCCAGCGCC

AGACATCCGTTCCGGCAATACGCGCCATCGTCTGAGGCTGCGCGTTTTGATGAT

GATCGGTTACGCCAGTGATGTAGACCCAGACACGCCGTATCGGGGAGTGTGTT

TCGTTTCCCTGCGGGTCGCGCCACCAAAAAGTGACCCGATAATTTCCGTCTTTT

TCCCGTATCCATTCCGGCCCGGTTTTCGTCCTCCACCAGGCCTCACTTCCCGTT

GCCAGCGCCTCTTTCATTATAACCCTGTGTTTATTATGAATTTTGTATATAAAAGGT

GAAATATATTGATAATATTATTGATAACTATTTGCATTTGCAATAGCGTATTGTAGCG

CTATGGGACGCGCGAACACAATTTCACCACCCGGCCAATGCCTTTGACGGGCG

CTTTGGCTTATGTGGCTAAAGAAAAGCAGGATATACAATGAACAAGAAGATTCAT TC

SEQ ID NO:33-PsufA (DNA sequence)

CTCATTCAGCACCTGAAATGCCGGAAAGAGTTTACATAAACCTATAGCTCAAACT

GAGTTATAGAACCGCAGCGGATTATAAAGAGCGCAACGCCAGGTATCCATACAA

AAAATGGGGTTCTGACCTCGCCGCCCGGCAATGTCGACAGCCTATTAATTAAAT

AGTCATTTTCTATACATCTTTTCGTTTTTGACCTGCCAGAACGGTTAATGTCTTATA

AATCATTACTTATCAAAAAGTTAAGTGGTTTTTTGTCTGTCGTATGACCTGGCGGA

CAGGGTCTATGCTTAATAAAAGGCGCTCAATATGACCATTTGTTGGAAAGCCCCT

GCGGTTAAGGGGTTGAAGTGATAATCATTATCACTAACATGCTGTTATATCCTGGT

GATTTAGAACGCGAGGTAACTCTATGGAATTGCATTCAGGCACGTTTAACCCGG

AGGACTTC SEQ ID NO:34-PsoxS (DNA sequence)

TTTCGCAGCGGACAGTCGCTACGCGATAAACAGCCGCAGCCGATACAACCGTC

CAGCTCATCGCGCAACGCCACCAGCGTATGAATACGTCGGTCTAACTCTTCGCG

CCACTGCGAGGAGAGCTGCTTCCACTCTTTCGCGCTTAACGTATGCCCTTCCG

GCAAGATACCAAACGCGTCGCCGATAGTTGCCAGCGGGATGCCGATACGCTGG

GCAATCTTGATAATCGCGACATAACGCAACACGTCACGCTTGTATCGCCGTTGG

TTACCGCTATTACGGATACTGGTAATTAGCCCTTTGCTTTCATAGAAGTGCAGGG

CGGACACAGCAACACCGCTACGTTTCGCAACTTCCCCCGGCGTCAGTAAGGCT

TTTAAACGGGGAGATTTTTTTTCCATAAATCGCTTTACCTCAAGTTAACTTGAGGA

ATTATACTCGCCCGCAGACAAAACGACGAATCGAATACTGTTTAAGAGGCAACAA

TATGTCGCATCAGCAGATAATTCAGACCCTTAT

SEQ ID NO:35-PiroN (DNA sequence)

AAGCGCCTGATAAATATTACCAGGCGCTTTGTATGTTGGTGCCAACATCACTTTC

ATCATCAAATATCGAATGGCTACAATCGTATCCGATCCCGCCATTACCCAGACGG

AAAGTCGCTGGCAAACTGTAAGAATGGTTCGCCGTCGGCAGGGAAGCGGCGG

TGAACCCTGAACCGTGCTATACCATCTTACCTGGGTGTTTCTTGTGATTAACGAT

CTGAAAAATAGTTTTATTTTATCTATTTCTGTTTTGTAAAACCTCCGTTCAGTAGGC

GCATTCTGCCCCCCTTCCCGGATTTACTGGCAAAGCGGAGCCCGGACAGAGAG

TCATATTGCAAAATCCCGTTTCCGTTTTTTTATTACCAGATTTTTGTGGTCGAAAG

ATTGCCTTTTCCTTAATTGAATGATAATTATTATCATTAGCATATGATAATAATTACTA

TATAGACGTAACCTGGCAAGGATGTGAGCTTGAGGGCAACAGCGCTACTTTAGA

CATTATTTAGGGAATGGGTATGAGAGTTAAGAAGTTC

SEQ ID NO:36-PfepB (DNA sequence)

CAAACTGCTGGCGCAATTTCTGCTGGAAAGGGCTATCCGGCGAATCTCCGGCA

ACCGCAGGCTCGGTATAACGGGCGAAGCACCCGGAAGTGGTAAAACTGCGATA

CGGCGACATGAAGAAAAAGCGATCGGGAGCAAGCGTTGCCATTGTCTCCTGAG

CGTCCTCGGCCAGTGACATATCCATATCATCCTCCAATGATAAAGGCTTATATACA

CTTCATCTTTCAAGCTGCCTCTTTGTTGGCTGCGATCAGAAACCCCGGTCACAC

AGTTATCTATGCCCGCGGGGATTCTTCCCTTGCCGTCGCGATGCTTCTTGAATG

ATTTTGTGTATAATAATGATTATTATTTCTATTTTTAGTGCGCTAACCTAAGAGTAAA

ACGTCGCTCTGTCAACTCTGGTCATGAGTTAAATGGCACTAAGGCTTGCATCTC

GCTGCCGCTGACATGAAAATGAGAAGCATTAACTTTATTAATAACAGGACGCTAT

TGTGAGACT

SEQ ID NO:37-LysE (amino acid sequence)

MVRWTLWDTLAFLLLLSLLLPSLLIMFIPSTFKRPVSSWKALNLRKTLLMASSVRLKP

LNCSRLPCVYAQETLTFLLTQKKTCVKNYVRKE SEQ ID NO:38-LysE (DNA sequence)

ATGGTACGCTGGACTTTGTGGGATACCCTCGCTTTCCTGCTCCTGTTGAGTTTAT

TGCTGCCGTCATTGCTTATTATGTTCATCCCGTCAACATTCAAACGGCCTGTCTC

ATCATGGAAGGCGCTGAATTTACGGAAAACATTATTAATGGCGTCGAGCGTCCG

GTTAAAGCCGCTGAATTGTTCGCGTTTACCTTGCGTGTACGCGCAGGAAACACT

GACGTTCTTACTGACGCAGAAGAAAACGTGCGTCAAAAATTACGTGCGGAAGG

AG

SEQ ID NO:39-kilR (amino acid sequence)

MIAHHFGTDEIPRQCVTPGDYVLHEGRTYIASANNIKKRKLYIRNLTTKTFITDRMIKV

F

SEQ ID NO:40-kilR (DNA sequence)

ATGATAGCACATCATTTCGGCACCGATGAAATCCCCCGCCAGTGCGTTACCCCT

GGTGATTACGTTCTGCATGAAGGTCGCACGTATATCGCGTCCGCCAACAACATC

AAAAAACGTAAACTTTACATCCGTAATCTTACAACAAAGACGTTCATCACTGATCG

TATGATTAAAGTATTT

SEQ ID NO:41-Kil (amino acid sequence)

MRKRFFVGIFAINLLVGCQANYIRDVQGGTIAPSSSSKLTGIAVQ*

SEQ ID NO:42-Kil (DNA sequence)

>

ATGCGCAAAAGGTTCTTCGTAGGTATTTTCGCCATCAACCTGTTGGTGGGATGT

CAAGCAAATTATATCCGTGACGTACAAGGTGGCACAATCGCGCCATCGAGTTCG

AGTAAATTAACCGGAATCGCTGTTCAATAG

SEQ ID NO:43-BRP (amino acid sequence)

MKATKLVLGAVILGSTLLAGCQANYIRDVQGGTVAPSSSSELTGIAVN

SEQ ID NO:44-BRP (DNA sequence)

ATGAAAGCGACCAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCT

GGCAGGCTGCCAGGCAAACTATATCCGGGATGTTCAGGGTGGAACGGTGGCAC

CATCGTCCTCCTCTGAACTGACGGGGATCGCGGTTAAC

SEQ ID NO: 45- (STN3250) (DNA sequence)

CAGAAGTCATAGGTATTGGAAGCGCCGCGACTGCTTACAGTTACGCCCGGCGT

GTAACCCAACGCTTCTTTTACTGACTGGAATTGATGCATCTGCATCTCTTCGTTA

GTGACCACCGAAACCGACTGTGGCGTTTTTTCGATAGATGTATCAGTTTTGGTG GTAGTGGCGGAACGCTTCGCGGCGATGGTCGGAGCCGGTCCCCAGGCACTTT

CCTGCGGCGCAGGCGCTGCGGTTACGGTAATGGTTTCTTCTTTCGGTTGAACC

GCCGCCTGTGCATAGACAGACATGCCGCTAACCGCTGTGGCTACTACAACTGC

GATTTTACGCAGTGAGGAGTTTGGCTGAGCAATTTTAAGACGCGCCATTGGTATA

TCTCTGATGAAAGTGAATGATAACGTAAACGATAATTATTATTATAACCGCAGCAT

GATAGGCACAGCACAACGG

SEQ ID NO: 46- (mtgC) (DNA sequence)

CGTTTAGCATCCCTTTTCTGGTGGAACCCATTTTTTCCTCGTCATGTTGTTTTATT

TTTTTACGTGCAGGCATCATAACAGAGCTATCGCCGGCATTAAGCAGGAATTTAT

TGTTTAATGATTTCAGACGAGCCTGTTATTGACATAATATTGTCATTTTTTTGTCAC

GGGAAATATCAAACAAACTTAAACAAATCGTCACTATCCCCGCCTTTGCACTTTA

CAGAACATATTGACTGACTATAATAAGCGCAAATTCATGCAGGAGTAATATGTTGG

ACAGTCACTTTTACGTAAATCATCTGGCAAGTTAACGCACGCTATTCCTGCGCTG

CTTGCCGAACCGGTGGGCAGCAATCTCCCCTTGTGACGATTGTCATCCCAATAA

TGTTACAACACGCGCATTGTCGCGAGGTAATCGTCATGTTCATGTTTAAACACGC

TTTATTTCCTCCGCCGTTAACACGACGCTAATTGCCTCAGGGCAGAAATTTGTCG

TGTGCTAAATATAGCACGTACTTATTCTTCCAGAAAAAATGGAGGAACGTATGTTA

ATGTTTCCTTATATTTTAAATTTACTGGCCGCTATGCT

SEQ ID NO: 47- (sseJ) (DNA sequence)

CACATAAAACACTAGCACTTTAGCAATAATAGTCGGATGATAAGTTTGTCTGTTTT

TCCTGAGTATCAAGCCAGCTCATACTCACGCCAGCACACTAAAATCAGGAGTGG

CTTCTTTTTTAGATCTTTGCCTTAGCCAGGCGCACACTCAATAATGATAGCAGTC

AGATAATATGTACCAGGCATTAACCTCACGTTGTTGATGATATATTTACTTCGTTGA

AAAACAATAAACATTGTATGTATTTTATTGGCGACGAAAAACTGTTAAAGAAGCGT

AATTCCATATACACCATTTACCTGATTACTTTTCTTGCTAATATTTGCTAATTAATTAT

TTGCTAAAGCGTGTTTAATAAAGTAAGGAGGAGGCACAGCACAACGG

SEQ ID NO: 48- (ssaG) (DNA sequence)

TATTGCCATCGCGGATGTCGCCTGTCTTATCTACCATCATAAACATCATTTGCCTA

TGGCTCACGACAGTATAGGCAATGCCGTTTTTTATATTGCTAATTGTTTCGCCAAT

CAACGCAAAAGTATGGCGATTGCTAAAGCCGTCTCCCTGGGCGGTAGATTAGCC

TTAACCGCGACGGTAATGACTCATTCATACTGGAGTGGTAGTTTGGGACTACAG

CCTCATTTATTAGAGCGTCTTAATGATATTACCTATGGACTAATGAGTTTTACTCGC

TTCGGTATGGATGGGATGGCAATGACCGGTATGCAGGTCAGCAGCCCATTATAT

CGTTTGCTGGCTCAGGTAACGCCAGAACAACGTGCGCCGGAGTAATCGTTTTC

AGGTATATACCGGATGTTCATTGCTTTCTAAATTTTGCTATGTTGCCAGTATCCTTA

CGATGTATTTATTTTAAGGAAAAGC SEQ ID NO: 49- (sseA) (DNA sequence)

CAGAGGCGTATTCTTGATTTTCATCGGTGGAATGGGTGTCCTGTTAAGTATTAGT

GGTCAGCCTGAAACGGTAAATGACTTACCTTTGCGGGTTAAGTTTTTATTAGACA

AAAGCAATATTCATTATGTGCGGGCGCAATGGAAAGAAGATGGAAGCCTGCAGT

TGTCCGGTTATTGCTCGTCAAGCGAACAGATGCAAAAGGTGAGAGCGACTCTC

GAATCATGGGGGGTCATGTATCGGGATGGTGTAATCTGTGATGACTTATTGATAC

GAGAAGTGCAGGATGTTTTGATAAAAATGGGTTACCCGCATGCTGAAGTATCCA

GCGAAGGGCCGGGGAGCGTGTTAATTCATGATGATATACAAATGGATCAGCAAT

GGCGTAAGGTTCAACCATTACTTGCAGATATTCCCGGGTTATTGCACTGGCAGAT

TAGTCATTCTCATCAGTCTCAGGGGGATGATATTATTTCTGCGATAATAGAGAACG

GTTTAGTGGGGCTTGTCAATGTTACGCCAATGCGGCGCTCTTTTGTTATCAGTG

GTGTACTGGATGAATCTCATCAACGCATTTTGCAAGAAACGTTAGCAGCATTAAA

GAAAAAGGACCCCGCTCTTTCTTTAATTTATCAGGATATTGCGCCTTCTCATGAT

GAAAGCAAGTATCTGCCTGCGCCAGTGGCTGGCTTTGTACAGAGTCGCCATGG

TAATTACTTATTACTGACGAATAAAGAGCGTTTACGTGTAGGGGCATTGTTACCCA

ATGGGGGAGAAATTGTCCATCTGAGTGCCGATGTGGTAACGATTAAACATAATGA

TACTTTGATTAACTATCCATTAGATTTTAAGTGAGTGGAAAATGACAACTCTGACC

CGGTTAGAAGATTTGCTGCTTCATTCGCGTGAAGAGGCCAAAGGCATAATTTTAC

AATTAAGGGCTGCCCGGAAACAGTTAGAAGAGAACAACGGTAGGTTACAGGATC

CGCAGCAATATCAGCAAAACACCTTATTGCTTGAAGCGA

SEQ ID NO: 50- (sifA) (DNA sequence)

ATAAGCGATTAATTGCGCAACGCTAACAAATCCACACGCATCCAGGCATGAAGT

TTATTCAAGGGTAAACTTCATGCCTTCGGCATAAAAAACGCATGAAAGAAGTTG

CCGCCAGTATTGCAAATCTACAACATCATCCGCGGTAGTCCTTCTTTTATTTTTA

CCTGTAGCGACGCTATCACAGACAGTAATGCGTTTATACGCGAAGCTCTCAGG

TTTTATACTGATTGCCAGTCTCTTTTAAAAATTATATTACATCCGATGCGCCCGC

AGTTGAGATAAAAAGGGTCGATTTAATCAATTATGTAGTCATTTTTACTCCAGTA

TAAGTGAGATTAAG

Claims

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

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

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

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

6. The live attenuated Gram-negative bacterium of any preceding claim, wherein a sigma factor of the RNA polymerase binds to the promoter operably linked to the heterologous polynucleotide.

7. The live attenuated Gram-negative bacterium of claim 6, wherein the sigma factor of the RNA polymerase is a CGG sigma factor RNA polymerase, a T3 sigma factor RNA polymerase or a K1 F sigma factor RNA polymerase.

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

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

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

11. The live attenuated Gram-negative bacterium of any preceding claim, wherein the RNA molecule is an mRNA molecule, a siRNA molecule or a shRNA molecule.

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

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

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

15. The live attenuated Gram-negative bacterium of any preceding claim, wherein the live attenuated Gram-negative bacterium is a Salmonella spp.

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

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

18. The live attenuated Gram-negative bacterium of any preceding claim, wherein the live attenuated Gram-negative bacterium is selected from the group comprising Ty21a, CVD 908-htrA, CVD 909, Ty800, M01ZH09, ZH9PA, x9633, x639, X9640, x8444, DTY88, MD58, WT05, ZH26, SL7838, SL7207, VNP20009, A1-R, or any combinations thereof, preferably wherein the live attenuated Gramnegative bacterium is M01ZH09.

19. The live attenuated Gram-negative bacterium of any preceding claim, wherein the RNA molecule is mammalian.

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

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

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

23. The live attenuated Gram-negative bacterium of any preceding claim, wherein the live attenuated Gram-negative bacterium is intratumourally administered, intravenously administered, intraperitoneally administered, or orally administered, preferably wherein the Gram-negative bacterium is intratumourally administered.

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

25. The live attenuated Gram-negative bacterium of any preceding claim for therapeutic use.

26. The live attenuated Gram-negative bacterium for use of claim 23, wherein the live attenuated Gram-negative bacterium is for use in the treatment, reduction, 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 disorder, preferably the live attenuated Gram-negative bacterium is for use in the treatment, reduction, inhibition, prevention of recurrence, or control of a neoplastic disease or an infectious disease.

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

28. The live attenuated Gram-negative bacterium for use of claim 25, wherein the solid cancer and/or the haematological malignancy is a cancer selected from prostate cancer, oesophageal cancer, liver cancer, renal cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, brain cancer, mesothelioma, hepatocellular cancer, lymphoma, leukaemia, gastric cancer, prostate cancer, endometrial cancer, endometrial cancer, vulvar/vaginal cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer or sarcoma, preferably wherein the neoplastic disease is associated with a cancer selected from bladder cancer, lung cancer, mesothelioma, hepatocellular cancer, melanoma, oesophageal 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, wherein the Gram-negative bacterium comprises i) a heterologous polynucleotide encoding a functional mRNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter and the mRNA molecule encodes a therapeutic protein and/or therapeutic peptide; and ii) a polynucleotide encoding an RNA polymerase which is orthogonal to components required for endogenous gene expression in the Gram-negative bacterium, wherein the RNA polymerase binds to the promoter, and wherein upon invasion of the Gram-negative bacteria to a eukaryotic cell, the mRNA molecule is transcribed and is capable of being transferred to the cytoplasm of the eukaryotic cell.

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

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

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

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

34. A method of treating, preventing, inhibiting, preventing recurrence or controlling a disease in a subject, wherein the method comprises administering to the subject a live attenuated Gram-negative bacterium comprising i) a heterologous polynucleotide encoding an RNA molecule, wherein the heterologous polynucleotide is operably linked to a promoter; and ii) a polynucleotide encoding an RNA polymerase which is orthogonal to components required for endogenous gene expression in the Gram-negative bacterium, wherein the RNA polymerase binds to the promoter, and wherein upon invasion of the Gram-negative bacteria to a eukaryotic cell, the RNA molecule is transcribed and is capable of being transferred to the cytoplasm of the eukaryotic cell.

35. The method of treating, preventing, inhibiting, preventing recurrence or controlling a disease in a subject of claims 33 or 34, wherein the method comprises the live attenuated Gram-negative bacterium for use of any of claims 1 to 24.

36. The method of treating, preventing, inhibiting, preventing recurrence or controlling a disease in a subject of any of claims 33-35, wherein the disease is a neoplastic disease or an infectious disease.

37. A method for delivering an RNA molecule into a eukaryotic cell, said method comprising 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; ii) contacting the Gram-negative bacterium with the eukaryotic cell such that the Gram-negative bacterium replicates within said eukaryotic cell, such that the heterologous polynucleotide is transcribed and subsequently transferred from the Gram-negative bacterium to the cytoplasm of the eukaryotic cell.

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