A cross-kingdom platform for therapeutic nucleic acid delivery

Abstract

A cross-kingdom platform for delivering therapeutic nucleic acids to epithelial tissues in which the nucleic acids are engineered to have enhanced stability. [Solution] This platform offers numerous improvements over previous delivery platforms, including expression of the double-stranded RNA binding domain (dsRBD) domain of TAR RNA binding protein (TRBP), knockout of RNase R activity in bacterial delivery vehicles, and expression of a methyltransferase gene, HEN1, for co-packaging with therapeutic nucleic acid delivery vehicles.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 480,875, filed on April 3, 2017.

Background Art

[0002] The present invention relates to the treatment and prevention of diseases. More specifically, the present invention relates to an improved invasive cross - kingdom platform for delivering therapeutic nucleic acids to epithelial tissues, wherein the cross - kingdom platform is engineered to produce nucleic acids with enhanced stability.

[0003] The therapeutic applications of nucleic acids (NA) have strong potential for a wide range of clinical targets. However, the delivery of NA to specific cells and tissues, and their transient targeting effects remain a major obstacle in harnessing their capabilities and clinical applications.

[0004] RNA interference (RNAi) is a technique that specifically inhibits gene expression. It is often carried out by small double - stranded interfering RNAs (siRNAs) or small hairpin RNAs (shRNAs) having a length of about 21 - 27 nucleotides.

[0005] The effectiveness of siRNA and shRNA can be limited by a number of factors. First, siRNA and shRNA are easily degraded by ribonuclease (RNase), thereby reducing their effectiveness. Second, they cannot penetrate the cell membrane and have very low bioavailability. Since siRNA cannot pass through the cell membrane independently, delivery vehicles such as viral vectors and synthetic carriers are often used. These viral and synthetic siRNA vehicles pose significant limitations and concerns for cell death, RNAi / microRNA saturation, and clinical effectiveness such as hepatotoxicity and tumorigenesis. Synthetic vehicles often have low delivery efficiency and require high doses to achieve clinical effectiveness, which is costly and can often be toxic. Furthermore, siRNA-based applications have historically been associated with transient RNAi effects, meaning that the silencing effect required for clinical targeting is transient. Although chemical modification of shRNA / siRNA and the application of numerous nanotechnology-based delivery techniques have been pursued, limited delivery efficiency to a limited number of target tissues and the transient nature of RNAi effects still remain major obstacles. Delivery platforms for the intracellular delivery of RNAi mediators and other nucleic acids, including deoxyribonucleic acid (DNA), ribonucleic acid (RNA), small interfering RNA / small hairpin RNA (siRNA / shRNA), microRNA (miRNA), antagomiR, aptamer, messenger RNA (mRNA), splice-switching oligonucleotide, interfering defective particles, and antisense oligonucleotides, to specific tissues are essential for harnessing the therapeutic potential held by nucleic acid-based technologies for translational medicine. The present invention provides a delivery platform as presented in the following disclosure. Summary of the Invention

[0006] Biological inter-kingdom delivery vehicles have advanced significantly by using several unique functions taught herein. The present invention provides modified biological inter-kingdom delivery vehicles with a number of important and substantial features and / or improvements over conventional delivery platforms. A first improvement is to express the double-stranded RNA-binding domain (dsRBD) of a double-stranded RNA-binding protein (dsRBP), such as the trans-activation response (TAR) RBP (TRBP), for co-packaging within the biological inter-kingdom delivery vehicle. The rationale behind this innovation is that dsRBPs such as TRBP bind dsRNA with high affinity, provide protection against RNA degradation, provide stabilization of miRNA precursors, and function as important components of siRNA- and miRNA-mediated gene silencing. A second improvement is to knock out the RNase R activity of the bacterial delivery vehicle. The rationale behind this modification is that following exponential growth of bacterial cells, when shRNA production is maximized, bacterial growth is arrested, and incubating the culture at less than 37°C inhibits the decay of double-stranded (ds) RNA, thereby improving the stability of the encoded therapeutic shRNA. A third improvement is to express a methyltransferase gene, such as HEN1, for co-packaging within the delivery vehicle. The rationale behind this modification is that methylation of the 3'-terminal nucleotide provided by a methyltransferase such as HEN1 protects against 3'-5' degradation and 3'-uridylation of siRNA, ultimately increasing siRNA stability and enhancing RNAi. Additional improvements and results are described below. These proposed improvements each represent unique approaches to improving shRNA stability and therapeutic lifespan of siRNA.

[0007] The technologies taught in this specification provide many advantages over current state-of-the-art platforms and other NA-based delivery applications, including the following (1)-(10): (1) Targeted delivery to mucosal epithelial tissues with efficient intracellular delivery and endosomal escape. (2) Stable delivery of shRNA payloads for catalytic and sustained RNAi silencing. (3) Elimination of costly synthetic siRNA production. (4) Clinical administration adaptable to oral, intranasal, intraocular, intravaginal, rectal, and respiratory administration. (5) Utilization of a natural-derived vehicle with a safety record confirmed for clinical purposes (not irritating delicate mucosal tissues). (6) Rapid scale-up of disease preparedness in the event of an emergency (i.e., influenza pandemic). (7) Feasible therapeutic applications for infectious diseases, immune disorders, and allergic diseases. (8) Ability to be used in combination with existing vaccination, antibiotic, antiviral, etc. regimens. (9) Elimination of the need for penetration enhancers that may increase the risk of toxin or pathogen entry. (10) NA is produced by prokaryotic RNA polymerase and delivered in a sequence-independent manner, making it a versatile and high platform for delivering NA-based therapeutics alone or in combination (i.e., siRNA / shRNA, miRNA, antagomiR, aptamer, mRNA, splice-switching oligonucleotide, interfering defective particle, antisense oligonucleotide).

[0008] This new approach provides a new perspective on NA delivery and addresses many of the drawbacks associated with current NA delivery methods. In general, the improvements taught herein enhance the function of the inter-kingdom delivery vehicle in many applications, including the therapeutic applications of (a)-(d) below. (a) Improving the stability of the NA produced and delivered, and providing RNase R knockout, by, for example, expression of HEN1 and TRBP. (b) Improving the stability of the inter-kingdom delivery vehicle itself, such as by expressing the gene of a particular gene (e.g., inv, hlyA, HEN1, TRBP, and / or HA-1 gene) on the chromosome rather than on a plasmid, thereby improving the stability of the delivery vehicle and facilitating manufacture. (c) Improving the invasiveness of the delivery vehicle (e.g., by including HA-1 and expressing both HA-1 and inv from the chromosome rather than from a plasmid. However, expression of these genes from a plasmid is also contemplated herein). (d) Eliminating the need for antibiotic selection, which is not ideal for manufacturing / regulation or therapeutic applications. All of these modifications act synergistically to improve the function of the inter-kingdom NA delivery system for therapeutic applications.

[0009] In a first aspect, the present invention provides a non-pathogenic bacterium or yeast that optionally contains a prokaryotic or eukaryotic vector. The vector includes a DNA molecule encoding one or more therapeutic nucleic acids (e.g., short double-stranded RNA) and a promoter that controls the transcription of the short double-stranded RNA. The short double-stranded RNA interferes with one or more target RNA molecules, and the bacterium is engineered to express one or more double-stranded RNA-binding proteins (dsRBPs). Many dsRBPs are known to bind dsRNA with high affinity to prevent degradation, bind miRNA precursors to stabilize them, and play important roles in siRNA- and miRNA-induced gene silencing. Expression of dsRBPs has been proposed to enhance stability and increase the concentration of available therapeutic nucleic acids by limiting degradation, increasing the concentration of miRNA precursors and their resulting mature products, and providing components necessary for involvement in target gene silencing. In an advantageous embodiment, the dsRBP is the trans-activation response (TAR) RNA-binding protein (TRBP). Furthermore, this platform has the ability to encode / deliver a wide range of other NA molecules such as siRNA / shRNA, miRNA mimics, inhibitors, etc. Other possible double-stranded RNA-binding proteins include nuclear factor 90 (NF90), ZNF346, Sid-1, and Ku70 (and combinations thereof).

[0010] In a second aspect, the present invention provides a non-pathogenic bacterium or yeast that contains a prokaryotic or eukaryotic vector. The vector includes a DNA molecule encoding one or more NA molecules, such as short double-stranded RNA (e.g., siRNA / shRNA), and a promoter that controls the transcription of the short double-stranded RNA. The short double-stranded RNA interferes with one or more target RNA molecules, and the bacterium is engineered to be deficient in RNase activity. In an advantageous embodiment, the bacterium lacks a functional rnr gene. In addition to RNase R knockout, other RNase proteins that can be targeted for knockout include RNase E, RNase I, RNase H, RNase J, and combinations thereof.

[0011] In a third aspect, the present invention provides a non-pathogenic bacterium comprising a prokaryotic vector, said vector comprising, for example, a DNA molecule encoding one or more NA molecules comprising short double-stranded RNA (e.g., siRNA / shRNA), and a promoter that controls the transcription of the short double-stranded RNA, said short double-stranded RNA interfering with one or more target RNA molecules, said bacterium being engineered such that a methyltransferase promotes methylation of the 3'-terminal nucleotide of the siRNA. More specifically, the bacterial system expresses a methyltransferase gene, which comprises HEN1 or another methyltransferase gene having 2'-O-methyltransferase activity, whereby the methyltransferase promotes methylation of the 3'-terminal nucleotide of the siRNA. Methylation of the 3'-terminal nucleotide provides protection against the major causes of RNA degradation, including 3-5' degradation and 3'-uridylation, ultimately enhancing stability and / or increasing the concentration of therapeutic NA available for participation in the RNAi pathway.

[0012] In a fourth aspect, the present invention provides a non-pathogenic bacterium or yeast comprising a prokaryotic or eukaryotic vector, said vector comprising, for example, a DNA molecule encoding one or more NA molecules comprising short double-stranded RNA (e.g., siRNA / shRNA), and a promoter that controls the transcription of the short double-stranded RNA, said short double-stranded RNA interfering with one or more target NA molecules, said bacterium being engineered to express a dsRNA-binding protein (dsRBP) and the HEN1 gene. In an advantageous embodiment, the Escherichia coli (E. coli) bacterium of the fourth aspect is engineered to be deficient in RNase activity. In a particularly advantageous embodiment, said bacterium lacks a functional rnr gene.

[0013] In a fifth aspect, the present invention provides a non-pathogenic bacterium or non-pathogenic yeast comprising a prokaryotic or eukaryotic vector, said vector comprising a DNA molecule encoding one or more nucleic acids and a prokaryotic promoter that controls the transcription of said therapeutic nucleic acid, said non-pathogenic bacterium or yeast being engineered to exhibit a reduced nucleic acid degradation phenotype.

[0014] In a broad sense, the one or more nucleic acids interact with one or more molecules or compounds. This can be for the purpose of upregulating or downregulating an effect in a target cell or organism, such as for treating an organism for a disease state or for studying the effect of a gene or gene product of an organism. In an advantageous embodiment, the one or more nucleic acids are therapeutic nucleic acids. The therapeutic nucleic acids can interfere with one or more targets such as a pathogen nucleic acid sequence. Therapeutic NA (TNA) can interfere with many types of target molecules (RNA, DNA, other oligonucleotides, etc.) depending on the nature and identity of the therapeutic nucleic acid. TNA can interact (interfere with, target, modify, alter) with one or more host factors or pathogen factors, and the host factors can be oligonucleotides, proteins, genes, etc. By way of example, TNA can interact with an oncogene or the product of an oncogene to regulate cancer and to study cancer or cancer treatment. In a further advantageous embodiment, the decreased nucleic acid degradation phenotype increases the stability, size, and / or duration of the desired therapeutic effect or activity. Non-pathogenic yeast is thought to use appropriate eukaryotic plasmids / episomes and promoter sequences.

[0015] In a sixth aspect, the present invention provides a non-pathogenic bacterium or non-pathogenic yeast comprising a prokaryotic or eukaryotic vector, said vector comprising a DNA molecule encoding one or more nucleic acids and a prokaryotic or eukaryotic promoter that controls the transcription of a therapeutic nucleic acid, wherein said non-pathogenic bacterium or yeast is engineered to have chromosomal integration of the inv, hlyA, HEN1, TRBP, and / or HA-1 genes. Chromosomal integration of the inv, hlyA, HEN1, TRBP, and / or HA-1 genes provides a way to enhance the expression of these genes (more stable and greater expression when expressed from the bacterial genome versus plasmid), improving the stability of the delivery system. It creates a system where the bacterium more readily expresses from the genome as compared to a system where the bacterium has to maintain a plasmid and express the gene from the plasmid. In other words, expression from the bacterial genome rather than the plasmid is more stable and results in greater expression. The non-pathogenic yeast is thought to use appropriate eukaryotic plasmid / episome and promoter sequences.

Brief Description of the Drawings

[0016] For a more complete understanding of the present invention, reference should be made to the following detailed description taken in conjunction with the accompanying drawings below.

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BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The present invention facilitates the delivery of therapeutic nucleic acids by providing a versatile delivery platform that can be tailored to target specific tissues for a wide range of therapeutic applications in various different eukaryotic species. Therapeutic nucleic acids initially showed great promise for the treatment of diseases, but due to the drawbacks in the delivery of these nucleic acids to target tissues and cells, their potential has not been fully realized. There may be significant improvements in terms of the magnitude and duration of the silencing effect, and the use of a delivery platform that targets multiple relevant tissues for the future development of a wide range of clinical applications. In a more focused sense, the improvements can be made such that both the delivery vehicle and the nucleic acids produced by the delivery vehicle have enhanced stability, improved invasiveness of the vehicle, improved safety, and improved characteristics for manufacturing processes and legal considerations.

[0018] Exosomes, liposomes, and other lipid vesicles have been used as NA delivery platforms to carry RNA payloads for delivery to distant tissues. Delivery vehicles such as liposomes have drawbacks including leakage of vesicle contents, batch-to-batch variability, high production costs, and limited targeting ability. This inter-kingdom delivery vehicle is based on the use of a non-pathogenic bacterium-mediated RNAi delivery vehicle that uses natural receptor-mediated endocytosis for specific intracellular delivery at the site of action of tissues, resulting in the accumulation of shRNA in endosomes and the efficient release of the shRNA payload into the cytoplasm of target cells for RNAi silencing. These inter-kingdom vehicles were Escherichia coli (E. coli) cells engineered to specifically target mucosal epithelial tissues and deliver a constitutively produced shRNA payload in a sequence-independent manner.

[0019] RNA interference across biological kingdoms using an engineered RNAi delivery platform that uses non-pathogenic bacteria containing shRNA expression plasmids has been used to dramatically reduce the severity of AIV infection and inhibit AIV shedding from avian epithelial cells. (Linke, Lyndsey M et al. “Inhibiting Avian Influenza Virus Shedding Using a Novel RNAi Antiviral Vector Technology: Proof of Concept in an Avian Cell Model.” AMB Express 6 (2016): 16. PMC. Web. 27 Mar. 2018.) An RNA system for delivery of shRNA across biological kingdoms is taught in Linke et al.'s US2016 / 0177296A1, the contents of which are incorporated by reference in their entirety. Nucleic acid delivery systems across biological kingdoms use non-pathogenic bacteria such as Escherichia coli (E. coli) engineered to transcribe shRNA from a plasmid. Nucleic acid interference across biological kingdoms has become relevant for clinical applications by including additional genes in the shRNA expression plasmid that facilitate both bacterial entry into target cells and release of the contents of the non-pathogenic bacteria into the target cells after their entry. Specifically, a system encoding two factors or genes has been developed that enables delivery of shRNA to mucosal epithelial cells (invasin gene (Inv) and listeriolysin O gene (hylA). The Inv gene is required for the expression of the invasin protein on the surface of Escherichia coli (E. coli) that interacts with the β(1)-integrin receptor present on mucosal epithelial cells and causes receptor-mediated endocytosis (Xiang et al 2006) (Conte et al 1994; Isberg and Barnes 2001; Isberg and Leong 1990). The hlyA gene encodes listeriolysin O (LLO), a pore-forming toxin that promotes rupture of the endosomal membrane and subsequent release of shRNA into the cytoplasm of the cell (Grillot-Courvalin et al 1998; Mathew et al 2003; Nguyen and Fruehauf 2009; Radford et al 2002; Xiang et al 2006).The nucleic acid delivery medium between biological kingdoms used in the above research was a diaminopimelic acid (Dap) auxotrophic mutant and was kanamycin resistant.

[0020] These media express listeriolysin O (LLO), a pore-forming toxin that enables Escherichia coli (E. coli) to escape from the host endosome for release in the cytosol. The lack of nutrients inside the endosome promotes the release of the LLO toxin, which lyses and destroys the endosomal membrane. The released shRNA is then processed into siRNA and incorporated into the RNA-induced silencing complex (RISC).

[0021] These bacteria can regulate the delivery of shRNA. When they enter the host endosome, they become non-viable and stop producing shRNA, thereby eliminating the possibility of overloading the RISC mechanism and causing unwanted side effects in the host.

[0022] The inter-kingdom delivery vehicle can be transformed with an shRNA (or other therapeutic nucleic acid) expression cassette under the control of a prokaryotic RNA polymerase, such as T7 polymerase, which can constitutively generate the shRNA (or other therapeutic nucleic acid) under the control of the polymerase for the target clinical gene. Including the prokaryotic RNA polymerase, bacteria are involved as an expression source of the product of interest (e.g., therapeutic nucleic acid), rather than the target cells. In the case of viral targeting, this platform technology can encode several shRNAs to target multiple viral RNA targets. Further, instead of, or in addition to, the sequence producing the shRNA in the expression cassette, other therapeutic nucleic acids can be included, and this system can target additional pathogens or disease-related molecules. This mixture approach limits the risk of viral escape by mutation. Further, this platform has the function of encoding a wide range of other NA molecules, including miRNA mimics and inhibitors. In other words, the proposed system has the ability to deliver other nucleic acids in addition to siRNA / shRNA, which is a significant improvement over current state-of-the-art technologies that are not adapted for the delivery, encoding, and delivery of molecules such as micro RNA mimics or micro RNA inhibitors.

[0023] The inter-kingdom delivery platform uses non-pathogenic bacteria that are diaminopimelic acid (Dap)-auxotrophic. Certain bacteria, including Escherichia coli (E. coli), require Dap as a component of their cell wall, and auxotrophic strains cannot grow or survive outside of Dap-supplemented media. As non-pathogenic and non-colony-forming bacteria, these vehicles pose no known risk to the host and have an established safety record. Further, as non-conjugative vectors, there is no risk of integration into the host genome and tumor formation. Previous studies (unpublished data) have demonstrated that these bacterial vehicles are not associated with the clinical disease, toxicity, or tissue etiology observed in the species tested. Thus, the bacteria are "non-pathogenic" to the host cells or target species and do not cause disease.

[0024] We propose to improve current inter-kingdom delivery vehicles with unique modifications centered around improving the stability of delivered nucleic acid (NA), improving the resistance of double-stranded interfering RNA to cellular breakdown mechanisms, improving invasiveness, and / or improving manufacturing processes, safety, and regulatory issues related to delivery systems. The rationale for increasing the stability of dsRNA is that stabilization of the payload of shRNA (and other NA molecules that can be generated and delivered using this delivery platform) increases the availability of shRNA and the processing of more siRNA. The rationale for improving invasiveness is that the inter-kingdom delivery vehicle has a greater chance of attaching to a wider range of host cells, thereby increasing the number of inter-kingdom delivery vehicles that are taken up and intracellularized. These improvements will provide a stronger and longer-lasting RNAi effect for optimal therapeutic applications. These improvements are made individually or in any combination and facilitate the translation of technology for therapeutic and clinical applications.

[0025] Improvements in the invasiveness and stability of delivered NA can be achieved through a plurality of techniques taught herein. A first technique for improving the delivery vehicle is to include a double-stranded RNA-binding protein (dsRBP) that increases the stability of dsRNA. A second technique for improving the delivery vehicle is to eliminate the activity of key bacterial enzymes involved in RNA degradation. A third technique for improving the delivery vehicle is to include a gene / protein that causes stable methylation of dsRNA. In one embodiment, the TAN RNA-binding protein (TRBP) and the C-terminal methylase domain of the HEN1 gene are encoded and the RNase R activity in an E. coli bacterial delivery vector is knocked out. Improvements in these formulations are utilized to deliver nucleic acids, including shRNA, to a wide range of mucosal epithelial tissues, including the upper / lower respiratory, oral, gastrointestinal (GI), vaginal, rectal, and ocular epithelia. Methods of administering these improved inter-kingdom NA delivery vehicles include intranasal administration to the nasal cavity for local action, aerosolization for upper and lower respiratory targeting, oral absorption for buccal delivery, ingestion for GI uptake, application to delicate genital mucosal epithelia, and topical administration for intraocular delivery. These improved delivery vehicles can be used to prevent and / or treat a wide range of diseases (infectious, allergic, cancerous, immune) in a wide range of species (human, avian, porcine, bovine, canine, equine, feline).

[0026] We propose to enhance the targeting of a delivery vehicle to epithelial cells expressing sialic acid receptors by engineering the delivery vehicle to express influenza virus hemagglutinin (HA-1) protein. HA-1 can be expressed either alone or in combination with invasin protein in the delivery vehicle. Expression of HA-1 in the delivery vehicle not only provides an appropriate means for uptake by bacterial cells and endocytosis into host cells, but also mechanically prevents uptake and infection by competing pathogens (e.g., influenza virus and other pathogens that use sialic acid receptors for attachment / invasion) by blocking access to receptor sites in target cells. In the case of viral and bacterial infections targeting intestinal, ocular, and respiratory mucosal epithelial cells, the inclusion of HA-1 protein for vehicle delivery provides greater affinity for targeting those tissues for maximal protection against local delivery and invading pathogens, both therapeutically and prophylactically.

[0027] Example 1 - Engineered Escherichia coli (E. coli) for expressing dsRBP In a first aspect, improvement of the delivery vehicle can be achieved by cloning a dsRNA binding protein domain into a therapeutic nucleic acid-producing plasmid for expression under the control of a prokaryotic RNA polymerase, or by chromosomally expressing a dsRNA binding protein (dsRBP) in Escherichia coli (E. coli) to increase the stability of dsRNA produced by non-pathogenic bacteria by including the dsRBP.

[0028] The family of dsRBPs contains one or more evolutionarily conserved double-stranded RNA binding domains (dsRBDs) of 65 - 68 amino acids found in products encoded by eukaryotes, prokaryotes, and viruses (Ryter et al. 1998). These dsRBDs were first recognized in 1992 as factors in many interactions between proteins and RNA duplexes (Johnston et al. 1992; McCormack et al. 1992). The dsRBP family includes the TAR RNA binding protein (TRBP), which interacts only with dsRNA in a sequence-nonspecific manner (Ryter et al.; Manche et al.). TRBP contains three highly conserved dsRBDs, with the first and second having high affinity for binding and protecting against dsRNA degradation (Yamishita et al.; Daviet et al.; Tian et al.). TRBP can be involved in RNAi (Gatignol et al., Gredell et al., Lee and Ambros, Tian et al.; Daniels et al.; Parker et al.). These two TRBP binding domains can be used for binding and preventing shRNA siRNA degradation in vitro and in vivo, play a role in gene silencing induced by siRNA and microRNA (miRNA), bind miRNA hairpin precursors, and play a physiological role in cells by stabilizing the pathway for maturation (Chendrimada et al.; Koh et al.; Gregory et al.; Dar et al.; Yamashita et al.; Koh et al.). Thus, only two domains can be preferentially used, or all three can be included in vectors across biological kingdoms. In summary, TRBP functions like a dsRNA gatekeeper. Encoding this dsRNA binding protein simultaneously in vectors across biological kingdoms represents a challenging approach to improving the delivery vehicle of nucleic acids across biological kingdoms. Instead of, or in addition to, TRBP, other dsRBPs whose expression can be considered include nuclear factor 90 (NF90), ZNF346, SiD-1, and Ku70 (and combinations thereof).

[0029] Example 2 - RNase R Knockout In a second aspect, improvement of the properties of the delivery medium can be achieved by removing or reducing the activity of the enzyme responsible for RNA degradation. In bacteria such as Escherichia coli (E. coli), structured RNA duplex decay is carried out only by RNase R, a hydrolytic 3’→5’ exoribonuclease (Matos 2009; Khemici and Carpousis; Cheng and Deutscher 2005; Vincent and Deutscher 2009; Awano et al. 2007, Awano 2010; Chen et al. 1998; Hossain 2015; Hossain 2016). Similar to the case of shRNA / siRNA molecules, RNase R can degrade these dsRNAs by specifically binding to the 3’ single-stranded overhangs (Cheng and Deutscher, Vincent and Deutscher 2006). In fact, RNase R is the only 3’→5’ exoribonuclease that can degrade (chew) by a wide range of secondary RNA structures (Vincent and Deutscher 2009). RNase R is a cold shock protein encoded by the rnr gene, and its activity can be regulated by changing the growth conditions of bacterial cells (Chen et al. 1998; Cheng and Deutscher; Cairrao and Arraiano 2006; Cairrao et al. 2003). When the exponential growth temperature of Escherichia coli (E. coli) shifts from 37°C to 15°C, the RNase R cold shock response is stimulated (Awano et al. 2007). That is, the RNase R activity increases and the dsRNA decay becomes larger. Studies have demonstrated that, simultaneously, when the concentration of a more stable dsRNA substrate is high, especially at low incubation temperatures, the RNase R helicase activity plays a catalytic role necessary for effective nuclease activity against dsRNA (Hossain et al. 2015, Hossain et al. 2016, Awano et al. 2007; Awano et al. 2010).E. coli mutants lacking the rnr gene were able to replicate efficiently at the standard growth temperature of 37 °C but were unable to digest dsRNA (Chen et al. 1998; Khemici and Carpousis; Cheng and Deutscher; Vincent and Deutscher 2009; Vincent and Deutscher 2006; Awano et al. 2007; Hossain et al. 2015; Hossain et al. 2016 (both); Awano et al. 2010). Since the stability of shRNA and siRNA molecules is improved at low temperature, it may be desirable to lower the culture temperature to maintain a high NA concentration only if cold shock induction of RNase R activity can be avoided. Thus, knockout of RNase R activity in an E. coli bacterial delivery vehicle can lead to enhanced delivery of NA. In a modified E. coli bacterial delivery vehicle with RNase R knockout, after exponential growth of bacterial cells when shRNA production is maximized, bacterial growth is stopped and the culture is incubated at a temperature below 37 °C, thereby suppressing the decay of dsRNA and enhancing the stability of the encoded therapeutic shRNA. Thus, a cross-kingdom system can be thought to include non-pathogenic bacteria such as E. coli lacking the rnr gene transformed with a special shRNA (or other therapeutic nucleic acid) production plasmid. Furthermore, these rnr-deficient E. coli bacteria can be thought to be able to grow at low temperatures (i.e., below 37 °C) such as 33 °C or below, 30 °C or below, 27 °C or below, 25 °C or below, 22 °C or below, 20 °C or below, 17 °C or below, preferably 25 °C or below, most preferably 17 °C or below. Growth at such low temperatures allows production of the desired shRNA while limiting degradation. Furthermore, the shRNA production plasmid used in the nucleic acid delivery system across kingdoms can be thought to be able to contain a gene encoding a dsRBD such as TRBP. One approach to knockout occurs by recombining deletions, thereby preventing E. coli from producing RNase R protein and essentially creating an E. coli mutant.Other methods of eliminating the function of the rnr gene include gene editing via Cre-lox, FLP-FRT, selection-counterselection, and CRISPR / Cas. Since there are other genes that can be knocked out to remove the RNase activity of E. coli, the knockout is considered to be not only the rnr gene but also the target. In general, the removal of RNase catalytic activity, including the removal of RNase R in Escherichia coli (E. coli), can enhance the effectiveness of E. coli. Other targets for knockout include, but are not limited to, RNase E, RNase I, RNase H, RNase J, and combinations thereof.

[0030] Example 3 - Methylation of the 3'-terminal nucleotide of siRNA In a third aspect, by including a gene that causes stabilization of methylation of shRNA in a therapeutic NA production plasmid or the like, improvement in the characteristics of the delivery medium can be achieved. As described above, degradation of dsRNA is mainly caused by exonucleases (Czauderna et al. 2003). Methylation of the 3'-terminal nucleotide of siRNA provides a stabilizing effect against exonucleases to protect against 3'-5' degradation and 3'-uridylation of siRNA (Ji & Chen 2012; Lifang et al. 2010; Kurth & Mochizuki 2009; Chan et al. 2009). RNAi activity is maintained when the siRNA molecule is modified only at the end of the sense strand or, in the case of an siRNA hairpin, at the 3'-end (Czauderna et al. 2003). 3'-terminal methylation is required for the function of all plant siRNAs and is also observed in some mammalian and bacterial siRNAs. The HEN1 gene encodes a methyltransferase, Methyltransferase Hen1, which catalyzes the S-adenosylmethionine-dependent transfer of a methyl group to the 2'-hydroxyl of the 3'-terminal nucleotide (Jain & Shuman 2010; Baranauske et al. 2015). The Hen1 domains of several bacterial species have been shown to be functionally equivalent to eukaryotic homologs (Chan et al. 2009). Using a methylation assay, Chan et al. (2009) showed that human and bacterial (Clostridium thermocellum) recombinant HEN1 proteins exhibit enzymatically equivalent methylation activity with small single-stranded RNAs (21 - 30 nt). However, the C-terminal methylase domain of Hen1 from Clostridium thermocellum (C. thermocellum) showed improved enzymatic activity compared to its full-length counterpart and human Hen1, indicating that this domain can function independently. Thus, packaging of the HEN1 gene, or in a preferred embodiment, the C-terminal methylase domain of the HEN1 gene, in a delivery medium across biological kingdoms provides a stabilizing effect of siRNA against exonucleases.In other words, HEN1 actively methylates and enhances the stability of the encoded shRNA, thereby providing an shRNA that is optimally suitable for therapeutic applications in vivo.

[0031] Example 4 - Non-pathogenic bacteria engineered to express influenza hemagglutinin-1 (HA-1) protein In a fourth aspect, bacteria are engineered to express influenza hemagglutinin-1 (HA-1) protein either by genomic (chromosomal) expression or plasmid expression, thereby improving local and targeted delivery to the mucosal tissue of the delivery vehicle, particularly the respiratory mucosal tissue. HA-1 is a glycoprotein found on the surface of influenza virus and promotes virus attachment and entry into target cells by receptor-mediated endocytosis (White et al. 1997). HA-1 represents a globular head containing a receptor site with high affinity for sialic acid, which is present on the surface of target cells including respiratory mucosal epithelial cells (Russell et al. 2008). In a preferred embodiment, HA-1 is expressed on the surface of Escherichia coli (E. coli) and interacts with sialic acid surface receptors on mucosal epithelial cells for bacterial uptake. The HA-1 protein is derived from either influenza A or B virus subtypes. The full-length HA-1 gene is cloned into an shRNA production plasmid (or other plasmid constructed to produce therapeutic nucleic acids) using standard cloning methodologies, e.g., Gibson assembly method and commercially available Gibson assembly cloning kits, flippase flippase recognition target (FLP / FRT) site-specific recombination technology (Posfai et al. 1994, Bertram et al. 2009), selection-counter-selection strategies, or CRISPR Cas manipulation (Jiang et al. 2013, Jiang et al. 2015, Pyne et al. 2015, Reisch et al. 2015, Zhao et al. 2016) and integrated into the E. coli chromosome.

[0032] Adding the HA-1 protein to the surface of E. coli cells improves over the sole expression of Inv alone. In one embodiment, HA-1 is expressed independently or in another embodiment, expressed in combination with Inv, providing a unique facet not provided by Inv alone. In the case of viral and bacterial pathogens that utilize sialic acid receptors for intracellular invasion into target cells, such as influenza viruses that infect respiratory tissues, binding E. coli cells via the HA-1 protein physically blocks the essential binding interaction between the infectious pathogen (i.e., influenza virus) and the sialic acid receptor on the target cell. This will thereby not only provide an appropriate means for the uptake of bacterial cells and their internalization into host cells, but also mechanically prevent the uptake and infection of competing pathogens (i.e., influenza virus). In the case of viral and bacterial infections targeting respiratory mucosal epithelial cells, including HA-1 protein for delivery in the medium provides high affinity for targeting these tissues for local delivery, and maximum protection against invading pathogens therapeutically and prophylactically. Other pathogens including coronaviruses, norovirus, rotavirus, mumps virus, parainfluenza, Clostridium botulinum, and Vibrio cholerae (Mastrovich et al 2013; Varki 2008) are known to produce proteins that bind to sialic acid on host cells and promote invasion or infection. Furthermore, these proteins, or binding protein domains from these pathogen proteins, can be used as an alternative to the influenza HA-1 protein (or sialic acid residue binding domain from HA-1).

[0033] Example 5: Removal of Antibiotic Resistance Genes from Non-Pathogenic Bacteria In a fifth aspect, by removing an antibiotic resistance gene, including but not limited to kanamycin resistance, which can be included in a plasmid as a selectable marker, the manufacturing process, safety, and regulatory considerations of the delivery system are improved. Antibiotic resistance genes are included as selectable markers in most bacterial expression vectors (Peubez et al. 2010). Antibiotic resistance is a major concern for human and animal health, and regulatory requirements for biological agents are becoming more stringent to reflect this (Mignon 2015). By eliminating the need to include antibiotics in the manufacturing process, costs are reduced and the concern that antibiotic resistance in unintended hosts will be induced by the bacterial delivery system is eliminated. In an advantageous embodiment, this delivery vehicle uses a system free of antibiotics, such as auxotrophy and complementation systems described in Example 6 below, to specifically select the desired plasmid-containing bacteria.

[0034] Example 6 - Generation of non-pathogenic auxotrophic mutants The functionality and stability of the delivery system are improved by including metabolic or nutritional selectable markers, including but not limited to auxotrophy and complementation, and thus the manufacturing process, safety, and regulatory considerations of the delivery system are improved.

[0035] In a sixth aspect, the functionality and stability of the delivery system are improved by including metabolic or nutritional selection markers including, but not limited to, amino acid auxotrophy and complementation (e.g., targeting essential amino acids including, but not limited to, histidine). One or more important genes in the amino acid synthesis pathway are targeted on the E. coli chromosome to create auxotrophic mutants that cannot produce the required amino acids. Complementation of the knockout gene or gene cluster on the plasmid enables the survival of only E. coli containing the plasmid and allows for efficient and reliable selection in media lacking amino acids (Peubez et al. 2010; Mignon et al. 2105; Fiedler et al. 2017). The same principle applies to other nutrients or nutrient processing pathways essential for bacterial survival. This approach facilitates antibiotic-free production, further supporting the improvements described in Example 5 above. In E. coli, the hisD gene product, the bifunctional enzyme histidinol dehydrogenase, which is synthesized in a ten-step pathway, catalyzes the last two oxidation steps (Ramage et al. 2012; Matte et al. 2003), and the hisB product, the bifunctional enzyme histidinol phosphatase, catalyzes the seventh and ninth steps (Winkler & Ramos-Montanez 2009; Chiariotti et al. 1986). The hisA, hisB, hisC, hisD, hisE, hisG, hisH, and hisl gene products are required for histidine synthesis and thus for the growth of E. coli in minimal media (Joyce et al. 2006), but computational models and published experiments have shown that hisD or hisB knockout generates growth strains that are completely dependent on the presence or absence of histidine and not on other amino acids or metabolites present in the culture medium (Bertels et al. 2012; Tepper & Shlomi 2011).

[0036] In a preferred embodiment, E. coli is engineered to be histidine auxotrophic, the bacteria lack a functional hisD gene (product: histidinol dehydrogenase), and the shRNA-producing plasmid (or other plasmid constructed to produce therapeutic NA) is engineered to express the hisD gene to complement the histidine biosynthetic pathway and permit bacterial replication in histidine-deficient media. In other embodiments, E. coli is engineered to lack a functional hisA, hisB, hisC, hisE, hisG, or hisH gene, and the prokaryotic vector is engineered to express the missing gene. In the case of genes involved in histidine synthesis, the gene can be expressed on the plasmid under the control of the bacterial hisp1 promoter or another prokaryotic promoter (Alifano et al 1996).

[0037] Example 7 - Expression of Multiple Therapeutic Nucleic Acids by a Transkingdom Delivery Vehicle In a seventh aspect, by enabling the expression of individual or multiple therapeutic NAs on a single plasmid, the functionality and stability of the delivery system are improved. Many different configurations are possible, including the following 1) to 3). 1) NAs expressed in tandem in a single expression cassette under the control of a single promoter. 2) Independent expression cassettes under the control of their own promoters and terminators, but with competing expression due to the use of the same promoter sequence. 3) NAs in independent expression cassettes under the control of different promoters for different expressions. As demonstrated in numerous published studies, the specific details of plasmid construction, including spacer length and sequence, promoter sequence and strength, therapeutic NA type (e.g., shRNA) and size, and the number of therapeutic NAs expressed, all affect NA expression levels, siRNA folding / processing, silencing specificity, and RNAi activity. Much of this work has been done on HIV models, but good methods are available to appropriately guide plasmid design and optimization for each approach (Liu et al. 2007; McIntyre et al. 2011; Spanavello et al. 2016; Choi et al. 2015; Wang et al. 2013). The expression of multiple NAs in a single plasmid achieves several important improvements. That is, rather than each NA expression plasmid in Escherichia coli (E. coli) bacteria, it is regarded as a single "active" component by certain regulatory authorities, and manufacturing is simplified by culturing a single strain of E. coli containing the relevant NA target, expanding the targeting ability, reducing the risk of pathogen mutation, and enabling the treatment of multiple diseases with a single therapy.

[0038] Tandem expression of multiple specific nucleic acid sequences on a plasmid provides a system that is easier and cheaper to manufacture, even when expressing multiple nucleic acids for different targets (e.g., it can be manufactured as a single culture). This reduces variability and makes the manufacturing process more predictable, thus improving the stability of the system. It also facilitates a simplified regulatory pathway (in the United States and internationally) as a single rather than multiple active / therapeutic agents, reducing cost and source requirements.

[0039] Example 8 - Chromosomal expression of Inv, LLO, HA-1, HEN1, and / or TRBP In the eighth aspect, the stability of Inv, LLO, HA-1, HEN1, and / or TRBP expression, the expression (total abundance) of Inv, LLO, HA-1, HEN1, Inv, LLO, HA-1, HEN1, and / or TRBP, and the manufacturing process of the delivery vehicle are improved by incorporating the expression of these genes onto the E. coli chromosome (or other non-pathogenic bacteria or yeast). This produces bacterial strains that stably express the Inv, LLO, HA-1, HEN1, and / or TRBP genes. When constructing the strains, regions along the chromosome of the bacteria into which the expression cassette is to be incorporated into the engineered bacteria can be found. For example, using the Gibson Assembly method and commercially available Gibson Assembly cloning kits, flippase / flippase recognition target (FLP / FRT) site-specific recombination technology (Posfai et al. 1994, Bertram et al. 2009), selection-counter-selection strategies, or CRISPR / Cas manipulation (Jiang AEM, Jiang NBT 2013, Pyne AEM, Reisch et al. 2015, Zhao et al. 2016), the full-length sequences of any combination of these genes (Inv, LLO, HA-1, HEN1, and / or TRBP), or shortened portions thereof, can be incorporated into the E. coli chromosome. Genomic expression is an improved approach over plasmid expression for several reasons. The carriage and maintenance of plasmids places E. coli cells at a selectively disadvantageous state. Reliance on expression from plasmids requires growing E. coli cells under specific environmental conditions to maintain the presence of the plasmid within the bacteria. This complicates manufacturing and can increase bacterial stress. During manufacturing, the various conditions of large-scale fermentation affect the replication of plasmid DNA in E. coli cells, thus greatly influencing the efficiency (stability and abundance) of gene expression. In this embodiment, genomic recombination of these functional genes eliminates the need for the bacteria to maintain plasmid replication, particularly when growth conditions fluctuate.Furthermore, when these genes are removed from the plasmid and integrated into the E. coli chromosome for expression, the E. coli cells grow faster (G Wegrzyn and A Wegrzyn 2002). In this embodiment, genomic expression of Inv, LLO, HA-1, HEN1, and / or TRBP from the E. coli chromosome also results in a greater abundance of these genes compared to plasmid expression. The reliance on plasmid expression means that it is often necessary to replicate the plasmid in order to generate a sufficient number of copies to be distributed to both daughter cells after division of the mother cell. This results in a decrease in the expression of plasmid-derived genes in the progeny cells (decrease in abundance) compared to stable and consistent genome-derived expression. Overall, recombining Inv, LLO, HA-1, HEN1, and / or TRBP into the E. coli chromosome improves stability and increases the expression of these genes, particularly in large-scale manufacturing processes.

[0040] In one embodiment of the above method of the present invention, the bacterium is non-pathogenic or apathogenic. In another aspect of this embodiment, the bacterium is therapeutic. In another aspect of this embodiment, the bacterium is invasive or engineered to be invasive and invade host cells. Examples of such bacteria include Listeria, Yersinia, Rickettsia, Shigella, E. coli, Salmonella, Legionella, Chlamydia, Brucella, Neisseria, Burkolderia, Bordetella, Borrelia, Coxiella, Mycobacterium, Helicobacter, Staphylococcus, Streptococcus, Porphyromonas, Vibrio, Treponema, Lactobacillus, and Bifidobacteriae. In another aspect of this embodiment, the delivery vehicle is a fungal cell such as Saccharomyce and Candida yeasts.

[0041] In a preferred embodiment of the present invention, the invasive delivery vehicle containing the NA molecule is introduced into the host by intravenous, intramuscular, intradermal, intraperitoneal, oral, intranasal, intraocular, rectal, vaginal, intraosseous, oral, immersion, topical, intraurethral, and aerosol administration inoculation routes.

[0042] In advantageous embodiments, the present invention relates to a cross-kingdom platform for the delivery of RNA agents and other nucleic acids into target cells. Such RNAi agents and NAs include, but are not limited to, deoxyribonucleases (DNA), RNA, siRNA / shRNA, miRNA, antagomiR, aptamers, mRNA, splice-switching oligonucleotides, interfering defective particles, and antisense oligonucleotides.

[0043] Pathways of pathogen entry across mucosae The surfaces most likely to be invaded by pathogens in the human body, including the pleura, peritoneum, and skin, are protected by the mucosal immune system (Janeway et al. 2001). Mucosal surfaces are the first line of defense against pathogens into the body's interior and allergens in the external environment. Epithelial cells form tight junctions and form a dynamic layer over all mucosal surfaces (Parham 2014; Presland & Jurevic 2002). The moist mucus layer covering mucosal surfaces contains a mixture of "defense compounds" secreted by epithelial cells and leukocytes, forming a physical barrier but may also act directly on selected microorganisms (Linden et al. 2008). Other organs such as the respiratory tract, gastrointestinal tract, urogenital tract, and eyes represent major systems that include mucosal epithelial surfaces (Parham 2014). The gastrointestinal (GI) tract alone has an impressive surface area of over 400 square meters (Guandalini et al. 2008). When a pathogen or allergen succeeds in breaching mucosal defenses, the disease response is usually contained within that compartment of the body (Janeway et al. 2001).

[0044] Exemplary viruses that can be targeted by the proposed cross-kingdom NA delivery system: Multiple diseases affect multiple tissue types, including mucosal epithelia. Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) replicate in mucosal epithelial cells, including those of the mouth, eyes, and genitalia (Chentoufi & Ben ohamed 2012; Karasneh & Shukla 2011; NIAID). In the United States, approximately 65% of people are seropositive for HSV-1, but the worldwide prevalence is much higher, with an estimated 90% of people infected with HSV-1 and / or HSV-2 (Wald & Corey 2007). In developed countries, HSV-1 is a major cause of corneal blindness and viral encephalitis (Chentoufi & BenMohamed 2012). Perinatal infection with HSV-2 during childbirth is involved in approximately 85% of neonatal herpes cases (Chentoufi & BenMohamed 2012). Influenza is a globally important and highly contagious virus that infects respiratory tissues, including the mucosae of the eyes, nose, lungs, and throat. Despite the availability of vaccination, millions of people become ill with seasonal influenza each year, and emerging and pandemic strains of the virus remain a threat (NIAID). Human papillomavirus (HPV) can infect mucosal epithelial cells of the mouth, throat, and upper respiratory tract (Nguyen et al. 2014; Rautava & Syrjanen 2011). HPV also infects the anal-genital mucosal epithelium and is one of the most common sexually transmitted infections in the world, with an estimated 1 million to 5.5 million new cases occurring each year in the United States alone (Burd 2003; NIAID).

[0045] Other important diseases that affect the mucosal epithelial cells of the respiratory system include Hansen's disease, tuberculosis, and highly infectious diseases such as whooping cough, respiratory syncytial virus, MERS, and SARS. Emphysema, asthma, chronic obstructive pulmonary disease, cystic fibrosis, and bronchitis are other lung diseases. In the GI mucosa, a wide range of diseases such as cholera are known to cause 3 to 5 million new cases annually (NIAID). Sexually transmitted infections such as gonorrhea and chlamydia (Becker 1996), and syphilis are also major concerns in the health of the urogenital mucosal epithelial cells, including vaginal and rectal tissues (NIAID). The inter-kingdom NA delivery system taught herein is considered to be usable for the treatment of these diseases through the delivery of target shRNA.

[0046] Many different diseases specifically affect the mucosal epithelium of different tissue types, and addressing pathogen replication at these sites provides treatment options. This delivery platform has the potential to deliver therapeutic NA to a wide range of mucosal epithelial tissues. Therefore, in addition to improving the properties of the inter-kingdom NA delivery vehicles, the feasibility of delivering these vehicles to several clinically relevant mucosal epithelial tissues prone to disease is demonstrated.

[0047] These tissues include the upper / lower respiratory tract, oral cavity, GI, vagina, rectum, and ocular epithelium.

[0048] The platform taught herein promotes the delivery of NA using efficient / appropriate dosing applications that target very specific and clinically relevant mucosal epithelial tissues, leading to significant advancements in the field of NA-based human medicine. The delivery vehicle can be administered nasally for local action, aerosolized for upper and lower respiratory diseases, absorbed orally for buccal delivery, ingested for GI uptake, applied to delicate genital mucosal epithelia, and administered for local intraocular delivery. This technology provides a safe and effective NA delivery vehicle to mucosal epithelial tissues with enhanced stability, therapeutic efficacy, and overall improved functional characteristics. This inter-kingdom NA delivery system platform has made significant progress in the field of NA therapy and has numerous therapeutic applications while addressing a vast number of diseases that affect a wide range of clinically relevant tissues.

[0049] Glossary of Terms in the Claims As used herein, the term "administer" and variations thereof (e.g., "administering" a compound) in reference to a compound of the invention mean introducing the compound into the system of a subject in need of treatment. When a compound of the invention is provided in combination with one or more other active agents (e.g., an AIV vaccine, etc.), "administer" and variations thereof are understood to include, respectively, the simultaneous and sequential introduction of the compound and the other agent(s).

[0050] As used herein, the term "composition" is intended to encompass a product comprising the specified amount of the specified ingredients, as well as a product that results directly or indirectly from the combination of the specified amounts of the specified ingredients.

[0051] As used herein, the term "therapeutically effective amount" means the amount of an active compound or pharmaceutical agent that elicits a biological or medical response in a tissue, system, animal, or human that is sought by a researcher, veterinarian, physician, or other clinician. For viral infections, an effective amount is an amount sufficient to prevent disease or reduce the severity of disease, as evidenced by clinical disease, clinical symptoms, viral titer, or viral shedding from the subject, or as evidenced by the ability to prevent or reduce transmission between animals. In some embodiments, an effective amount is an amount sufficient to delay the onset of clinical disease and / or symptoms or to prevent the disease. In some embodiments, an effective amount is an amount sufficient to reduce viral titer and / or reduce viral shedding. An effective amount can be administered in one or more doses.

[0052] As used herein, "treatment" refers to obtaining a beneficial or desirable clinical outcome. Beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction in the degree of viral infection, stable (i.e., non-worsening) state of viral infection, prevention or delay of the spread of viral infection (e.g., shedding), prevention, delay, slowing, and / or maintenance of body weight / weight gain. The methods of the invention contemplate any one or more of these aspects of treatment.

[0053] A "pharmaceutically acceptable" component is a component suitable for use in animals without undue adverse side effects (such as toxicity, irritation, allergic reactions, etc.) commensurate with a reasonable benefit / risk ratio.

[0054] A "safe and effective amount" refers to the amount of a component sufficient to provide the desired therapeutic response without undue adverse side effects (such as toxicity, irritation, allergic reactions, etc.) commensurate with an appropriate benefit / risk ratio when used in the methods of the invention.

[0055] As used throughout the application, the terms "a" and "an" are used in the sense of meaning "at least one," "at least first," "one or more," or "a plurality" of the referenced component or step, unless the context clearly dictates otherwise. For example, the term "cell" includes a plurality of cells including mixtures thereof.

[0056] The term "and / or" as used herein always includes the meanings of "and," "or," and "all or any other combination of the elements connected by this term" when used herein.

[0057] As used herein, the term "comprising" is intended to mean that the product, composition, and method include the referenced components or steps but do not exclude others. "Consisting essentially of," when used to define a product, composition, and method, is intended to mean excluding other components or steps that are essentially important. Thus, a composition consisting essentially of the recited components will not exclude trace contaminants and pharmaceutically acceptable carriers. "Consisting of" means excluding trace elements of other components or steps.

[0058] As used herein, the term "invasive" when referring to a microorganism, such as a bacterium or a bacterial therapeutic particle (BTP), refers to a microorganism capable of delivering at least one molecule, such as an RNA or a DNA molecule encoding an RNA, to a target cell. An invasive microorganism can pass through the cell membrane, thereby entering the cytoplasm of the cell and being able to deliver at least a part of its contents, such as an RNA or a DNA encoding an RNA, to the target cell. The process of delivering at least one molecule to a target cell preferably does not significantly alter the invasive apparatus.

[0059] As used herein, the term "across the biological kingdom" refers to a delivery system that generates nucleic acids using bacteria (or other invasive microorganisms) in a target tissue for processing without host genome recombination and delivers the nucleic acids intracellularly (i.e., across kingdoms: from prokaryotes to eukaryotes, or across phyla: from invertebrates to vertebrates).

[0060] Examples of invasive microorganisms include microorganisms that can naturally deliver at least one molecule to a target cell by, for example, passing through a cell membrane such as a eukaryotic cell membrane and entering the cytoplasm, as well as microorganisms that are not originally invasive but are modified by genetic recombination or the like to become invasive. In other preferred embodiments, a bacterium or BTP can be made invasive by linking it to an "invasion factor", also referred to as an "invasion factor" or "cytoplasmic targeting factor". As used herein, an "invasion factor" is a factor such as a protein or group of proteins that, when expressed by a non-invasive bacterium or BTP, makes the bacterium or BTP invasive. As used herein, an "invasion factor" is encoded by a "cytoplasmic targeting gene". Invasive microorganisms are generally described in the art (e.g., U.S. Pat. Pub. Nos. US20100189691A1 and US20100092438A1, and Xiang, S. et al., Nature Biotechnology 24, 697 - 702 (2006).). Each is incorporated by reference in its entirety for all purposes. In a preferred embodiment, as taught in the examples of the present application, the invasive microorganism is Escherichia coli (E. coli). However, it is contemplated that additional microorganisms can potentially be adapted to function as inter-kingdom delivery vehicles for the delivery of NA. These non-pathogenic and invasive bacteria and BTPs are invasive or modified to be invasive and can enter host cells via various mechanisms. Typically, invasive bacterial or BTP strains have the ability to enter non-phagocytic host cells, as opposed to uptake by specialized phagocytic cells that result in the destruction of the bacteria or BTPs within specialized lysosomes.Examples of such naturally occurring intracellular bacteria include Yersinia, Rickettsia, Legionella, Brucella, Mycobacterium, Helicobacter, Coxiella, Chlamydia, Neisseria, Burkolderia, Bordetella, Borrelia, Listeria, Shigella, Salmonella, Staphylococcus, Streptococcus, Porphyromonas, Treponema, and Vibrio. However, this property can also be introduced into other bacteria or BTPs, including Escherichia coli, Lactobacillus, Lactococcus, or Bifidobacteriae, including probiotics, by the transfer of invasion-related genes (P. Courvalin, S. Goussard, C. Grillot-Courvalin, C.R. Acad. Sci. Paris 318, 1207 (1995)). Factors to be considered or addressed when evaluating additional bacterial species as candidates for use as inter-kingdom NA delivery vehicles include the pathogenicity of the candidate, or its absence, the tropism of the candidate bacterium for the target cell, or the degree to which the bacterium can be manipulated to deliver NA inside the target cell, and the value of the synergistic effect provided by the candidate bacterium by inducing the host's innate immunity.

[0061] Nucleic acids are defined as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or any closely related compound. They can be coding or non-coding, synthetic or naturally occurring, single- or double-stranded segments, and often consist of molecules of many (two or more) nucleotides linked together. Examples include small interfering RNA small hairpin-type RNA (siRNA / shRNA), microRNA (miRNA), antagomiR, RNA or DNA aptamers, messenger RNA (mRNA), splice-switching oligonucleotides, antisense oligonucleotides, antigene oligonucleotides, DNAzymes, RNA decoys, ribozymes, peptide nucleic acids, oligomers, and interfering defective particles.

[0062] Therapeutic nucleic acids are the NAs described herein, or closely related compounds used in the treatment of diseases, in the study of diseases, or for the achievement of a desired genetic modification, or for the purpose of gene delivery. They are used when the specific inhibition or disruption or alteration of the function of a particular gene or other molecule involved in a disease is considered therapeutically desirable.

[0063] As used herein, (1) a pharmaceutical composition is administered internally (by ingestion, inhalation, injection, etc.), topically (to the skin for absorption into the body), or vice versa to a subject, and (2) the pharmaceutical composition prevents a disease when the subject is exposed to the disease before and after exposure, if the subject is at risk of developing the disease and experiencing the symptoms / clinical conditions normally associated with the disease. Or, if the subject has the disease and experiences some or all of the symptoms / clinical conditions normally associated with the disease, with some difference in the degree of severity, the subject recovers from the disease to a normal state of health.

[0064] There is further provided a kit for carrying out the method of the present invention. By "kit" is intended any product (e.g., a package or container) containing at least one reagent, such as the pH buffer of the present invention. The kit can be advertised, distributed, or sold as a means for carrying out the method of the present invention. Further, the kit can include an accompanying document that describes the kit and its method of use. Any or all of the kit reagents can be provided in a container that protects them from the external environment, such as a sealed container or a pouch.

[0065] In an advantageous embodiment, the kit container can further contain a pharmaceutically acceptable carrier. The kit can further contain a sterile diluent, which is preferably stored in a separate additional container. In another embodiment, the kit further includes an accompanying document that includes printed instructions indicating the use of the pH buffer in combination therapy with an anti-pathogen agent as a method of treating and / or preventing a disease of a subject. The kit can also include an additional container containing an additional anti-pathogen agent (e.g., amantadine, rimantadine, and oseltamivir), an agent that enhances the effect of such an agent, or other compounds that improve the effectiveness or tolerance of the treatment. The kit can also contain at least one reagent used to carry out certain conventional techniques within the scope of the art (i.e., nucleic acid extraction).

[0066] The practice of the present invention, unless otherwise specified, can use conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant technology), cell biology, biochemistry, and immunology, which are within the scope of the art. Such conventional techniques include polymer array synthesis, hybridization, ligation, and detection of hybridization using labels. By referring to the above examples described herein, suitable techniques can be specifically described. However, other equivalent conventional procedures can of course also be used. Such conventional techniques and descriptions can be found in Genome Analysis: A Laboratory Manual Series (Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCR Primer: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press), Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, N.Y., Gait, “Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press, London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3 rd Ed., W.H. Freeman Pub., New York, N.Y. and Berg et al. (2002) and other standard laboratory manuals.

[0067] Biochemistry, 5 th Ed., W.H. Freeman Pub., New York, N.Y. are all hereby incorporated by reference in their entirety for all purposes.

[0068] All references cited in this application are hereby incorporated by reference in their entirety to the extent not inconsistent with this specification.

[0069] The advantages described above, and those that become apparent from the foregoing description, are efficiently achieved, and since specific changes can be made to the above configuration without departing from the scope of the present invention, it will be understood that all matters are intended to be included. Matters included in the foregoing description or shown in the accompanying drawings are to be construed as illustrative rather than in a limiting sense.

[0070] Also, it should be understood that the following claims are intended to embrace all of the general and specific features of the invention described herein, and all statements of the scope of the invention that are said to lie therebetween, as a matter of language. The present invention has been described.

Claims

**Claim 1**: A nucleic acid delivery vehicle, wherein the nucleic acid delivery vehicle is a non-pathogenic bacterium containing a prokaryotic vector, the vector containing a DNA molecule encoding one or more therapeutic nucleic acids and a prokaryotic promoter that controls transcription of the therapeutic nucleic acid, the bacterium expressing invasin and exhibiting a reduced therapeutic nucleic acid degradation phenotype, and being engineered to increase the stability, size, or duration of the desired effect or activity of the therapeutic nucleic acid, the bacterium being a) engineered to express Clostridium thermocellum HEN1 protein or the C-terminal methylase domain of Clostridium thermocellum HEN1, and / or b) a dsRNA-binding protein (dsRBP) or one or more binding domains or fragments of a dsRBP, A nucleic acid delivery vehicle thus engineered. **Claim 2**: The nucleic acid delivery vehicle according to claim 1, wherein the bacterium is engineered to express a dsRBP or one or more binding domains or fragments of a dsRBP. **Claim 3**: The nucleic acid delivery vehicle according to claim 1 or 2, wherein the dsRBP or one or more binding domains or fragments of the dsRBP are encoded in a therapeutic nucleic acid-producing plasmid. **Claim 4**: The nucleic acid delivery vehicle according to claim 1 or 2, wherein the bacterium is engineered to express a TAR RNA-binding protein (TRBP) or a dsRBP-binding domain derived from TRBP. **Claim 5**: The nucleic acid delivery vehicle according to any one of claims 1 to 4, wherein the bacterium is engineered to express Clostridium thermocellum HEN1 protein or its active methyltransferase fragment or domain, and a dsRNA-binding protein (dsRBP) or one or more binding domains or fragments of a dsRBP. **Claim 6**: The nucleic acid delivery medium according to any one of claims 1 to 5, wherein the bacterium is engineered to express influenza virus hemagglutinin-1 (HA-1) protein.

7. The nucleic acid delivery medium according to any one of claims 1, 2, and 4 to 6, wherein the bacterium is engineered to express hlyA, inv, HA-1, TRBP, and / or HEN1 from the chromosome.

8. The nucleic acid delivery medium according to any one of claims 1 to 7, wherein the therapeutic nucleic acid interferes with one or more target influenza virus RNA molecules.

9. The nucleic acid delivery medium according to any one of claims 1 to 8, wherein the therapeutic nucleic acid is a nucleic acid selected from small interfering RNA / small hairpin RNA (siRNA / shRNA), microRNA (miRNA), antagomiR, RNA or DNA aptamer, messenger RNA (mRNA), splice-switching oligonucleotide, antisense oligonucleotide, antigene oligonucleotide, DNAzyme, RNA decoy, ribozyme, peptide nucleic acid, oligomer, and interfering defective particle.

10. The nucleic acid delivery medium according to any one of claims 1 to 9, wherein the bacterium is an engineered bacterium from the group of bacteria consisting of Listeria, Yersinia, Rickettsia, Shigella, Escherichia coli, Salmonella, Legionella, Chlamydia, Brucella, Neisseria, Bordetella, Borrelia, Coxiella, Mycobacterium, Helicobacter, Staphylococcus, Streptococcus, Porphyromonas, Vibrio, Treponema, and Bifidobacteria.

11. The nucleic acid delivery medium according to any one of claims 1 to 10, wherein the bacterium is engineered Escherichia coli (E. coli).

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