A cross-kingdom platform for therapeutic nucleic acid delivery

Abstract

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

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 480,875, filed April 3, 2017. [Background technology]

[0002] The present invention relates to the treatment and prevention of diseases. More specifically, the present invention relates to a cross-kingdom platform with improved invasiveness for the delivery of therapeutic nucleic acids to epithelial tissues, said cross-kingdom platform being engineered to produce nucleic acids with enhanced stability.

[0003] The therapeutic application of nucleic acids (NAs) has strong potential for a wide range of clinical targets. However, the delivery of NAs to specific cells and tissues, and their transient targeting effect, remain major obstacles in exploiting their potential and clinical applications.

[0004] Ribonucleic acid (RNA) interference (RNAi) is a technique for specifically inhibiting gene expression, often by using small double-stranded short interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) with a length of approximately 21-27 nucleotides.

[0005] The efficacy of siRNA and shRNA may be limited by a number of factors. First, siRNA and shRNA are easily degraded by ribonucleases (RNases), thereby reducing their efficacy. Second, they cannot penetrate cell membranes, resulting in very low bioavailability. Because siRNA cannot independently cross cell membranes, delivery vehicles such as viral vectors and synthetic carriers are often used. These viral and synthetic siRNA vehicles pose significant limitations and concerns for clinical efficacy, such as cell death, RNAi / microRNA saturation, and hepatotoxicity and tumor formation. Synthetic vehicles often have low delivery efficiency and require high doses to achieve clinical efficacy, which can be costly and often toxic. Furthermore, the application of siRNA systems has historically been associated with transient RNAi effects, meaning that the silencing effect required for clinical targeting is temporary. Although chemical modifications of shRNA / siRNA and the application of numerous nanotechnology-based delivery methods have been pursued, the limited delivery efficiency to a limited number of target tissues and the temporary effect of RNAi remain major obstacles. Delivery platforms for targeted intracellular delivery of RNAi agents and other nucleic acids, including deoxyribonucleic acid (DNA), ribonucleic acid (RNA), small interfering RNA / small hairpin RNA (siRNA / shRNA), microRNA (miRNA), antagomir, aptamers, messenger RNA (mRNA), splice-switching oligonucleotides, defective interfering particles, and antisense oligonucleotides, to specific tissues are essential to harness the therapeutic potential that nucleic acid-based technologies hold for translational medicine. The present invention provides such delivery platforms as presented in the following disclosure. Summary of the Invention

[0006] Interkingdom NA delivery vehicles have been significantly advanced by using several unique features taught herein. The present invention provides modified interkingdom delivery vehicles with a number of important and substantial features and / or advances to conventional delivery platforms. The first advance is to express the double-stranded RNA-binding domain (dsRBD) of a double-stranded RNA-binding protein (dsRBP), such as transactivation response (TAR) RBP (TRBP), for co-packaging within the interkingdom 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 a key component of siRNA and miRNA-mediated gene silencing. The second advance 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, once shRNA production is maximized, arresting bacterial growth and incubating the culture below 37° C. inhibits the decay of double-stranded (ds)RNA, thereby improving the stability of the encoded therapeutic shRNA. The third advancement 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 the siRNA, ultimately increasing siRNA stability and enhancing RNAi. Additional improvements and accomplishments are described below. Each of these proposed improvements represents a unique approach to improving shRNA stability and therapeutic longevity of siRNA.

[0007] The technology taught herein offers many advantages over current state-of-the-art platforms and other NA-based delivery applications, including: (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 expensive synthetic siRNA production; (4) clinical administration adaptable for oral, intranasal, ocular, vaginal, rectal, and respiratory administration; (5) utilization of a naturally derived vehicle with a confirmed safety record for clinical purposes (non-irritating to delicate mucosal tissues); (6) rapid scale-up for disease preparedness in the event of an emergency (i.e., influenza pandemic); (7) feasible therapeutic applications for infectious, immune, and allergic diseases; (8) compatibility with existing vaccination, antibiotic, antiviral, etc. regimens; and (9) elimination of the need for penetration enhancers that may increase the risk of toxin and pathogen entry. (10) NAs are generated by prokaryotic RNA polymerases and delivered in a sequence-independent manner, making them a highly versatile platform for delivering NA-based therapeutics alone or in combination (i.e., siRNA / shRNA, miRNA, antagomir, aptamers, mRNA, splice-switching oligonucleotides, defective interfering particles, and antisense oligonucleotides).

[0008] This new approach provides a new perspective on NA delivery and addresses many of the shortcomings associated with current NA delivery approaches. In general, the improvements taught herein enhance the functionality of the interkingdom delivery vehicle for many applications, including therapeutic applications, including: (a) improving the stability of the NA produced and delivered, such as by expressing HEN1 and TRBP, and providing an RNase R knockout; (b) improving the stability of the interkingdom delivery vehicle itself, such as by expressing certain genes (e.g., inv, hlyA, HEN1, TRBP, and / or HA-1 genes) on the chromosome rather than on a plasmid, thereby improving the stability of the delivery vehicle and making it easier to 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 on a plasmid, although expression of these genes from a plasmid is also contemplated herein); and (d) removing the need for antibiotic selection, which is not ideal for manufacturing / regulation or therapeutic applications. All of these modifications act synergistically to improve the functionality of the interkingdom NA delivery system for therapeutic applications.

[0009] In a first aspect, the invention provides a non-pathogenic bacterium or yeast comprising a prokaryotic or eukaryotic vector, as appropriate. The vector comprises a DNA molecule encoding one or more therapeutic nucleic acids (e.g., short double-stranded RNAs) and a promoter controlling the transcription of the short double-stranded RNAs, which interfere with one or more target RNA molecules, and the bacterium is engineered to express one or more dsRNA-binding proteins (dsRBPs). Many dsRBPs are known to bind dsRNA with high affinity to prevent degradation, bind and stabilize miRNA precursors, and play important roles in siRNA and miRNA-induced gene silencing. Expression of dsRBPs has been proposed to increase stability and concentration of available therapeutic nucleic acids by limiting degradation, increasing the concentration of miRNA precursors and their mature products, and providing the components required for participation in target gene silencing. In an advantageous embodiment, the dsRBP is a TAR RNA-binding protein (TRBP). Furthermore, this platform has the capacity to encode / deliver a wide range of other NA molecules, such as siRNA / shRNA, miRNA mimics, inhibitors, etc. Other possible dsRNA-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 comprising a prokaryotic or eukaryotic vector, the vector comprising a DNA molecule encoding one or more NA molecules, including, for example, short double-stranded RNA (e.g., siRNA / shRNA), and a promoter controlling the transcription of the short double-stranded RNA, the short double-stranded RNA interfering with one or more target RNA molecules, the bacterium being 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, the vector comprising, for example, a DNA molecule encoding one or more NA molecules, including short double-stranded RNAs (e.g., siRNA / shRNA), and a promoter controlling the transcription of the short double-stranded RNA, the short double-stranded RNAs interfering with one or more target RNA molecules, the 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, including HEN1 or another methyltransferase gene having 2'-0-methyltransferase activity, whereby the methyltransferase promotes methylation of the 3'-terminal nucleotide of the siRNA. Methylation of the 3'-terminal nucleotide provides protection against major causes of RNA degradation, including 3-5' degradation and 3' uridylation, ultimately increasing the stability and / or concentration of the therapeutic NA available for engagement in the RNAi pathway.

[0012] In a fourth aspect, the invention provides a non-pathogenic bacterium or yeast comprising a prokaryotic or eukaryotic vector, the vector comprising a DNA molecule encoding one or more NA molecules, including, for example, short double-stranded RNA (e.g., siRNA / shRNA), and a promoter controlling the transcription of the short double-stranded RNA, the short double-stranded RNA interfering with one or more target NA molecules, the bacterium being engineered to express a dsRNA binding protein (dsRBP) and the HEN1 gene. In an advantageous embodiment, the E. coli bacterium of the fourth aspect is engineered to be deficient in RNase activity. In a particularly advantageous embodiment, the 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, the vector comprising a DNA molecule encoding one or more nucleic acids and a prokaryotic promoter controlling transcription of said therapeutic nucleic acid, wherein the non-pathogenic bacterium or yeast has been 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 to treat an organism for a disease state or to study the effect of a gene or gene product of the 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 pathogen nucleic acid sequences. Therapeutic NAs (TNAs) can interfere with many types of target molecules (RNA, DNA, other oligonucleotides, etc.), depending on the nature and identity of the therapeutic nucleic acid. TNAs can interact (interfere, target, modify, alter) one or more host or pathogen factors, which can be oligonucleotides, proteins, genes, etc. As an example, TNAs can interact with oncogenes or products of oncogenes to modulate cancer and study cancer or cancer treatment. In a further advantageous embodiment, the reduced nucleic acid degradation phenotype increases the stability, magnitude, and / or duration of the therapeutic effect or activity of interest. The non-pathogenic yeasts are believed to use appropriate eukaryotic plasmids / episomes and promoter sequences.

[0015] In a sixth aspect, the present invention provides a non-pathogenic bacterium or yeast comprising a prokaryotic or eukaryotic vector, the vector comprising a DNA molecule encoding one or more nucleic acids and a prokaryotic or eukaryotic promoter controlling transcription of the therapeutic nucleic acid, the non-pathogenic bacterium or yeast being 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 increase expression of these genes (more stable and greater expression when expressed from the bacterial genome versus a plasmid) and improves the stability of the delivery system. It creates a system where the bacteria is more likely to express from the genome compared to a system where the bacteria must maintain a plasmid and express genes from a plasmid. In other words, it results in more stability and greater expression when expressed from the bacterial genome rather than a plasmid. It is believed that the non-pathogenic yeast will use the appropriate eukaryotic plasmid / episomal 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, in which: [Figure 1A] Figure 1 is a diagrammatic representation of two versions of the therapeutic NA-producing plasmid. (A) Plasmid version A contains all genes required for targeting and delivery in addition to enhancers of therapeutic NA stability and is very large and unstable at over 12,100 bp. (B) Plasmid version B contains only the therapeutic NA sequence and the HisD antibiotic-free selection marker since all other required genes have been transferred to the delivery vehicle chromosome for improved efficiency and stability as well as a small plasmid size of approximately 4,700 bp. [Figure 1B] Same as above explanation. [Diagram 2]Figure 2 shows the removal of RNase R activity. Eliminating RNase R function in the delivery vehicle means that this heat shock protein is not induced at low incubation temperatures. NAs are more stable at low temperatures, for example, when incubated at 17°C in the absence of RNase R, resulting in higher concentrations of therapeutic NAs for delivery to disease sites. [Diagram 3] FIG. 3 is a diagrammatic representation of the mechanism of action of the inter-kingdom nucleic acid delivery vehicle in respiratory epithelial cells with enhancement as taught herein. [Figure 4] Figure 4 illustrates an enhanced delivery vehicle (shown on the left) that expresses the cell surface proteins invasin and HA-1, which bind to β1 integrin and sialic acid receptors, respectively, for specific delivery to disease-relevant sites. This allows for greater invasiveness compared to the current state of the art (shown on the right), which expresses only the invasin protein. The enhanced delivery vehicle can also compete with pathogens that bind to the same host cell surface receptors, thereby reducing pathogen uptake. [Diagram 5] Figure 5 shows the delivery vehicle expression of dsRBP such as TRBP. It binds to dsRNA and stabilizes it through processing by Dicer to generate siRNA, which is then loaded into the RISC complex for guide strand recognition and interaction with the target sequence. shRNA (and other dsRNA nucleic acids) that are not bound to TRBP are vulnerable to degradation by RNase. Greater availability and abundance of TRBP correlates with greater shRNA stability and higher concentration of guide strand siRNA sequences. [Figure 6]Figure 6 is a diagram and associated histogram. (Top diagram) Enhanced delivery vehicles express 3' methyltransferases such as HEN1 that methylate the 3' terminal nucleotides of the forward and reverse strands of siRNA, thereby providing protection from RNases that normally cause degradation starting from the 3' terminal nucleotide. The 3' methyl group also prevents uridylation, which is known to contribute to RNA degradation. (Bottom graph) Following production and delivery by enhanced delivery vehicles expressing HEN1, the graph shows the predicted copies of methylated siRNA (me-siRNA) per ng cDNA over time compared to unmethylated siRNA (siRNA). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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 a variety of different eukaryotic species. Therapeutic nucleic acids initially showed great potential for the treatment of disease, but the shortcomings of delivering these nucleic acids to target tissues and cells have not fully realized this potential. Significant improvements can be made to the magnitude and duration of silencing effects, and the use of delivery platforms that target multiple relevant tissues for future development of broad clinical applications. In a more focused sense, improvements can be made to have enhanced stability of both the delivery vehicle and the nucleic acid produced by the delivery vehicle, improved penetration of the vehicle, improved safety, and improved properties 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 content, batch-to-batch variability, high production costs, and limited targeting ability. This interkingdom delivery vehicle is based on the use of non-pathogenic bacteria-mediated RNAi delivery vehicles that use natural receptor-mediated endocytosis for specific intracellular delivery at the site of action in tissues, resulting in accumulation of shRNA in endosomes and efficient release of shRNA payload into the cytoplasm of target cells for RNAi silencing. These interkingdom vehicles were Escherichia coli (E. coli) cells engineered to specifically target mucosal epithelial tissues and deliver a payload of constitutively generated shRNA in a sequence-independent manner.

[0019] Cross-kingdom RNA interference using an engineered RNAi delivery platform using non-pathogenic bacteria containing shRNA expression plasmids has been used to dramatically reduce the severity of AIV infection and inhibit AIV shedding in 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.) A cross-kingdom RNA system for shRNA delivery is taught in US 2016 / 0177296 A1 by Linke et al., the contents of which are incorporated by reference in their entirety. Cross-kingdom nucleic acid delivery systems use non-pathogenic bacteria such as E. coli engineered to transcribe shRNA from a plasmid. Interkingdom nucleic acid interference has become relevant for clinical applications through the inclusion of additional genes in the shRNA expression plasmids that promote both bacterial entry into target cells and the release of non-pathogenic bacterial contents after their entry into the target cells. Specifically, a system encoding two factors or genes has been developed to enable the delivery of shRNA to mucosal epithelial cells (the invasin gene (Inv) and the listeriolysin O gene (hylA). The Inv gene is required for the expression of the invasin protein on the surface of Escherichia coli (E. coli) (Xiang et al. 2006), which interacts with the β(1) integrin receptor present on mucosal epithelial cells and triggers receptor-mediated endocytosis (Conte et al. 1994; Isberg and Barnes 2001; Isberg and Leong 1990). The hlyA gene encodes the pore-forming toxin, listeriolysin O (LLO), which promotes the rupture of the endosomal membrane and the subsequent release of the 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. 2003). 2006).The interkingdom nucleic acid delivery vehicle used in the above studies was a diaminopimelic acid (Dap) auxotroph and kanamycin resistant mutant.

[0020] These vehicles express Listeriolysin O (LLO), a membrane pore-forming toxin that allows E. coli to escape host endosomes for release in the cytosol. Lack of nutrients inside the endosome promotes lysis and release of the LLO toxin, which disrupts the endosomal membrane. The released shRNA is then processed into siRNA and incorporated into the RNA-induced silencing complex (RISC).

[0021] These bacteria can modulate the delivery of shRNA: once inside the host endosome, they become nonviable and no longer produce shRNA, thereby eliminating the possibility of overloading the RISC machinery and causing unwanted side effects in the host.

[0022] The interkingdom 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, that can constitutively produce the shRNA (or other therapeutic nucleic acid) under the control of a prokaryotic RNA polymerase against a clinical target gene of interest. The inclusion of a prokaryotic RNA polymerase involves the bacterium as the expression source of the product of interest (e.g., therapeutic nucleic acid) rather than the target cell. In the case of viral targeting, this platform technology can encode several shRNAs to target multiple viral RNA targets. Furthermore, other therapeutic nucleic acids can be included in place of or in addition to the sequence that produces the shRNA in the expression cassette, allowing the system to target additional pathogen or disease-related molecules. This mixture approach limits the risk of viral escape through mutation. Furthermore, this platform has the capability to encode a wide range of other NA molecules, including miRNA mimics and inhibitors. In other words, the proposed system has the capacity to deliver other nucleic acids in addition to siRNA / shRNA, which is a significant improvement over the current state of the art, which is not adapted to deliver, encode and deliver molecules such as microRNA mimics or microRNA inhibitors.

[0023] The interkingdom delivery platform employs non-pathogenic bacteria that are auxotrophic for diaminopimelic acid (Dap). Certain bacteria, including 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, non-colonizing bacteria, these vehicles pose no known risk to the host and have a confirmed safety record. Furthermore, 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 clinical disease, virulence, or tissue pathogenesis observed in the species tested. Thus, the bacteria are "non-pathogenic" and do not cause disease to the host cells or target species.

[0024] We propose to improve current interkingdom delivery vehicles with unique modifications centered on improving the stability of the delivered nucleic acid (NA), improving the resistance of double-stranded interfering RNA to cytolytic mechanisms, improving invasiveness, and / or improving the manufacturing process, safety, and regulatory issues associated with the delivery system. 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) will increase the availability of shRNA and the processing of more siRNA. The rationale for improving invasiveness is that the interkingdom delivery vehicle will have a greater chance of attaching to a wider range of host cells, thereby increasing the number of interkingdom delivery vehicles that are taken up and intracellularized. These improvements will provide stronger and longer-lasting RNAi effects for optimal therapeutic application. These improvements, taken individually or in any combination, will facilitate the translational potential of the technology for therapeutic and clinical applications.

[0025] Improved penetration and stability of delivered NA can be achieved through multiple approaches taught herein. The first approach to improve the delivery vehicle is to include dsRNA binding proteins (dsRBPs) that increase the stability of dsRNA. The second approach to improve the delivery vehicle is to eliminate the activity of key bacterial enzymes involved in RNA degradation. The third approach to improve the delivery vehicle is to include genes / proteins that cause stable methylation of dsRNA. In one embodiment, TAN RNA binding protein (TRBP) and the C-terminal methylase domain of the HEN1 gene are encoded, and RNase R activity is knocked out in the Escherichia coli (E. coli) bacterial delivery vector. These formulation improvements are utilized to deliver nucleic acids, including shRNA, to a wide range of mucosal epithelial tissues, including upper / lower respiratory, oral, gastrointestinal (GI), vaginal, rectal, and ocular epithelia. Methods of administering these improved interkingdom NA delivery vehicles include intranasal administration to the nasal cavity for local action, aerosolization for upper and lower respiratory targeting, absorption in the oral cavity for buccal delivery, ingestion for GI adsorption, application to the delicate genital mucosal epithelium, 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, cancer, immunological) in a wide range of species (human, avian, porcine, bovine, canine, equine, feline).

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

[0027] Example 1 - Engineering E. coli to express dsRBP In a first aspect, improved delivery vehicles 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 E. coli to increase the stability of dsRNA produced by non-pathogenic bacteria by including dsRBP.

[0028] The family of dsRBPs contains one or more evolutionarily conserved dsRNA-binding domains (dsRBDs) of 65–68 amino acids found in eukaryotic, prokaryotic, and virally encoded products (Ryter et al. 1998). These dsRBDs were first recognized in 1992 as responsible for 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 exclusively with dsRNA in a non-sequence-specific manner (Ryter et al.; Manche et al.). TRBP contains three highly conserved dsRBDs, the first and second of which have high affinity to bind and protect dsRNA from 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 to bind and prevent shRNA siRNA degradation in vitro and in vivo, play a role in siRNA and microRNA (miRNA)-induced gene silencing, and play a physiological role in cells by binding miRNA hairpin precursors and 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 interkingdom vectors. In summary, TRBP functions like a dsRNA gatekeeper. Co-encoding this dsRNA binding protein in interkingdom vectors represents a formidable approach to improve delivery vehicles of interkingdom NAs. Other dsRBPs whose expression can be examined instead of or in addition to TRBP include nuclear factor 90 (NF90), ZNF346, SiD-1, and Ku70 (and combinations thereof).

[0029] Example 2 - RNase R knockout In a second aspect, the improvement of the properties of the delivery vehicle can be achieved by removing or reducing the activity of the enzymes responsible for RNA degradation. In bacteria such as E. coli, structured RNA duplex destruction is carried out exclusively 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). As in the case of shRNA / siRNA molecules, RNase R can degrade these dsRNAs by specifically binding the 3' single-stranded overhangs (Cheng and Deutscher, Vincent and Deutscher 2006). In fact, RNase R is the only 3'→5' exoribonuclease that can chew extensive secondary RNA structures (Vincent and Deutscher 2009). RNase R is a cold shock protein encoded by the rnr gene, and its activity can be modulated by changing the growth conditions of bacterial cells (Chen et al. 1998; Cheng and Deutscher; Cairrao and Arraiano 2006; Cairrao et al. 2003). Shifting the exponential growth temperature of E. coli from 37°C to 15°C stimulates the RNase R cold shock response (Awano et al. 2007), which increases RNase R activity and leads to greater dsRNA decay. Concurrent studies have demonstrated that RNase R helicase activity plays a necessary catalytic role for effective nuclease activity against dsRNA when the concentration of more stable dsRNA substrates is high, especially at low incubation temperatures (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 37 °C growth temperature 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 temperatures, it would be desirable to lower the culture temperature to maintain high NA concentrations only if cold shock induction of RNase R activity can be avoided. Thus, knockout of RNase R activity in E. coli bacterial delivery vehicles could lead to enhanced delivery of NAs. In modified E. coli bacterial delivery vehicles with RNase R knockout, after exponential growth of bacterial cells when shRNA production is maximized, bacterial growth is stopped and the culture is grown at less than 37°C to inhibit dsRNA decay, thereby enhancing stability of the encoded therapeutic shRNA. Thus, it is believed that an interkingdom system can include non-pathogenic bacteria such as E. coli lacking the rnr gene transformed with a specific shRNA (or other therapeutic nucleic acid) producing plasmid. Furthermore, it is believed that these rnr-deficient E. coli bacteria can be grown at low temperatures (i.e., less than 37°C), such as 33°C or less, 30°C or less, 27°C or less, 25°C or less, 22°C or less, 20°C or less, 17°C or less, preferably 25°C or less, and most preferably 17°C or less. Growth at such low temperatures allows production of the desired shRNA while limiting degradation. Additionally, shRNA-producing plasmids for use in rnr-deficient interkingdom nucleic acid delivery systems could contain genes encoding dsRBDs such as TRBP. One approach to knockout occurs by recombineering a deletion, which essentially creates an E. coli mutant that no longer produces the RNase R protein.Other methods to eliminate the function of the rnr gene include Cre-lox, FLP-FRT, selection-counterselection, and CRISPR / Cas-mediated gene editing. It is believed that knockouts will be targeted beyond just the rnr gene, as there are other genes that can be knocked out to eliminate RNase activity in E. coli. In general, elimination of RNase catalytic activity, including elimination of RNase R in E. coli, can increase 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, improved properties of the delivery vehicle can be achieved by including a gene that causes stabilization of shRNA methylation in a therapeutic NA generating plasmid or the like. As mentioned 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, protecting the siRNA from 3'-5' degradation and 3' uridylation (Ji&Chen 2012; Lifang et al. 2010; Kurth&Mochizuki 2009; Chan et al. 2009). RNAi activity is maintained when siRNA molecules are modified only at the end of the sense strand, or at the 3' end in the case of siRNA hairpins (Czauderna et al. 2003). Methylation of the 3' end is necessary for the function of all plant siRNAs and is observed in some mammalian and bacterial siRNAs as well. The HEN1 gene encodes the methyltransferase Hen1, a methylase that 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 domain of several bacterial species has been shown to be functionally equivalent to eukaryotic homologues (Chan et al. 2009). Using methylation assays, Chan et al. (2009) showed that human and bacterial (Clostridium thermocellum) recombinant HEN1 proteins displayed enzymatically equivalent methylation activity on small single-stranded RNAs (21-30 nt). However, the C-terminal methylase domain of C. thermocellum Hen1 showed improved enzymatic activity over its full-length counterpart and human Hen1, indicating that this domain can function independently. Thus, packaging of the HEN1 gene, or in an advantageous embodiment the C-terminal methylase domain of the HEN1 gene, in an interkingdom delivery vehicle provides a stabilizing effect of the siRNA against exonucleases.In other words, HEN1 actively methylates and stabilizes the encoded shRNA, thereby providing an optimally suitable shRNA for therapeutic application in vivo.

[0031] Example 4 - Non-pathogenic bacteria engineered to express influenza hemagglutinin-1 (HA-1) protein In a fourth aspect, bacteria can be engineered to express influenza hemagglutinin-1 (HA-1) protein, either by genomic (chromosomal) or plasmid expression, to improve localized and targeted delivery of delivery vehicles to mucosal tissues, particularly respiratory mucosal tissues. HA-1 is a glycoprotein found on the surface of influenza viruses, which facilitates viral 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, 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 type A or B viral subtype. The full-length HA-1 gene can be cloned into an shRNA-generating plasmid (or other plasmid constructed to generate therapeutic nucleic acids) using standard cloning methodologies or integrated into the E. coli chromosome using, for example, the Gibson assembly method and commercially available Gibson assembly cloning kits, flippase / flippase recognition site (FLP / FRT) site-specific recombination techniques (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).

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

[0033] Example 5: Removal of antibiotic resistance genes from non-pathogenic bacteria In a fifth aspect, the manufacturing process, safety, and regulatory considerations of the delivery system are improved by removing antibiotic resistance genes, including but not limited to kanamycin resistance, which can be included in the plasmid as a selection marker. Antibiotic resistance genes are included as selection markers in the majority of bacterial expression vectors (Peubez et al. 2010). Antibiotic resistance is a major concern for human and animal health, and regulatory requirements for biologics are becoming more stringent to reflect this (Mignon 2015). Eliminating the need to include antibiotics in the manufacturing process reduces costs and eliminates concerns that the bacterial delivery system may induce antibiotic resistance in unintended hosts. In an advantageous embodiment, the delivery vehicle uses an antibiotic-free system, such as the amino acid auxotrophy and complementation system described in Example 6 below, to specifically select for the desired plasmid-containing bacteria.

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

[0035] In a sixth aspect, the functionality and stability of the delivery system is 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 key genes of an amino acid synthesis pathway are targeted on the E. coli chromosome to create auxotrophic mutants unable to produce the required amino acid. Supplementation of a knockout gene or genes on a plasmid allows survival of only E. coli containing the plasmid, allowing efficient and reliable selection on media lacking amino acids (Peubez et al. 2010; Mignon et al. 2105; Fiedler et al. 2017). Similar principles apply 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, it is synthesised in a 10-step pathway, with the hisD gene product, a bifunctional enzyme histidinol dehydrogenase, catalysing the last two oxidation steps (Ramage et al. 2012; Matte et al. 2003), and the hisB product, a bifunctional enzyme histidinol phosphatase, catalysing the seventh and ninth steps (Winkler & Ramos-Montanez 2009; Chiariotti et al. 1986). The hisA, hisB, hisC, hisD, hisE, hisG, hisH, and hissl gene products are required for histidine synthesis and therefore growth of E. coli in minimal medium (Joyce et al. 2006), but computational modeling and published experiments have shown that hisD or hisB knockout generates strains whose growth is entirely 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 an advantageous embodiment, E. coli is engineered to be histidine auxotrophic, the bacteria lacking a functional hisD gene (product: histidinol dehydrogenase), and an shRNA-producing plasmid (or other plasmid constructed to produce therapeutic NAs) is engineered to express the hisD gene, thereby complementing the histidine biosynthetic pathway and allowing bacterial replication in histidine-deficient media. In another embodiment, E. coli is engineered to lack a functional hisA, hisB, hisC, hisE, hisG, or hisH gene, and a prokaryotic vector is engineered to express the missing gene. In the case of genes involved in histidine synthesis, the gene can be expressed on a 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 interkingdom delivery vehicles In a seventh aspect, the functionality and stability of the delivery system is improved by allowing expression of individual or multiple therapeutic NAs on a single plasmid. Many different configurations are possible, including 1) to 3) below. 1) NAs expressed in a single expression cassette in tandem under the control of a single promoter. 2) Independent expression cassettes, each under the control of its own promoter and terminator, but with competitive expression by using the same promoter sequence. 3) NAs in independent expression cassettes, each under the control of a different promoter for different expression. As demonstrated in numerous published studies, the specific details of the plasmid configuration, including spacer length and sequence, promoter sequence and strength, therapeutic NA type (e.g. shRNA) and size, and 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 with the HIV model, 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). Expression of multiple NAs on a single plasmid achieves several important improvements: simply culturing a single strain of E. coli containing the relevant NA targets, which are viewed separately as a single "active" component for a given regulatory agency, rather than each NA-expressing plasmid within the E. coli bacterium, simplifies manufacturing, expands targeting capacity, reduces the risk of pathogen mutation, and allows for the treatment of multiple diseases with a single treatment.

[0038] Tandem expression of multiple specific nucleic acid sequences on a plasmid provides a system that is easier and cheaper to manufacture (e.g., can be manufactured as a monoculture), even when expressing multiple nucleic acids with different targets. This increases the stability of the system by reducing variability and making the manufacturing process more predictable. This also facilitates simplified regulatory pathways (U.S. and international) as a single active / therapeutic drug rather than multiple, reducing costs and supply requirements.

[0039] Example 8 - Chromosomal expression of Inv, LLO, HA-1, HEN1, and / or TRBP In an 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 expression of these genes onto the E. coli chromosome (or other non-pathogenic bacteria or yeast). This produces a bacterial strain that stably expresses the Inv, LLO, HA-1, HEN1, and / or TRBP genes. In constructing the strain, one can find a region along the bacterial chromosome where the expression cassette is integrated into the engineered bacteria. For example, using the Gibson assembly method and commercially available Gibson assembly cloning kits, flippase / flippase recognition site (FLP / FRT) site-specific recombination techniques (Posfai et al. 1994, Bertram et al. 2009), selection-counter-selection strategies, or CRISPR / Cas engineering (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 truncated portions thereof, can be integrated into the E. coli chromosome. Genomic expression is an improved approach over plasmid expression for several reasons. Plasmid delivery and maintenance puts E. coli cells at a selective disadvantage. The reliance on expression from plasmids requires E. coli cells to be grown under specific environmental conditions to maintain the presence of the plasmid inside the bacteria. This can complicate production and increase bacterial stress. During production, the various conditions of large-scale fermentation can affect the replication of plasmid DNA in E. coli cells and therefore greatly affect 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, especially when growth conditions fluctuate.Furthermore, E. coli cells grow faster when these genes are removed from the plasmid and integrated into the E. coli chromosome for expression (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 greater abundance of these genes compared to plasmid expression. The reliance on plasmid expression means that the plasmid must often be replicated to generate a sufficient number of copies to distribute to both daughter cells after division of the mother cell. This results in lower expression (lower abundance) of the plasmid-derived genes in the progeny cells compared to stable and consistent genome-derived expression. Overall, recombining Inv, LLO, HA-1, HEN1, and / or TRBP into the E. coli chromosome improves the stability and increases the expression of these genes, especially in large-scale manufacturing processes.

[0040] In one embodiment of the above method of the invention, the bacteria is non-pathogenic or non-pathogenic. In another aspect of this embodiment, the bacteria is therapeutic. In another aspect of this embodiment, the bacteria is invasive or is engineered to be invasive and invade host cells. Examples of such bacteria include Listeria, Yersinia, Rickettsia, Shigella, E. coli, Salmonella, Legionella, Chlamydia, Brucella, Neisseria, Burkholderia, Bordetella, Borrelia. Examples of the microbial organisms that may be used include 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 an advantageous embodiment of the invention, the invasive delivery vehicle containing the NA molecule is introduced into the host by intravenous, intramuscular, intradermal, intraperitoneal, oral, intranasal, intraocular, intrarectal, intravaginal, intraosseous, oral, immersion, topical, intraurethral, ​​and aerosol administration inoculation routes.

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

[0043] Routes of pathogen entry across mucosa The surfaces through which pathogens are most likely to invade 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 from pathogens to the interior of the body and allergens in the external environment. Epithelial cells form tight junctions, forming a dynamic layer on all mucosal surfaces (Parham 2014; Presland & Jurevic 2002). The moist mucus layer that covers mucosal surfaces contains a mixture of "defensive 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, digestive tract, genitourinary tract, and eyes, represent major systems that contain 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 successfully breaches the mucosal defenses, a disease response is usually contained to that compartment of the body (Janeway et al. 2001).

[0044] Exemplary viruses that can be targeted by the proposed interkingdom NA delivery system: Numerous diseases affect multiple tissue types, including mucosal epithelium. Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) replicate in mucosal epithelial cells, including the mouth, eyes, and genital tract (Chentoufi&Ben ohamed 2012; Karasneh&Shukla 2011; NIAID). In the United States, approximately 65% ​​of people are seropositive for HSV-1, but the global prevalence is much higher, with approximately 90% of people thought to be infected with HSV-1 and / or HSV-2 (Wald&Corey 2007). In developed countries, HSV-1 is the leading cause of corneal blindness and viral encephalitis (Chentoufi&BenMohamed 2012). Perinatal infection with HSV-2 in the birth canal is responsible for approximately 85% of neonatal herpes cases (Chentoufi&BenMohamed 2012). Influenza is a globally significant and highly contagious virus that infects respiratory tissues, including the mucous membranes of the eyes, nose, lungs, and throat. Despite the availability of vaccinations, millions of people become sick with seasonal influenza each year, and emerging and pandemic strains of the virus remain a threat (NIAID). Human papillomaviruses (HPV) can infect mucosal epithelial cells of the mouth, throat, and upper respiratory tract (Nguyen et al. 2014; Rautava & Syrjanen 2011). HPV can also infect the anogenital epithelial mucosa and is one of the most common sexually transmitted diseases in the world, with an estimated 1 to 5.5 million new cases occurring annually in the United States alone (Burd 2003; NIAID).

[0045] Other important diseases that affect mucosal epithelial cells of the respiratory system include leprosy, tuberculosis, and highly contagious 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 per year (NIAID). Sexually transmitted diseases such as gonorrhea, chlamydia (Becker 1996), and syphilis are also major concerns in the health of urogenital mucosal epithelial cells, including vaginal and rectal tissues (NIAID). It is believed that the interkingdom NA delivery system taught herein can be used to treat these diseases through the delivery of targeted shRNA.

[0046] Many different diseases specifically affect mucosal epithelia of different tissue types, and addressing pathogen replication at these sites provides therapeutic treatment options. This delivery platform has the potential to deliver therapeutic NAs to a wide range of mucosal epithelial tissues. Thus, in addition to improving the properties of interkingdom NA delivery vehicles, we demonstrate the feasibility of delivering these vehicles to several clinically relevant mucosal epithelial tissues that are susceptible to disease.

[0047] These tissues include the upper / lower respiratory tract, oral, GI, vaginal, rectal, and ocular epithelia.

[0048] The platform taught herein utilizes efficient / suitable administration applications that target highly specific and clinically relevant mucosal epithelial tissues to facilitate the delivery of NAs, providing a major advancement in the field of human medicine for NA systems. The delivery vehicle can be administered to the nasal cavity for local action, aerosolized for upper and lower respiratory tract diseases, absorbed in the oral cavity for buccal delivery, ingested for GI adsorption, applied to delicate genital mucosal epithelium, and administered for topical 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 properties. This inter-kingdom NA delivery system platform represents a major advancement in the field of NA therapy and has a vast number of therapeutic applications, addressing a vast number of diseases that affect a wide range of clinically relevant tissues.

[0049] Glossary of Claim Terms The term "administration" and variations thereof with respect to the compounds of the invention (e.g., "administering" a compound) refers to the introduction of the compound into the system of a subject in need of treatment. When the compounds of the invention are provided in combination with one or more other active agents (e.g., an AIV vaccine, etc.), "administration" and variations thereof are understood to include simultaneous and sequential introduction of the compound and the other agent, respectively.

[0050] As used herein, the term "composition" is intended to encompass products that contain specified ingredients in specified amounts, as well as products that result directly or indirectly from combinations of the specified ingredients in the specified amounts.

[0051] As used herein, the term "therapeutically effective amount" refers to an amount of an active compound or pharmaceutical agent that elicits a biological or medical response in a tissue, system, animal or human as desired by a researcher, veterinarian, physician or other clinician. With respect to viral infections, an effective amount includes an amount sufficient to prevent contracting disease or reduce the severity of disease, as evidenced by clinical disease, clinical symptoms, viral titer or viral shedding from a subject, or 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 prevent 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 beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviation of symptoms, reduction in the extent of viral infection, stable (i.e., not worsening) viral infection, prevention or slowing of spread of viral infection (e.g., shedding), prevention, slowing, slowing of progression of viral infection, and / or maintenance of weight / weight gain. The methods of the invention contemplate any one or more of these aspects of treatment.

[0053] A "pharmacologically acceptable" ingredient is one that is suitable for use in animals without undue adverse side effects (e.g., toxicity, irritation, allergic response) commensurate with a reasonable benefit / risk ratio.

[0054] A "safe and effective amount" refers to the amount of a component that is sufficient, when used in the methods of the invention, to produce the desired therapeutic response without undue adverse side effects (toxicity, irritation, allergic response, etc.) commensurate with a reasonable benefit-risk ratio.

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

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

[0057] As used herein, the term "comprising" is intended to mean that the products, compositions, and methods include the referenced components or steps, but do not exclude others. "Consisting essentially of" when used to define products, compositions, and methods shall mean excluding other components or steps of significant essential importance. Thus, a composition consisting essentially of the recited components would not exclude trace contaminants and pharma- ceutically 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, e.g., a bacterium or a bacterial therapeutic particle (BTP), refers to a microorganism that is capable of delivering at least one molecule, e.g., an RNA or a DNA molecule encoding an RNA, to a target cell. An invasive microorganism can be a microorganism that is capable of passing through a cell membrane and thereby entering the cytoplasm of said cell and delivering at least a portion of its contents, e.g., an RNA or a DNA molecule encoding an RNA, to a target cell. The process of delivering at least one molecule to a target cell preferably does not significantly alter the invasive apparatus.

[0059] The term "interkingdom" as used herein refers to a delivery system that uses bacteria (or other invasive microorganisms) to produce and deliver nucleic acids intracellularly (i.e., across kingdoms: prokaryotes to eukaryotes, or phyla: invertebrates to vertebrates) within a target tissue for processing without recombining the host genome.

[0060] Invasive microorganisms include microorganisms that can naturally deliver at least one molecule to a target cell, such as by penetrating a cell membrane, e.g., a eukaryotic cell membrane, and entering the cytoplasm, as well as microorganisms that are not naturally invasive and are modified, e.g., genetically modified, to become invasive. In another preferred embodiment, a naturally non-invasive microorganism can be modified to become invasive by linking the bacterium or BTP to an "invasion factor," also referred to as an "invasion factor" or "cytoplasmic targeting factor." As used herein, an "invasion factor" is a factor, e.g., a protein or group of proteins, that, when expressed by a non-invasive bacterium or BTP, renders 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., US 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 this application, the invasive microorganism is Escherichia coli (E. coli). However, it is believed that additional microorganisms could potentially be adapted to serve as interkingdom delivery vehicles for the delivery of NAs. These non-pathogenic, invasive bacteria and BTPs are invasive or modified to be invasive and can invade host cells via a variety of mechanisms. In contrast to the uptake of bacteria or BTPs by professional phagocytes, which usually results in the destruction of the bacteria or BTPs in specialized lysosomes, invasive bacteria or BTP strains have the ability to invade non-phagocyte host cells.Naturally occurring examples of such intracellular bacteria include Yersinia, Rickettsia, Legionella, Brucella, Mycobacterium, Helicobacter, Coxiella, Chlamydia, Neisseria, Burkholderia, Bordetella, Borrelia, Listeria, Shigella, Salmonella, and others. la, Staphylococcus, Streptococcus, Porphyromonas, Treponema, and Vibrio, but this property can also be transferred to other bacteria or BTPs, such as E. coli, Lactobacillus, Lactococcus, or Bifidobacteriae, including probiotics, by the transfer of invasion-related genes (P. Courvalin, S. Goussard, C. Grillot-Courvalin, CRAcad. Sci. Paris 318, 1207 (1995)). Factors that may be considered or addressed when evaluating additional bacterial species as candidates for use as interkingdom NA delivery vehicles include the candidate's pathogenicity, or lack thereof, the tropism of the candidate bacterium for target cells or the extent to which the bacterium can be engineered to deliver NAs inside target cells, and the value of any synergistic effects the candidate bacterium may provide by triggering 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 many (two or more) molecules of nucleotides linked together. Examples include small interfering RNA / small hairpin RNA (siRNA / shRNA), micro RNA (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 defective interfering particles.

[0062] Therapeutic nucleic acids are NAs as described herein, or closely related compounds used to treat disease, study disease, or achieve desired genetic modifications, or for gene transfer purposes, where specific inhibition, interruption, or alteration of the function of a particular gene or other molecule involved in the disease is deemed therapeutically desirable.

[0063] As used herein, (1) a pharmaceutical composition is administered to a subject internally (by ingestion, inhalation, injection, etc.), topically (to the skin for absorption into the body), or vice versa, and (2) the pharmaceutical composition prevents a subject from contracting the disease and experiencing symptoms / clinical illness normally associated with the disease, either before or after exposure to the disease, or restores a subject to normal health from a disease, when the subject contracts the disease and experiences, to a greater or lesser degree of severity, some or all of the symptoms / clinical illness normally associated with the disease.

[0064] Further provided are kits for carrying out the methods of the invention. By "kit" is intended any article of manufacture (e.g., package or container) that contains at least one reagent, such as a pH buffer of the invention. The kit can be promoted, distributed, or sold as a means for carrying out the methods of the invention. Additionally, the kit can include a package insert that describes the kit and how to use it. 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 pouch.

[0065] In an advantageous embodiment, the kit container may further comprise a pharma- ceutically acceptable carrier. The kit may further comprise a sterile diluent, preferably stored in another additional container. In another embodiment, the kit further comprises a package insert comprising printed instructions directing the use of the combined treatment of the pH buffer and the anti-pathogen agent as a method for treating and / or preventing a disease in a subject. The kit may also comprise 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 another compound that improves the efficacy or tolerability of the treatment. The kit may also comprise at least one reagent used to perform certain conventional techniques within the skill of the art (i.e., nucleic acid extraction).

[0066] The implementation of the present invention can use conventional techniques and explanations of organic chemistry, polymer technology, molecular biology (including recombinant technology), cell biology, biochemistry, and immunology, which are within the scope of the art, unless otherwise specified. Such conventional techniques include polymer array synthesis, hybridization, ligation, and detection of hybridization using labels. Suitable techniques can be specifically described by referring to the above examples described in this specification. However, other equivalent conventional procedures can also be used. Such prior art and explanations 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, NY, Gait, “Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press, London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3 rd Ed., WH Freeman Pub., New York, NY and Berg et al. (2002).

[0067] Biochemistry, 5 th Ed., WH Freeman Pub., New York, NY, all of which are incorporated herein 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 herewith.

[0069] It will be understood that all matters are intended to be included, as the advantages set forth above and those which will become apparent from the foregoing description are efficiently attained, and certain modifications may be made to the above-described configurations without departing from the scope of the invention. The matter contained in the foregoing description or shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

[0070] It is also to be understood that the following claims are intended to encompass all of the general and specific features of the invention described herein, and, as a matter of language, all statements of the scope of the invention that are said to lie therebetween.

Claims

1. A composition for nucleic acid delivery to eukaryotic cells, including non-pathogenic bacteria, wherein the bacterium has been engineered to express at least one invasion factor from a sequence in the bacterial chromosome, and the bacterium comprises a plasmid having a length of less than 4,700 base pairs, the plasmid comprises one or more sequences encoding one or more therapeutic nucleic acids and a prokaryotic promoter for controlling transcription of the therapeutic nucleic acids, and the bacterium has been engineered to express a HEN1 protein or an active methyltransferase fragment or domain thereof.

2. The composition for nucleic acid delivery to eukaryotic cells described in claim 1, wherein the plasmid essentially consists of one or more sequences encoding one or more therapeutic nucleic acids, a sequence encoding a selectable marker, an origin of replication, and one or more prokaryotic promoters.

3. The composition for nucleic acid delivery to eukaryotic cells described in claim 1, wherein the plasmid comprises multiple therapeutic nucleic acid sequences and multiple prokaryotic promoters, a first therapeutic nucleic acid sequence being under the control of a first prokaryotic promoter and a second therapeutic nucleic acid sequence being under the control of a second prokaryotic promoter.

4. The composition for nucleic acid delivery to eukaryotic cells described in claim 3, wherein the first prokaryotic promoter is a different promoter from the second prokaryotic promoter, thereby having different prokaryotic promoters and allowing different transcription of one or more of the multiple therapeutic nucleic acid sequences.

5. The composition for nucleic acid delivery to eukaryotic cells according to claim 1 , wherein the therapeutic nucleic acid is a small interfering RNA / small hairpin RNA (siRNA / shRNA).

6. 2. The composition for nucleic acid delivery to eukaryotic cells of claim 1, wherein the therapeutic nucleic acid is a nucleic acid selected from the group consisting of microRNA (miRNA), miRNA mimics and miRNA inhibitors, antagomir, RNA or DNA aptamers, messenger RNA (mRNA), splice-switching oligonucleotides, antisense oligonucleotides, antigene oligonucleotides, DNAzymes, RNA decoys, ribozymes, peptide nucleic acids, oligomers, and defective interfering particles.

7. The bacteria include Listeria, Yersinia, Rickettsia, Shigella, E. coli, Salmonella, Legionella, Chlamydia, Brucella, Neisseria, Burkholderia, Bordetella, Borrelia, and Coxiella.

2. The composition for nucleic acid delivery to eukaryotic cells of claim 1, wherein the eukaryotic cell is an engineered bacterium from the group of bacteria consisting of Mycobacterium, Helicobacter, Staphylococcus, Streptococcus, Porphyromonas, Vibrio, Treponema, Lactobacillus, and Bifidobacteriae.

8. The composition for nucleic acid delivery to a eukaryotic cell of claim 1, wherein the bacterium is engineered to express an Inv protein, or a fragment or binding domain thereof, from a sequence in the chromosome of the bacterium, or the bacterium is engineered to express an influenza hemagglutinin-1 (HA-1) protein, or a fragment or binding domain thereof, from a sequence in the chromosome of the bacterium.

9. The composition for nucleic acid delivery to eukaryotic cells according to claim 1, wherein the entry factor is encoded by a gene selected from the group consisting of an inv gene, an hlyA gene, an HA-1 gene, or a combination thereof.

10. The composition for nucleic acid delivery to eukaryotic cells of claim 1 , wherein the therapeutic nucleic acid interferes with one or more target influenza virus RNA molecules.

11. 2. The composition for nucleic acid delivery to eukaryotic cells of claim 1, wherein the therapeutic nucleic acid interferes with one or more viruses selected from the group consisting of coronavirus, herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), human papillomavirus (HPV), respiratory syncytial virus, MERS and SARS.

12. 2. A composition for nucleic acid delivery to a eukaryotic cell as described in claim 1, wherein the bacterium has been engineered to express a protein that binds sialic acid residues in a target cell from a sequence in the chromosome of the bacterium.

13. 2. The composition for nucleic acid delivery to eukaryotic cells of claim 1, wherein the bacterium is engineered to express binding domains from coronavirus, norovirus, rotavirus, mumps virus, parainfluenza, Clostridium botulinum, and Vibrio cholera.

14. A composition for nucleic acid delivery to eukaryotic cells, including non-pathogenic bacteria, wherein the bacterium has been engineered to express LLO, INV, or HA-1 from a sequence in the chromosome of the bacterium, and the bacterium comprises a plasmid having a length of less than 4,700 base pairs, the plasmid comprising one or more sequences encoding one or more therapeutic nucleic acids and a prokaryotic promoter for controlling transcription of the therapeutic nucleic acid, and the bacterium has been engineered to express a HEN1 protein or an active methyltransferase fragment or domain thereof.

15. The composition for nucleic acid delivery to eukaryotic cells described in claim 14, wherein the plasmid essentially consists of one or more sequences encoding one or more therapeutic nucleic acids, a sequence encoding a selectable marker, an origin of replication, and one or more prokaryotic promoters.

16. The composition for nucleic acid delivery to eukaryotic cells described in claim 14, wherein the plasmid comprises multiple therapeutic nucleic acid sequences and multiple prokaryotic promoters, a first therapeutic nucleic acid sequence being under the control of a first prokaryotic promoter and a second therapeutic nucleic acid sequence being under the control of a second prokaryotic promoter.

17. 17. A composition for nucleic acid delivery to eukaryotic cells as described in claim 16, wherein the first prokaryotic promoter is a different promoter from the second prokaryotic promoter, thereby having different prokaryotic promoters and allowing for different transcription of one or more of the multiple therapeutic nucleic acid sequences.

18. The composition for nucleic acid delivery to eukaryotic cells according to claim 14, wherein the therapeutic nucleic acid is a small interfering RNA / small hairpin RNA (siRNA / shRNA).

19. 15. The composition for nucleic acid delivery to eukaryotic cells of claim 14, wherein the therapeutic nucleic acid is a nucleic acid selected from the group consisting of microRNA (miRNA), miRNA mimics and miRNA inhibitors, antagomirs, RNA or DNA aptamers, messenger RNA (mRNA), splice-switching oligonucleotides, antisense oligonucleotides, antigene oligonucleotides, DNAzymes, RNA decoys, ribozymes, peptide nucleic acids, oligomers, and defective interfering particles.

20. The bacteria include Listeria, Yersinia, Rickettsia, Shigella, E. coli, Salmonella, Legionella, Chlamydia, Brucella, Neisseria, Burkholderia, Bordetella, Borrelia, and Coxiella.

15. The composition for nucleic acid delivery to eukaryotic cells of claim 14, wherein the bacterium is an engineered bacterium from the group of bacteria consisting of Mycobacterium, Helicobacter, Staphylococcus, Streptococcus, Porphyromonas, Vibrio, Treponema, Lactobacillus, and Bifidobacteriae.

21. The composition for nucleic acid delivery to a eukaryotic cell of claim 14, wherein the therapeutic nucleic acid interferes with one or more target influenza virus RNA molecules.

22. 15. The composition for nucleic acid delivery to eukaryotic cells of claim 14, wherein the therapeutic nucleic acid interferes with one or more viruses selected from the group consisting of coronavirus, herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), human papillomavirus (HPV), respiratory syncytial virus, MERS and SARS.

23. 15. The composition for nucleic acid delivery to eukaryotic cells of claim 14, wherein the bacterium is engineered to express binding domains from coronavirus, norovirus, rotavirus, mumps virus, parainfluenza, Clostridium botulinum, and Vibrio cholera.

24. A kit for nucleic acid delivery to eukaryotic cells, including non-pathogenic bacteria, wherein the bacterium has been engineered to express at least one invasion factor from a sequence in the bacterial chromosome, and the bacterium comprises a plasmid having a length of less than 4,700 base pairs, the plasmid comprises a cloning site for one or more sequences encoding one or more therapeutic nucleic acids, and a prokaryotic promoter, wherein the prokaryotic promoter controls transcription of the cloning site, and the bacterium has been engineered to express a HEN1 protein or an active methyltransferase fragment or domain thereof.

25. A composition for nucleic acid delivery to eukaryotic cells, including non-pathogenic bacteria, wherein the bacterium has been engineered to express at least one invasion factor from a sequence in the chromosome of the bacterium, and the bacterium comprises a plasmid consisting essentially of one or more sequences encoding one or more therapeutic nucleic acids, an origin of replication, a sequence encoding a selectable marker, and one or more prokaryotic promoters for controlling transcription of the nucleic acid, and the bacterium has been engineered to express a HEN1 protein or an active methyltransferase fragment or domain thereof.

26. 26. The composition for nucleic acid delivery to a eukaryotic cell of claim 25, wherein the plasmid has a length of less than 4,700 base pairs.

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