Chimeric invasive element system

Abstract

A transboundary platform for delivering a therapeutic agent to a target cell. The system retains the output and absorption functions of Inv, meanwhile, the targeting property of the system is changed from beta1 integrin to other proteins (namely cell surface proteins) or chemical parts (namely cell surface chemical parts) which are expressed on the surfaces of target eukaryotic cells in a targeting manner, and the method is as follows: D4 and D5 of Inv are replaced by binding domains from heterologous proteins through genetic engineering. These heterologous proteins may be from bacterial, fungal, animal or viral genomes. This modification will result in the construction of a chimeric Inv protein in which D1-D3 (i.e., non-binding domain) is fused to an alternative binding domain from a heterologous protein in a manner conforming to the reading frame. The alternative binding domains will interact with different cell surface proteins or chemical moieties, which may in some cases be referred to as receptors, of the surface of eukaryotic cells, allowing specifically targeting the cells independent of Inv intrinsic beta1 integrin binding.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 363,536, filed on April 25, 2022, and U.S. Provisional Application No. 63 / 367,518, filed on July 1, 2022. Technical Field

[0003] The present invention relates to bacterial delivery vectors for therapeutic applications. More specifically, the present invention relates to bacterial vectors that can target certain types of eukaryotic cells. Background Art

[0004] To date, it remains challenging to precisely deliver therapeutic modalities to specific targeted cells in vivo. Drug delivery involves two key components: the vector itself and the mechanism by which the vector specifically reaches the desired cell type with minimal off-target delivery. Most current delivery strategies are based primarily on mechanical methods (e.g., electroporation, hydrodynamic injection, and microinjection) and viral vector delivery (e.g., lentivirus, adenovirus, and adeno-associated virus). Non-viral delivery methods are also used, such as liposomes and nanoparticles, but they are extremely limited in the size and number of cargo moieties they can carry. Although useful in vitro, many of these methods cannot be easily applied clinically to animals or human patients. Bacterial delivery vectors have many advantages. One particular advantage is their ability to deliver therapeutic moieties into cells by invasion. Currently, bacterial delivery vectors reach their targets through passive mechanisms, which often rely on the biological characteristics of a specific niche (e.g., a hypoxic tumor microenvironment). Alternatively, bacteria are specifically targeted through ligand-receptor interactions with factors on the surface of target cells. To date, even the most specific targeting methods may have off-target effects on the expression of targeted ligands in multiple cell types in multiple tissues and organs. To realize the full potential of bacterial delivery vectors, more specific targeting mechanisms are needed to mitigate off-target effects (i.e., delivery to unwanted or undesirable tissues and cell types). Summary of the invention

[0005] The present invention provides systems and methods for specific targeting of cells in eukaryotic hosts using highly specific targeting of invasive, non-pathogenic bacterial delivery vectors that have been engineered to produce chimeric invadin (Inv) polypeptides with modified binding domains. The non-chimeric form of the Inv polypeptide has five domains, referred to as D1, D2, D3, D4, and D5, and a beta barrel that passes through the bacterial outer membrane.

[0006] In certain aspects, the present invention provides systems and methods to maintain the export (i.e., export to the surface of bacterial cells) and uptake functions of the Inv protein from Yersinia pseudotuberculosis, while replacing D4 and D5 of Inv with binding domains from heterologous proteins through genetic engineering, modifying its targeting from β1 integrin to other protein domains expressed on the surface of target eukaryotic cells (i.e., cell surface proteins) or chemical moieties expressed on the surface of target eukaryotic cells (i.e., cell surface chemical moieties). These heterologous proteins can be from bacterial, fungal, animal or viral genomes. Such engineering will result in the construction of chimeric Inv proteins in which D1 to D3 from Inv (i.e., non-binding domains) are fused in a frame-matched manner to alternative binding domains from heterologous proteins or synthetic binding domains. The alternative binding domains will interact with different cell surface proteins or chemical moieties (which may be referred to as receptors in some cases) on the surface of eukaryotic cells, thereby allowing specific targeting of cells without relying on the inherent β1 integrin binding of Inv. In some cases, the binding domain of a heterologous protein may be referred to as a ligand binding domain.

[0007] It is contemplated that a non-binding domain can be removed from Inv (e.g., D1, D2, and D3) or full-length Inv, a linker sequence as described immediately below can be added, and then a binding domain from one of the proteins listed in Table 1, Table 2, or Table 3 can be added. Thus, a sequence from Inv can be utilized, such as the sequence provided below, SEQ ID NO. 1, from about amino acid 1 up to about amino acid 795 or from about amino acid 1 to about amino acid 986, or a sequence that is 95% or 90% identical thereto.

[0008] In certain aspects, the present invention provides non-pathogenic bacteria that have been modified to express chimeric targeting ligands. The bacteria are modified to have a sequence encoding a non-binding domain of the Inv protein (e.g., D1, D2 and / or D3) fused to a sequence encoding a heterologous binding domain (e.g., see Tables 1-3 below for proteins that can utilize binding sites from the protein). Thus, a chimeric Inv protein with an altered binding domain can be generated when the sequence is expressed. This can enable cross-border delivery vectors to target tissues other than those that can be reached using a binding domain targeting β1 integrin. The following SEQ ID. NO. 1 discloses the amino acid sequence of the Inv polypeptide. It is envisioned that the D1, D2 and / or D3 regions of the sequence can be used to construct chimeric proteins, for example by conversion into corresponding nucleic acid sequences or sequences having 99%, 95% or 90% homology to the nucleic acid sequences of the D1, D2 and / or D3 regions (see SEQ ID NO. 38). Since D1-D3 favors binding to target cells but not invasion, bacteria expressing chimeric targeting ligands comprising D1-D3 fused to heterologous binding domains targeting specific factors on the surface of target cells can be used for cell labeling or detection.

[0009] In a further embodiment, the present invention provides a non-pathogenic bacterium that has been engineered to express a chimeric targeting ligand, wherein the bacterium is engineered to have a sequence encoding a complete Inv protein (i.e., D1, D2, D3, D4, D5) fused to a sequence encoding a binding domain from a heterologous protein or a synthetic binding domain (see, e.g., Tables 1-3 below for proteins that can utilize binding sites from the protein). Thus, a chimeric Inv protein with an altered binding domain can be generated when the sequence is expressed. This can enable cross-border delivery vectors to target cell types and tissues other than those that can be reached using a binding domain targeting β1 integrin. The amino acid sequence of the Inv polypeptide is disclosed in SEQ ID. NO. 1 below. It is envisioned that the D1, D2, D3, D4, and D5 regions of the sequence can be used to construct a chimeric protein, for example by conversion to a corresponding nucleic acid sequence or a sequence having 95% or 90% homology to the nucleic acid sequence encoding the D1, D2, D3, D4, and D5 regions of Inv.

[0010] In the construction of fusion (i.e., chimeric) proteins, various linker sequences (specific amino acid sequences) can be used to connect protein domains (i.e., independently folded amino acid sequences). Linker sequences are generally classified as rigid linkers or flexible linkers, some of which may contain cleavage sites as described herein. The physical characteristics of the linker can affect key properties of the fusion protein, including expression levels, biological activity, or other in vivo behaviors. Linker sequences containing glycine and serine are generally flexible, allowing the domains to move independently relative to each other. Linker sequences containing proline tend to be more rigid, thereby restricting the relative movement of the domains [see Chen X, Zaro JL, Shen WC. Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013 Oct;65(10):1357-69. doi:10.1016 / j.addr.2012.09.039. Epub 2012 Sep 29. PMID: 23026637; PMCID: PMC3726540. ]

[0011] In a further aspect, the chimeric polypeptide according to the invention utilizes a sequence comprising a linker sequence to link the non-binding domain of Inv fused to a sequence encoding a heterologous binding domain. The linker sequence may be a sequence selected from SEQ ID NOS. 2-20, as described below. In certain embodiments, the binding domain sequence is a sequence encoding a binding domain selected from any one of the polypeptides mentioned in Tables 1-3.

[0012] In advantageous embodiments, the chimeric targeting ligand utilizes a sequence comprising a composite linker sequence to connect the non-binding domain of Inv or full-length Inv to a sequence encoding a heterologous binding domain (BD), including a synthetic binding domain. The first portion (N-terminus) of the linker sequence can be a sequence selected from SEQ ID NOS. 2-20, as described below. The second portion of the linker can include a cleavage site or multiple cleavage sites for one or more peptidases or proteases provided in Table 4. The use of a composite linker is used to confirm that the correct target cell type has been reached because a peptidase or protease needs to be present on the surface of the target cell to allow invasion by removing the heterologous BD. In certain embodiments, the BD sequence is a sequence encoding a BD or a synthetic BD selected from any one of the polypeptides mentioned in Tables 1-3.

[0013] According to various aspects, non-pathogenic bacteria engineered to express chimeric Inv polypeptides may utilize a sequence encoding a non-binding domain of an Inv protein that encodes a polypeptide that is 90% (or 95% or even 99%) identical to amino acids 1-794 of SEQ ID NO. 1 presented below.

[0014] Non-pathogenic bacteria engineered to express chimeric targeting ligands may alternatively utilize a sequence encoding all domains of an Inv protein that is 90% (or 95% or 99%) identical to amino acids 1-986 of SEQ ID NO.1.

[0015] Non-pathogenic bacteria engineered to express chimeric Inv polypeptides can be further engineered to express therapeutic nucleic acids, proteins, antibodies, antibody derivatives, polypeptides, gene editing systems (CRISPR and other gene editing nucleases), eukaryotic translatable mRNAs, or combinations thereof. Therapeutic nucleic acids, proteins, antibodies, antibody derivatives, polypeptides, gene editing systems (CRISPR and other gene editing nucleases), eukaryotic translatable mRNAs, or combinations thereof can be expressed from sequences on bacterial chromosomes or plasmids.

[0016] On the other hand, the present invention provides non-pathogenic bacteria designed to express chimeric Inv polypeptides. The expressed chimeric Inv polypeptides can have non-binding domains (e.g., D1, D2 and / or D3) of Inv proteins or be fused with heterologous binding domains to produce full-length Inv (D1-D5) of chimeric Inv proteins. Chimeric targeting ligands produced by bacteria can be used to change the target cell or tissue type of bacterial delivery vectors.

[0017] In yet another aspect, the present invention provides a method for delivering a nucleic acid or delivering another molecule (e.g., a protein, an antibody, an antibody derivative, a polypeptide, a gene editing system (CRISPR and other gene editing nucleases), a eukaryotic translatable mRNA) to a bacterium containing a eukaryotic cell of a non-pathogenic bacterium, wherein the bacterium has been modified to express at least one invasion factor, and the invasion factor has an Inv protein or a non-binding domain of a full-length Inv fused to a heterologous binding domain to produce a chimeric Inv protein. The heterologous protein can be a protein that binds to a cell surface protein or a cell surface chemical moiety on a target eukaryotic cell. The binding domain can be a fragment of a heterologous protein, such as a fragment produced by not including a non-binding region of a heterologous protein. In an advantageous embodiment, the binding domain of the heterologous protein is translated from a sequence encoded in a bacterial, fungal, viral or animal genome, but is designed to be encoded and translated as part of a chimeric Inv protein by a bacterial delivery vector. Alternatively, the binding domain can be any synthetic (i.e., non-natural) protein that is conducive to binding to the target cell surface.

[0018] The chimeric Inv targeting ligand can be expressed from a sequence on the chromosome of the engineered bacterial delivery vector or from a plasmid carried by the engineered bacterial delivery vector.

[0019] In a preferred embodiment, the chimeric Inv targeting ligand has a peptide linker fused between the non-binding domain of the Inv protein and the binding domain from the heterologous protein. The peptide linker may have one or more amino acids fused in frame to the non-binding domain of the Inv protein and the binding domain from the heterologous protein. The non-binding domain of the Inv protein may be the D1, D2, and D3 domains of Inv or a combination or subset thereof or the full-length Inv.

[0020] In an advantageous embodiment, the chimeric Inv protein has a peptide linker fused between the non-binding domain of the Inv protein and the binding domain from the heterologous protein. The peptide linker may have one or more amino acids fused in frame with the full-length Inv protein and the heterologous binding domain.

[0021] Bacteria used to deliver nucleic acids to eukaryotic cells can be further engineered to express therapeutic nucleic acids from sequences on the bacterial chromosome or from plasmids.

[0022] Thus, in certain aspects, the present invention provides an expression cassette for producing a chimeric invadin (Inv) polypeptide. The expression cassette may include a prokaryotic promoter and a nucleic acid sequence encoding an Inv polypeptide fused to a linker polypeptide at the carboxyl terminus of the Inv polypeptide. Expression of the nucleic acid encoding the chimeric invadin polypeptide is controlled by the prokaryotic promoter. In certain advantageous embodiments, the expression cassette for producing a chimeric invadin polypeptide includes a sequence encoding a binding domain to bind to a surface moiety on a target cell. The binding domain is fused to the amine terminus of the linker polypeptide. The binding domain may be a binding domain from a protein listed in Tables 1-3, or the binding domain may be a synthetic binding domain. The nucleic acid sequence of the invadin region of the chimeric polypeptide may be 90% identical, 95% identical, or even 99% identical to nucleic acids 1-2958 of SEQ ID. NO. 37. Similarly, the nucleic acid sequence of the invadin region of the chimeric polypeptide may be 90% identical, 95% identical, or even 99% identical to nucleic acids 1-2382 of SEQ ID. NO. 37 or SEQ ID. NO. 38.

[0023] According to the first aspect, the expression cassette for producing the chimeric invadin polypeptide may further include a nucleic acid sequence encoding a linker polypeptide and / or a protease cleavage site. Exemplary linkers include SEQ.ID.NO.2 to SEQ ID.NO.20. Exemplary protease cleavage sites include SEQ.ID.NO.22 to SEQ ID.NO.36.

[0024] A sequence encoding a protease cleavage site for cleavage by a peptidase or proteinase may be located between the sequence encoding invasin and the sequence encoding the binding domain.

[0025] The expression cassette for producing the chimeric invasin polypeptide can use a prokaryotic promoter, such as T7, lacUV5, gapA, T5, recA, Ptac, Patac, pAl, lac, Sp6, araBad, and trp promoters. The prokaryotic promoter can also be a hybrid or synthetic prokaryotic promoter.

[0026] A bacterium expressing a chimeric invadin polypeptide can be constructed, wherein the bacterium is designed to include an expression cassette for producing a chimeric invadin polypeptide. The bacterium can be a bacterium selected from the group consisting of Clostridium difficile, Escherichia coli, Clostridium tetani, Helicobacter pylori, Fusobacterium nucleatum, Gardnerella vaginitis, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Listeria monocytogenes, Staphylococcus aureus, Campylobacter jejuni, Vibrio vulnificus, Salmonella typhi, Clostridium botulinum, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium leprae, Corynebacterium diphtheriae, Klebsiella pneumoniae, Baumannii, and Streptococcus aureus. Acinetobacter, Streptococcus mutans, Group B Streptococcus, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae, Enterococcus, Enterococcus faecalis, Listeria, Yersinia, Rickettsia, Shigella, Salmonella, Legionella, Chlamydia, Brucella, Neisseria, Burkholderia, Bordetella, Borrelia, Coxiella, Mycobacterium, Helicobacter, Staphylococcus, Streptococcus, Porphyromonas, Vibrio, Treponema, Lactobacillus and Bifidobacterium. In an advantageous embodiment, the bacterium expressing the chimeric invasin polypeptide is Escherichia coli.

[0027] In a further aspect, the invention provides a method of treating or preventing a disease in a subject by administering a bacterium expressing a chimeric invadin polypeptide, wherein the bacterium is further engineered to express a therapeutic nucleic acid produced by the bacterium. Similarly, in addition to expressing a chimeric invadin polypeptide, the bacterium can also be engineered to express a therapeutic nucleic acid, protein, antibody, antibody derivative, polypeptide, gene editing system (CRISPR and other gene editing nucleases), eukaryotic translatable mRNA, or a combination thereof from a sequence on a bacterial chromosome or plasmid.

[0028] The present invention also provides chimeric invadin polypeptides. The chimeric invadin polypeptides may include an Inv polypeptide and a linker polypeptide, wherein the linker polypeptide has a first end (N-terminus) and a second end (C-terminus), wherein the first end (N-terminus) of the linker polypeptide is attached to the C-terminus of the Inv polypeptide. The chimeric invadin polypeptide may also include a peptidase or protease cleavage site.

[0029] In an advantageous embodiment, the chimeric invadin polypeptide further comprises a binding domain of a heterologous protein or a synthetic binding domain attached to the second end (C-terminus) of the linker polypeptide. Advantageous binding domains may be binding domains from proteins listed in Tables 1-3.

[0030] The amino acid sequence of the Inv polypeptide of the chimeric invasin may have a sequence that is 90% (or 95% or 99%) identical to amino acids 1-986 of SEQ ID.NO.1 or a sequence that is 90% (or 95% or 99%) identical to amino acids 1-985 of SEQ ID.NO.39.

[0031] The amino acid sequence of the Inv polypeptide of the chimeric invasin may have a sequence that is 90% (or 95% or 99%) identical to amino acids 1-794 of SEQ ID.NO.1 or 90% (or 95% or 99%) identical to amino acids 1-794 of SEQ ID.NO.39.

[0032] In yet further aspects, the invention provides compositions for selectively binding a substrate to a target molecule. The compositions can utilize a chimeric invadin polypeptide bound to a biological and synthetic substrate surface at the amino terminus of the Inv polypeptide, wherein the substrate is selected from beads, viruses, exosomes, rigid substrates (e.g., for producing lateral flow strips), paper-based biosensors, plastic substrates (e.g., for producing plastic-based biosensors), graphene-based substrates, or nanomaterials (e.g., lipid nanoparticles, metal nanoparticles, mesoporous silica nanoparticles, nanowires, ITO, organic polymers).

[0033] In a further aspect, the invention provides a chimeric invadin polypeptide comprising the D1-D3 domains of an Inv polypeptide attached to a binding domain of a heterologous protein or a synthetic binding domain. The chimeric invadin polypeptide may include a linker, wherein the linker polypeptide has a first end (N-terminus) and a second end (C-terminus), wherein the first end of the linker polypeptide is attached to the C-terminal amino acid of the Inv polypeptide and the second end of the linker polypeptide is attached to the amino terminus of the binding domain of the heterologous protein or the amino terminus of the synthetic binding protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] 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:

[0035] Figure 1 is a series of four illustrations (labeled (A)-(D)) of the structure of full-length Inv (A) and the structures of various versions of the chimeric Inv protein described herein ((B)-(D)). The chimeric Inv protein includes D1-D3 of Inv fused to the binding domain of a heterologous protein ( Figure 2 B) or full-length Inv( Figure 2 C and Figure 2 D).

[0036] Figure 2 is a diagram showing the three-step chimeric ligand targeting and invasion paradigm described herein.

[0037] Figure 3This is a micrograph of A549 cells treated with FEC19 bacteria containing pSi_1fHER2scr.c. The image (A549 cells) demonstrates that the bacterial delivery vector can specifically target and invade HER2-positive, furin-positive A549 cells, but not HER2-negative HeLa cells.

[0038] Figure 4 are a diagram (A) and a schematic diagram (B) showing the two-factor chimeric Inv protein used in the following Examples. Figure 4 A shows a schematic diagram of a chimeric Inv protein comprising D1-D5 of Inv linked within the framework of a synthetic nanobody that binds to HER2. The linker contains a cleavage site for the cell surface protease furin. Figure 4 B provides a schematic diagram of the chimeric Inv protein sequence shown in (A). Only the C-terminus of Inv D5 is shown for illustration purposes.

[0039] Figure 5 is a pair of diagrams labeled (a) and (b) providing linear representations of annotated Inv amino acid sequences, with amino acids comprising D1–D4 / D5 labeled with domain names and functions. The chimeric Inv proteins described herein will comprise D1, D2, and D3, and D4 / D5 will be replaced by heterologous binding domains. The diagrams in (a) and (b) are identical, with (b) rotating the diagram in (a) 90 degrees to magnify the data presented therein. The sequences shown in (A) and (B) are identical and are presented as SEQ.ID.NO.1 below. DETAILED DESCRIPTION

[0040] The present invention provides a system for targeted intracellular delivery of therapeutic or non-therapeutic moieties into eukaryotic cells using a non-pathogenic bacterial delivery platform that expresses a bifunctional chimeric targeting-invasion factor ("chimeric targeting ligand") that interacts and binds to a factor ("receptor") on the surface of a target cell, and then triggers the internalization of the bacterial delivery vector by the target cell. The chimeric ligand comprises a constant region, including a Yersinia pseudotuberculosis invadin (Inv) protein (encoded by the Inv gene), and a variable region customized for targeting purposes. The variable region includes a peptide or protein that binds to a receptor on the target cell. For example, the variable region may include a single domain antibody, a nanobody, a camel IgG antibody, a llama IgG antibody, a peptibody, any other immune polypeptide, or any peptide consisting of amino acid residues that specifically bind to a receptor molecule on the outer membrane of a eukaryotic cell. This chimeric ligand can target bacteria to a specific cell type or a class of cell types that express the same receptor.

[0041] The Gram-negative genus Yersinia includes at least 17 species, three of which are human and animal pathogens: Yersinia enterocolitica, Yersinia pseudotuberculosis, and Yersinia pestis. The pathogenicity of these bacteria depends on factors that enable them to adhere to cells and cross the cell membrane to reach the cytoplasm of target cells. These organisms express a variety of such factors, including invasins (Inv), YadA, YadB, YadC, Ail, Pla, and Ph6 antigens. These different proteins are collectively known as adhesins, and each protein functions at a specific stage of the host-pathogen interaction. Importantly, each of these proteins binds to a range of host factors, including b1 integrins, collagen, fibronectin, laminin, and complement-related factors. In addition, all of these proteins are anchored to the outer membrane (OM) of the bacteria, where they form rod-shaped structures. Presentation on the OM allows the proteins to mediate interactions with target cell surface factors. Trafficking from the cytoplasm of the bacterial cell to the OM can also occur by a variety of mechanisms. It is contemplated that chimeric polypeptides can be constructed using the YadA, YadB, YadC, Ail, Pla, and Ph6 antigens by replacing the Inv nucleic acid sequence being expressed with a sequence encoding the YadA, YadB, YadC, Ail, Pla, and Ph6 antigens, which can produce an alternative chimeric targeted bacterium. Thus, for example, a YadA chimeric polypeptide can include a binding domain of a chimeric invasin as taught herein, and optionally also include a linker sequence and / or a cleavage site between the YadA amino acids and the BD amino acids. The sequences of these adhesins are known, such as YadA (UniProt P31489-YADA1_YEREN; UniProt P10858-YADA_YERPS, incorporated by reference). Sequences that are 90% identical, 95% identical, or 99% identical to the consensus sequence of the YadA, YadB, YadC, Ail, Pla, or Ph6 antigens can be used for the corresponding adhesins.

[0042] Invasins are the first adhesins expressed during invasion by enteropathogenic Yersinia spp. Their primary role is to initiate bacterial colonization and internalization of host epithelial cells by binding to β1 integrins. Invasins have a modular structure that includes several well-defined functional sequences. Most broadly, Inv contains structural elements associated with autotransporters: a β-barrel “transporter” structure at the amino (N) terminus and an extracellular “passenger” domain at the carboxyl (C) terminus. The passenger domain is autonomously transported from the periplasm to the outer membrane (OM) without the need for an energy source (e.g., ATP). For Inv, this transport is thought to be mediated by protein entry into the periplasm via an N-terminal signal peptide followed by insertion of the β-barrel domain into the OM to form a pore through which the passenger domain passes. The structure of the passenger domain is highly modular and contains five protein domains (D1-D5). The secondary structure of D1–D4 consists primarily of β sheets, whereas that of D5 consists of α helices / β helices. Together, D4 and D5 form a module that binds to integrins with high affinity.

[0043] In some aspects, the present invention provides bacterial-mediated delivery vectors, which include invasive, non-pathogenic bacteria that express chimeric ligands that are then exported to the outer membrane of bacterial cells. Bacteria can be contained in a prokaryotic expression cassette encoding chimeric ligands under the control of a prokaryotic promoter (synthetic or endogenous). The novel bacterial delivery platform expressing and presenting the ligand can provide cell-specific and tissue-specific delivery and cellular internalization of the delivery vector in any eukaryotic cell at any cell cycle stage (division, non-division, static), as long as the cell expresses homologous cell surface receptors. The targeting of desired eukaryotic cells can be controlled by selecting a variable region that has specificity for receptors on target eukaryotic cells.

[0044] In a further aspect, the present invention utilizes E. coli cross-kingdom delivery vectors to advance the delivery of therapeutic nucleic acids, proteins, antibodies, antibody derivatives, polypeptides, gene editing systems (CRISPR and other gene editing nucleases) and eukaryotic translatable mRNA by allowing precise targeting of bacteria to target eukaryotic cells expressing specific surface proteins or chemical moieties. Delivery of therapeutic modalities is discussed in, for example, U.S. Patent No. 11,312954B2 to Linke et al. and U.S. Patent No. 2022 / 0364122A1 to Linke et al., the contents of which are incorporated herein by reference. Cross-kingdom bacterial delivery vectors must target and invade specific cell types for intracellular cargo delivery; however, targeting and invasion are not trivial or passive processes, especially when the target cells do not naturally absorb bacteria by phagocytosis, etc.

[0045] Bacteria use various invasion factors to invade non-phagocytic cells, such as Yersinia pseudotuberculosis (Mikula et al., 2012). These bacteria rely on surface-presented invasion factor proteins, invadin (Inv), which bind to β1 integrins on the surface of target eukaryotic cells. Upon binding, the intrinsic properties of Inv stimulate the uptake of bacteria by non-phagocytic eukaryotic cells. This uptake process depends on three specific properties of Inv: 1) Inv export to the bacterial surface, 2) Inv binding to β1 integrins on the cell surface, and 3) stimulation of bacterial uptake.

[0046] The Yersinia pseudotuberculosis Inv protein is a multidomain protein containing five independently folded domains, namely D1, D2, D3, D4 and D5. The primary accession number of the Inv protein is UNIPROT P11922, the Inv gene is YPTB1668 (Isberg et al., 1987, Leong et al., 1990, Chain et al., 2004), and its complete sequence is shown in Table 5 below. The key invasion functions of Inv mentioned above are divided into these different domains. D1, D2 and D3 are responsible for the export of Inv to the bacterial surface and stimulate cellular uptake, while D4 and D5 are required for β1 integrin binding (Figures 7 and 8) (Dersch and Iseberg, 2000). Inv is an autotransporter (Leo et al., 2014), which means that its export to the bacterial cell surface is an intrinsic property of the protein, i.e., it does not require any separate export mechanism. Therefore, by separating the domains of Inv, one can also separate its export, uptake stimulation, and targeting functions and exploit these functions independently to achieve targeting and invasion of cross-domain delivery vectors.

[0047] The present invention describes a method that maintains the export and absorption functions of Inv by replacing D4 and D5 of Inv with binding domains from heterologous proteins or synthetic (i.e., non-natural) binding domains, or fusing the full-length Inv with binding domains from heterologous proteins or synthetic (i.e., non-natural) binding domains by genetic engineering, while changing its targeting from β1 integrin to other proteins (i.e., cell surface proteins) or chemical moieties (i.e., cell surface chemical moieties) expressed on the surface of target eukaryotic cells. Heterologous proteins can be from bacterial, fungal, animal or viral genomes. Alternatively, BDs can include synthetic proteins (i.e., non-naturally occurring proteins). The source of synthetic BDs can be laboratory procedures that are typically based on biochemical methods or computational discoveries (e.g., by computer modeling or artificial intelligence). The synthetic BD can be a single domain antibody, a nanobody, or any other ligand that binds to a target cell surface moiety. Such engineering will result in the construction of chimeric Inv proteins in which D1-D3 (i.e., non-binding domains) are fused in frame to alternative heterologous binding domains, or in which InvD1-D5 (i.e., full-length Inv) are fused in frame to heterologous binding domains. The alternative binding domains will interact with different cell surface proteins or chemical moieties on the surface of eukaryotic cells instead of the intrinsic binding domain of Inv (which may be referred to as a receptor in some cases), thereby allowing specific targeting of cells independent of the intrinsic β1 integrin binding of Inv. In some cases, the binding domain of the heterologous protein may be referred to as a ligand binding domain. Table 1 gives examples of bacterial heterologous proteins and their binding partners (proteins or chemicals). Table 2 gives examples of fungal heterologous proteins and their binding partners (proteins or chemicals). Table 3 gives examples of viral heterologous proteins and their binding partners (proteins or chemicals). Examples of animal heterologous proteins containing binding domains include glycan binding proteins and cell adhesion proteins (e.g., GalNAc binding proteins, lectins, cell adhesion molecule (CAM) group, sulfated glycosaminoglycan (GAG) binding protein group, selectins, integrins, laminins, cadherins, fibronectin, collagen, thrombospondin, vitronectin, tenascin, apolipoprotein B, E and AV, lipoprotein lipase, hepatic lipase, Siglecs, galectins, immunoglobulins and annexins, etc.).

[0048] Invasive agents (e.g., SARS-CoV2 virus) interact with and invade target cells through a multistep process in which the invasive agent first binds to receptors on the surface of the target cell through a specific binding moiety, which is then proteolytically processed to enable or enhance invasion. This proteolytic processing occurs when a protease or peptidase cleaves the protein at a specific cognate cleavage site (e.g., the SARS-CoV2 spike protein must be cleaved at the furin cleavage site). This strategy helps optimize ligand binding and invasion functions. [See, e.g., Jackson, CB, Farzan, M., Chen, B. et al. Mechanisms of SARS-CoV-2 entry into cells. Nat Rev Mol Cell Biol 23, 3–20 (2022); see also Pager CT, Dutch RE. Cathepsin L is involved in proteolytic processing of the Hendra virus fusion protein.] J Virol. 2005 Oct;79(20):12714-20. doi:10.1128 / JVI.79.20.12714-12720.2005. PMID:16188974; PMCID:PMC1235853; Carruthers VB, Blackman MJ. A new release on life: emerging concepts in proteolysis and parasite invasion. Mol Microbiol. 2005 Mar;55(6):1617-30. doi:10.1111 / j.1365-2958.2005.04483.x. PMID:15752188. ]. Similar paradigms can be applied to the bacterial delivery platforms described herein through genetic engineering. In order to construct such a bifunctional invasion system, a site that can be recognized and cleaved by a peptidase or protease (including but not limited to the peptidases or proteases of the proteins given in Table 4 below) is placed between the Inv sequence and the heterologous binding domain sequence in a reading frame manner (see Figure 1 The bacterial delivery vector then enters the target cell through a three-step process ( Figure 2): (1) Targeting: The heterologous binding domain recognizes and binds to receptors on the surface of target cells, thereby targeting the bacterial vector to specific cell types. (2) Conversion: The heterologous binding domain is cleaved from the chimeric Inv protein by specific peptidases or proteases found on the surface of target cells to activate the invasion function of the Inv protein. (3) Invasion: The activated Inv protein binds to β1 integrin on the surface of target cells, promoting the invasion of target cells.

[0049] The function of multidomain proteins such as Inv requires specific topological interactions between their own domains or with other binding partners (e.g., proteins or chemical moieties). A key feature of a protein that influences these topological interactions is the spacing between its internal domains, which is determined by specific amino acid sequences (i.e., linker peptides) (Chen et al., 2012). For chimeric Inv, this is the spacing between the non-binding and binding domains; therefore, when designing chimeric Inv proteins, it may be advantageous to modify the amino acid sequence of the linker peptides between the domains to modulate these interactions to optimize the binding of the chimeric Inv protein to its binding partners on the surface of eukaryotic cells. Such modifications can be made by altering the amino acid sequence of the interdomain linker peptide (i.e., peptide linker) to modulate flexibility and spacing. Examples of potentially useful peptide linker amino acid sequences include [SEQ.ID.NO.2] EAAAREAAAR, [SEQ.ID.NO.3] EAAAREAAAREAAAREAAAR, [SEQ.ID.NO.4] GSGSGS, [SEQ.ID.NO.5] GSGSGSGSGS, [SEQ.ID.NO.6] GGGS, [SEQ.ID.NO.7] GGGGS, [SEQ.ID.NO.8] GGGSGGGGSGGGS, [SEQ.ID.NO.9] GGSG, [SEQ.ID.NO.10] GGSGGGSG, [SEQ.ID.NO.11] GGSGGGSGGGSG, [SEQ.ID.NO.12] GGSGGGSGGGSG, [SEQ.ID.NO.13] GGSGGGSGGGSG, [SEQ.ID.NO.14] GGSGGGSGGGSG, [SEQ.ID.NO.15] GGSGGGSGGGSG, [SEQ.ID.NO.16] GGSGGGSGGGSG, [SEQ.ID.NO.17] GGSGGGSGGGSG, [SEQ.ID.NO.18] GGSGGGSGGGSG, [SEQ.ID.NO.19] GGSGGGSGGGS, [SEQ.ID.NO.20] GGSGGGSGGGS, [SEQ.ID.NO.21] GGSGGGSGGGS, [SEQ.ID.NO.22] GGSGGGSGGGS, [SEQ.ID.NO.23] GGSGGGSGGGS, [SEQ.ID.NO.24] GGSGGGSGGGS, [SEQ.ID.NO.25] GGSGGGSGGGS, [SEQ.ID.NO.26] GGSGGGSGGGS, [SEQ.ID.NO. [SEQ.ID.NO.12]GSGGS, [SEQ.ID.NO.13]GSSGS, [SEQ.ID.NO.14]ACGSLSCGSF, [SEQ.ID.NO.15]GENLYFQSGG, [SEQ.ID.NO.16]SACYCELS, [SEQ.ID.NO.17]RPACKIPNDLKQKVMNH, [SEQ.ID.NO.18]PPPYQPLGGGGS, [SEQ.ID.NO.19]WRKRLRKKRLRKKRRLKKRRRKKQRRKRR, LEGSGQGPGSGQGSGSPGSGQG and [SEQ.ID.NO.20]GS. It is contemplated that a non-binding domain can be removed from Inv (e.g., D1, D2 and D3) or full-length Inv, a linker sequence as described above can be added, and then a binding domain from one of the proteins listed in Table 1, Table 2 or Table 3 or a synthetic binding protein / binding domain can be added. Thus, the sequence as provided in the Figures, from about amino acid 1 up to about amino acid 795 or from about amino acid 1 to about amino acid 986, or a sequence that is 95% or 90% identical thereto, may be utilized.

[0050] The present invention facilitates the delivery of nucleic acids, proteins, antibodies, antibody derivatives, polypeptides, gene editing systems, and eukaryotic translatable mRNA by providing a bacterial delivery platform that can be further customized to target specific cell surface proteins and cell surface chemical moieties to achieve more precise delivery of nucleic acids, proteins, antibodies, antibody derivatives, polypeptides, gene editing systems, and eukaryotic translatable mRNA. The Inv output and uptake domains fused (or linked) to the binding domains of heterologous or synthetic proteins can be encoded in bacterial cells via genomic or plasmid expression. Similarly, it is often advantageous to express nucleic acid coding sequences from bacterial chromosomes rather than from plasmids for many reasons, including low metabolic burden on host cells, stable expression levels, genetic stability, and no need for selection agents (Ou et al., 2018).

[0051] It is also envisioned that the output and uptake domains of the Inv or full-length Inv (chimeric Inv) fused to the binding domains of heterologous proteins can be expressed, added or bound to other biological and synthetic surfaces. These include beads, viruses, exosomes, rigid matrices (e.g., for the production of lateral flow test strips), paper-based biosensors, plastic matrices (e.g., for the production of plastic-based biosensors), graphene-based matrices, or nanomaterials (e.g., lipid nanoparticles, metal nanoparticles, mesoporous silica nanoparticles, nanowires, ITO, organic polymers).

[0052] Exosomes, liposomes, and other lipid vesicles have been used as nucleic acid delivery platforms to carry RNA payloads for delivery to distant tissues. Delivery vehicles such as liposomes have disadvantages, including leakage of vesicle contents, batch-to-batch variability, high production costs, and limited targeting capabilities. This cross-kingdom delivery system is based on the use of non-pathogenic bacteria-mediated RNAi delivery vectors that use receptor-mediated phagocytosis for specific intracellular delivery at the site of tissue action, resulting in accumulation of shRNA in endosomes and efficient release of the shRNA payload into the cytoplasm of target cells for RNAi silencing. These cross-kingdom vectors are Escherichia coli (E. coli) cells that have been engineered to specifically target mucosal epithelial tissues and deliver constitutively generated shRNA payloads in a sequence-independent manner.

[0053] Example. Bacterial vectors invade HER2-positive cancer cells via chimeric Inv targeting ligands (two-factor invasion paradigm).

[0054] Invasion assays and laser scanning confocal microscopy demonstrated that the chimeric Inv targeting ligand successfully invaded HER2-positive cancer cells. The ligand included D1-D5 of Inv, a linker containing a furin cleavage site, and a specific nanoantibody against the target cell surface presenting protein HER2.

[0055] A plasmid ("pSi_1fHER2-scr.c") was constructed by molecular cloning, encoding a chimeric Inv protein comprising D1-D5 of Inv linked to a synthetic (i.e., non-natural) nanobody via an in-frame furin cleavage site, such as Figure 4 As shown. In this embodiment, the joint is a composite joint, i.e., a universal joint sequence with a fusion furin cleavage site. Nano antibodies specifically bind to HER2, which is a receptor expressed on the surface of eukaryotic target cells. The bacterial transcription of the chimeric Inv protein is turned on under the control of a modified lacUV5 promoter, and transcription is terminated by a standard bacterial transcription terminator. After bacterial transcription and translation of the chimeric Inv protein, the protein is transferred to the bacterial surface by the self-export activity of the Inv domain D1-D3. pSi_1fHER2-scr.c is transformed into Escherichia coli (FEC19), and in the absence of chimeric InvD1-D5 (contained in the pSi_1fHER2-scr.c plasmid), Escherichia coli (FEC19) is non-invasive. The transformed FEC19 is inoculated on brain heart infusion (BHI) agar containing appropriate antibiotics for selection. The cultures used for invasion validation in this study were prepared from two isolated colonies of each strain and incubated in BHI medium with appropriate antibiotics at 37°C until late logarithmic phase (OD 600 0.8-1.0).

[0056] A standard invasion assay was also used to demonstrate bacterial invasion of human alveolar basal epithelial cells (A549 cells), which are positive for both the HER2 receptor and furin.

[0057] The cells were then detached and transferred to glass slides for imaging by laser scanning confocal microscopy. The cells were mounted in 10% NBF and mounted under a coverslip with Fluoromount-G mounting medium containing DAPI. The slides were imaged with a Zeiss LSM510 meta microscope and images were collected at 40x magnification with an excitation wavelength of 488 nm.

[0058] Figure 3 The micrograph in shows that FEC19 / pSi_1fHER2-scr.c successfully invaded A549 cells, thus demonstrating the functionality of this chimeric invasion targeting system. FEC19 bacteria are visualized as small bacillus-like gray clumps next to larger gray clumps, which are the nuclei of eukaryotic cells.

[0059] In addition to the natural binding domain, various computational and biochemical methods can also be used to find proteins and polypeptides with synthetic (i.e. non-natural) binding domains (e.g. single domain antibodies or nano antibodies). The use of synthetic binding domains designed for targeting specific surface presentation binding partners on the surface of target cells will provide additional opportunities to target specific cell types. For example, as shown in the Examples, synthetic nano antibodies bound to human protein HER2 present on the surface of various cancer cells are fused with full-length Inv sequences (D1-D5) in a form that conforms to the reading frame to form a chimeric Inv protein that only invades HER2 positive (i.e., there is HER2 on the cell surface) cells. In this Example, A549 cells were used.

[0060] Claim Glossary

[0061] As used herein, the term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.

[0062] The term "administering" and variants thereof (e.g., "administering" a compound) with respect to a compound of the invention means 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., AIV vaccines, etc.), "administering" and variants thereof are to be understood to include simultaneous and sequential introduction of the compound and other agents, respectively.

[0063] As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

[0064] The term "therapeutically effective amount" as used herein refers to the amount of an active compound or agent that triggers a biological or medical response sought by a researcher, veterinarian, doctor or other clinician in a tissue, system, animal or human. For viral infections, an effective amount includes an amount sufficient to prevent infectious diseases or reduce the severity of the disease, as evidenced by clinical disease, clinical symptoms, viral titers, or viruses shed from a 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 clinical disease and / or symptom onset or prevent disease. In some embodiments, an effective amount is an amount sufficient to reduce viral titers and / or reduce viral shedding. The effective amount can be administered in one or more doses.

[0065] "Treatment" as used herein refers to obtaining a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to, any one or more of the following: alleviating one or more symptoms, reducing the extent of viral infection, stabilizing (i.e., not worsening) the viral infection state, preventing or delaying the spread (e.g., shedding) of viral infection, preventing, delaying, or slowing the progression of viral infection, and / or maintaining body weight / weight gain. The method of the present invention contemplates any one or more of these aspects of treatment.

[0066] A "pharmaceutically acceptable" component is one that is suitable for use in humans and / or animals without undue adverse side effects (eg, toxicity, irritation, and allergic response) and at a reasonable benefit / risk ratio.

[0067] "Safe and effective amount" refers to an amount of a component that is sufficient to produce the desired therapeutic response without undue adverse side effects (such as toxicity, irritation or allergic response) and has a reasonable benefit / risk ratio when used in the manner of the present invention.

[0068] Throughout the application, unless the context clearly dictates otherwise, the terms "a" and "an" are used to mean "at least one", "at least a first", "one or more" or "a plurality" of the referenced components or steps. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0069] The term "and / or" as used herein includes the meanings of "and", "or" and "all or any other combinations of the elements connected by the term".

[0070] As used herein, the term "comprising" means that the products, compositions and methods include the mentioned components or steps, but do not exclude other components or steps. When used to define products, compositions and methods, "consisting essentially of shall mean excluding other components or steps of any significance. Thus, a composition consisting essentially of the components will not exclude trace contaminants and pharmaceutically acceptable carriers. "Consisting of shall mean excluding other components or steps beyond trace elements.

[0071] 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 RNA, to a target cell. An invasive microorganism can be a microorganism that is capable of passing through a cell membrane, thereby entering the cytoplasm of the cell and delivering at least some of its contents, such as RNA or DNA encoding RNA, to a target cell. The process of delivering at least one molecule to a target cell preferably does not significantly change the invasion mechanism.

[0072] As used herein, the term "cross-kingdom" refers to a delivery system that utilizes bacteria (or another invasive microorganism) to produce nucleic acids, proteins, antibodies, antibody derivatives, polypeptides, gene editing systems (CRISPR and other gene editing nucleases), eukaryotic translatable mRNA, or a combination thereof, and delivers the nucleic acids, proteins, antibodies, antibody derivatives, polypeptides, gene editing systems (CRISPR and other gene editing nucleases), eukaryotic translatable mRNA, or a combination thereof into target tissues or cells (i.e., across kingdoms: from prokaryotes to eukaryotes, or across phyla: from invertebrates to vertebrates) for treatment without the need for host genome integration.

[0073] Invasive microorganisms include microorganisms that are naturally capable of delivering at least one molecule to a target cell, such as by passing through a cell membrane (e.g., a eukaryotic cell membrane) and entering the cytoplasm, as well as non-naturally invasive microorganisms that have been modified (e.g., genetically modified) to be invasive. In another preferred embodiment, non-naturally invasive microorganisms can be modified to be invasive by connecting bacteria or BTPs to "invasive factors" (also referred to as "entry factors" or "cytoplasm targeting factors"). As used herein, "invasive factor" is a factor, such as a protein or a group of proteins, that when expressed by non-invasive bacteria or BTPs, makes bacteria or BTPs invasive. As used herein, "invasive factor" is encoded by a "cytoplasm targeting gene". Invasive microorganisms have been widely described in the art, such as U.S. Patent Publication Nos. US20100189691A1 and US20100092438A1 and Xiang, S. et al., Nature Biotechnology 24, 697-702 (2006). Each is incorporated herein by reference in its entirety for all purposes.

[0074] In a preferred embodiment, the invasive microorganism is Escherichia coli, as taught in the examples of the present application. However, it is envisioned that other microorganisms may be suitable for use as cross-border delivery vectors to deliver NA. These non-toxic and invasive bacteria and BTPs will exhibit invasiveness, or will be modified to exhibit invasiveness, and can enter host cells by various mechanisms. In contrast to the absorption of bacteria or BTPs by professional phagocytes (which typically results in the destruction of bacteria or BTPs in specialized lysosomes), invasive bacteria or BTP strains have the ability to invade non-phagocytic host cells. Naturally occurring examples of such intracellular bacteria are Yersinia, Rickettsia, Legionella, Brucella, Mycobacterium, Helicobacter, Coxiella, Chlamydia, Neisseria, Burkholderia, Bordetella, Borrelia, Listeria, Shigella, Salmonella, Staphylococcus, Streptococcus, Porphyromonas, Treponema and Vibrio, but this property can also be transferred to other bacteria or BTPs, such as Escherichia coli, Lactobacillus, Lactococcus or Bifidobacterium, including probiotics, by transferring invasion-associated genes (P. Courvalin, S. Goussard, C. Grillot-Courvalin, CR Acad., Sci. Paris 318, 1207 (1995)). Factors that need to be considered or addressed when evaluating other bacterial species as candidates for cross-kingdom nucleic acid delivery vectors include the pathogenicity or lack thereof of the candidate, the tropism of the candidate bacteria for target cells, or alternatively, the extent to which the bacteria can be engineered to deliver nucleic acids inside target cells, and any synergistic value that the candidate bacteria may provide by triggering the host's innate immunity.

[0075] 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 derived, single-stranded or double-stranded fragments, usually composed of molecules connected by many (2 or more) nucleotides. Examples include, but are not limited to, small interfering RNA / short hairpin RNA (siRNA / shRNA), microRNA (miRNA), antagonists, RNA or DNA aptamers, messenger RNA (mRNA), splice switching oligonucleotides, antisense oligonucleotides, antigen oligonucleotides, DNA enzymes, RNA baits, ribozymes, peptide nucleic acids, oligomers, and defective interfering particles.

[0076] Therapeutic nucleic acids are NAs as described herein or closely related chemical compounds used to treat disease, study disease, or to achieve desired genetic modifications or for gene transfer purposes. They are used in situations where specific inhibition, interruption or alteration of the function of a specific gene or other molecule associated with a disease is considered therapeutically desirable.

[0077] Synthetic binding proteins are artificial proteins that are tailored to bind to a target molecule of interest. A synthetic binding domain is the binding domain of a synthetic binding protein. Synthetic binding proteins (SBPs) are smaller, more stable, less immunogenic, and have better tissue penetration than typical non-synthetic alternatives. SBPs include affibodies, antilysins, DARPins, i-bodies, monomers / adnectins, nanobodies, repebodies, scFabs, scFvs, and vNARs. It is anticipated that SBPs and / or their binding domains (including those mentioned above) will find use in chimeric invasin polypeptides. SBPs are discussed in: ShaF, Salzman G, GuptaA, Koide S. Monobodies and other synthetic binding proteins for expanding protein science. Protein Sci. 2017 May;26(5):910-924. doi:10.1002 / pro.3148. Epub 2017 Mar 24. PMID: 28249355; PMCID: PMC5405424, and Xiaona Wang, Fengcheng Li, Wenqi Qiu, Binbin Xu, Yanlin Li, Xichen Lian, Hongyan Yu, Zhao Zhang, Jianxin Wang, Zhaorong Li, Weiwei Xue, Feng Zhu, SYNBIP: synthetic binding proteins for research, diagnosis and therapy, Nucleic Acids Research, volume 50, issue D1, January 7, 2022, pages D560–D570, https: / / doi.org / 10.1093 / nar / gkab926.

[0078] Nanobodies, also known as single-domain antibodies (sdAbs), are antibody fragments composed of a single monomeric variable antibody domain.

[0079] Affibody molecules are small, robust proteins that have been engineered to bind with high affinity to a large number of target proteins or peptides, mimicking monoclonal antibodies and thus belonging to the family of antibody mimics. These molecules can be used for molecular recognition in diagnostic and therapeutic applications.

[0080] DARPins (engineered ankyrin repeat proteins) are genetically engineered antibody-mimicking proteins that typically exhibit highly specific and high-affinity binding to target proteins. They are derived from natural ankyrin repeat proteins, one of the most common classes of binding proteins in nature, responsible for a variety of functions such as cell signaling, regulation, and cellular structural integrity. DARPins consist of at least three repeating motifs or modules, of which the most N-terminal and most C-terminal modules are called "caps" because they protect the hydrophobic core of the protein.

[0081] Anticalins are artificial proteins that can bind to antigens (proteins or small molecules). They are structurally unrelated to antibodies, making them an antibody mimetic. Instead, they are derived from human lipocalins, a class of natural binding proteins. Anticalins are used to replace monoclonal antibodies, but are about eight times smaller than monoclonal antibodies, with a size of about 180 amino acids and a mass of about 20kDa.

[0082] As used herein, a disease is prevented if (1) a pharmaceutical composition is administered to a subject internally (by ingestion, inhalation, injection, etc.), topically (absorbed into the body through the skin), or otherwise, before or after exposure to the disease, and (2) the pharmaceutical composition prevents the subject from contracting the disease and developing symptoms / clinical illness normally associated with the disease, or if the subject contracts the disease and develops or does not develop some or all of the symptoms / clinical illness normally associated with the disease of varying severity, the subject recovers from the disease to a normal state of health.

[0083] Also provided are kits for practicing the methods of the invention. "Kit" refers to any article (e.g., package or container) comprising at least one reagent (e.g., pH buffer of the invention). The kit can be promoted, distributed, or sold as a unit for practicing the methods of the invention. In addition, the kit can include a package insert describing the kit and its use. Any or all of the reagents of the kit can be provided in a container that protects them from the external environment, such as in a sealed container or bag.

[0084] In an advantageous embodiment, the kit container may further include a pharmaceutically acceptable carrier. The kit may further include a sterile diluent, which is preferably stored in a separate additional container. In another embodiment, the kit further includes a package insert containing printed instructions for using a combination of a pH buffer and an antipathogen as a method for treating and / or preventing a subject's disease. The kit may also include an additional container containing additional antipathogen agents (e.g., amantadine, rimantadine, and oseltamivir), agents that enhance the effects of such agents, or other compounds that improve the therapeutic effect or tolerability. The kit may also include at least one reagent for performing a specific conventional technique (i.e., nucleic acid extraction) within the skill of the art.

[0085] Sequence identity / similarity: Identity / similarity between two or more nucleic acid sequences or two or more amino acid sequences is expressed as the identity or similarity between the sequences. Sequence identity can be measured as a percentage identity; the higher the percentage, the more identical the sequences are.

[0086] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, Comput. Appl. Biosci. 5:151-3, 1989; Corpet et al., Nucl. Acids Res. 16:10881-90, 1988; Huang et al. Comput. Appl. Biosci. 8, 155-65, 1992; and Pearson ... Gene, 73:237-44, 1988; Higgins et al., Comput. Appl. Biosci. 5:151-3, 1989; Corpet et al., Nucl. Acids Res. 16:10881-90, 1988; Huang et al., Comput. Appl. Biosci. 8, 155-65, 1992; and Pearson et al., Gene, 73:237-44, 1988; Higgins et al., Comput. Appl. Biosci. 5:151-3, 1989; al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, provide a detailed introduction to sequence alignment methods and homology calculations.

[0087] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from a number of sources, including the National Center for Biological Information (NCBI, U.S. National Library of Medicine, Building 38A, Room 8N805, Bethesda, Maryland 20894) and the Internet, and can be used in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Blastn is used to compare nucleic acid sequences, while blastp is used to compare amino acid sequences. More information can be found on the NCBI website.

[0088] After alignment, the number of matches is determined by counting the number of positions where identical nucleotides or amino acid residues are present in the two sequences. The percentage of sequence identity is determined by dividing the number of matches by the length of the sequence as shown in the identified sequence, or by a defined length (e.g., 100 consecutive nucleotides or amino acid residues of the sequence as shown in the identified sequence), and then multiplying the resulting value by 100.

[0089] Unless otherwise indicated, the implementation of the present invention can adopt the conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant technology), cell biology, biochemistry and immunology, which belong to the technical scope of this field. Such conventional techniques include polymer array synthesis, hybridization, connection (ligation) and use of labels to detect hybridization. Suitable techniques can be specifically described with reference to the above examples. However, other equivalent conventional procedures can certainly be used. Such routine techniques and descriptions can be found in standard laboratory manuals, e.g., 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," IRL Press, London, 1984, Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3rd Ed., 1995, Freeman, NY, Gait, "Oligonucleotide Synthesis: A Practical Approach," IRL Press, London, 1984, Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3rd Ed., 1995, Freeman, NY, Gait, "Oligonucleotide Synthesis: A Practical Approach," IRL Press, London, 1984, Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3rd Ed., 1995, Freeman, NY, Gait, "Oligonucleotide Synthesis: A Practical Approach," IRL Press, London, 1984, Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3rd Ed., 1995, Freeman, NY, Gait, "Oligonucleotide Synthesis: A Practical Approach," IRL Press, London, 1984, Ed. (Principles of Biochemistry 3rd ed.), WH Freeman Pub., New York, New York and Berg et al. (2002) Biochemistry, 5th ed., WH Freeman Pub., New York, New York, all of which are incorporated herein by reference in their entirety.

[0090] References

[0091] Isberg et al., 1987: Isberg RR, Voorhis DL, Falkow S. Identification of Invasin: a protein that allows enteric bacteria to penetrate cultured mammalian cells. Cell. 1987 Aug 28;50(5):769-78. doi: 10.1016 / 0092-8674(87)90335-7. PMID: 3304658.

[0092] Chain et al. 2004: Chain PS, Carniel E, Larimer FW, Lamerdin J, Stoutland PO, Regala WM, Georgescu AM, Vergez LM, Land ML, Motin VL, Brubaker RR, Fowler J, Hinnebusch J, Marceau M, Medigue C, Simonet M, Chenal-Francisque V, Souza B, Dacheux D, Elliott JM, Derbise A, Hauser LJ, Garcia E. Insights into the evolution of Yersinia pestis through whole-genome comparison with Yersinia pseudotuberculosis. Proc Natl Acad Sci U S A. 2004 Sep 21;101(38):13826-31. doi: 10.1073 / pnas.0404012101. Epub 2004 Sep 9. PMID: 15358858; PMCID: PMC518763.

[0093] Leong et al., 1990: Leong JM, Fournier RS, Isberg RR. Identification of the integrin binding domain of the Yersinia pseudotuberculosis Invasin protein. EMBO J. 1990 Jun;9(6):1979-89. PMID: 1693333; PMCID: PMC551907.

[0094] All references cited in this application are hereby incorporated by reference in their entirety to the extent not inconsistent herewith.

[0095] It will be seen that the above advantages and those apparent from the above description can be effectively achieved, and since certain changes can be made in the above construction without departing from the scope of the invention, all matter contained in the above description or shown in the accompanying drawings should be interpreted as illustrative and not restrictive.

[0096] It is also to be understood that the following claims are intended to cover all generic and specific features of the invention described herein, together with all statements of the scope of the invention which, as language might be said, might come therebetween.The invention having now been described.

[0097] Table 1. Bacterial derived heterologous proteins with binding domains.

[0098]

[0099]

[0100] Table 2. Fungal derived heterologous proteins with binding domains.

[0101]

[0102] Table 3. Virus-derived heterologous proteins with binding domains.

[0103]

[0104] Table 4. Peptidases and proteases.

[0105]

[0106]

[0107]

[0108] Invasin amino acids (1-986) [SEQ ID NO.1]

[0109] D1=AA1-595

[0110] D2=AA596-694

[0111] D3=AA695-794D4 / D5=795-986

[0112]

[0113]

[0114] Amino acid sequence of Yersinia pseudotuberculosis [SEQ ID NO: 39]

[0115] D1=AA1-595

[0116] D2=AA596-694

[0117] D3=AA695-794

[0118] D4 / D5=795-985

[0119]

[0120]

[0121] Invasin D1-D5 nucleic acid sequence [SEQ ID NO.37]

[0122]

[0123]

[0124]

[0125] Invasin D1-D3 nucleic acid sequence [SEQ ID NO.38]

[0126]

[0127]

[0128] pSi_1fHER2-scr.c (complete plasmid nucleic acid sequence) [SEQ ID NO.40]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135] Chimeric invasin-furin-her2nb fusion protein (nucleic acid sequence) [SEQ ID NO.41]

[0136]

[0137]

[0138] HER2 synthetic nanobody (nucleic acid sequence) [SEQ ID NO.42]

[0139]

Claims

1. An expression cassette for producing a chimeric invadin (Inv) polypeptide, comprising a prokaryotic promoter and a nucleic acid sequence encoding an Inv polypeptide fused to a linker polypeptide at the carboxyl terminus of the Inv polypeptide, and wherein expression of the nucleic acid encoding the chimeric invadin polypeptide is controlled by the prokaryotic promoter.

2. The expression cassette for producing a chimeric invasin polypeptide according to claim 1, further comprising a sequence encoding a binding domain, wherein the binding domain is fused to the amine terminus of the linker polypeptide.

3. An expression cassette for producing a chimeric invasin polypeptide according to claim 2, wherein the binding domain is a binding domain from a protein listed in Tables 1-3, or the binding domain is a synthetic binding domain.

4. The expression cassette for producing a chimeric invadin polypeptide according to claim 1, wherein the nucleic acid sequence of the invadin region of the chimeric polypeptide has 90% identity with nucleic acids 1-2958 of SEQ ID. NO.

37.

5. The expression cassette for producing a chimeric invadin polypeptide according to claim 1, wherein the nucleic acid sequence of the invadin region of the chimeric polypeptide has 95% identity with nucleic acids 1-2958 of SEQ ID. NO.

37.

6. The expression cassette for producing a chimeric invadin polypeptide according to claim 1, wherein the nucleic acid sequence of the invadin region of the chimeric polypeptide has 99% identity with nucleic acids 1-2958 of SEQ ID. NO.

37.

7. An expression cassette for producing a chimeric invadin polypeptide according to claim 1, wherein the nucleic acid sequence of the invadin region of the chimeric polypeptide has 90% identity with nucleic acids 1-2382 of SEQ ID. NO. 37 or SEQ ID. NO.

38.

8. The expression cassette for producing a chimeric invadin polypeptide according to claim 1, wherein the nucleic acid sequence of the invadin region of the chimeric polypeptide has 95% identity with nucleic acids 1-2382 of SEQ ID. NO.

37.

9. The expression cassette for producing a chimeric invadin polypeptide according to claim 1, wherein the nucleic acid sequence of the invadin region of the chimeric polypeptide has 99% identity with nucleic acids 1-2382 of SEQ ID. NO.

37.

10. An expression cassette for producing a chimeric invadin polypeptide according to claim 1, wherein the nucleic acid sequence encodes a linker polypeptide selected from [SEQ.ID.NO.2] to [SEQ ID.NO.20] and / or a protease cleavage site selected from [SEQ.ID.NO.22] to [SEQID.NO.36].

11. The expression cassette for producing a chimeric invasin polypeptide according to claim 1, wherein the nucleic acid sequence encodes a protease cleavage site that is cleaved by a peptidase or a protease.

12. The expression cassette for producing a chimeric invasin polypeptide according to claim 1, wherein the nucleic acid sequence encodes a protease cleavage site for cleavage by a peptidase or protease listed in Table 4.

13. The expression cassette for producing a chimeric invasin polypeptide according to claim 2, wherein a nucleic acid sequence encoding a protease cleavage site for cleavage by a peptidase or a proteinase is located between the sequence encoding the invasin and the sequence encoding the binding domain.

14. The expression cassette for producing a chimeric invasin polypeptide of claim 1, wherein the prokaryotic promoter is a promoter selected from the group consisting of T7, lacUV5, gapA, T5, recA, Ptac, Patac, pAl, lac, Sp6, araBad and trp.

15. The expression cassette for producing a chimeric invasin polypeptide of claim 1, wherein the prokaryotic promoter is a hybrid or synthetic prokaryotic promoter.

16. A bacterium expressing a chimeric invasin polypeptide, wherein the bacterium comprises an expression cassette according to any one of claims 1 to 15.

17. The bacterium expressing a chimeric invasin polypeptide according to claim 16, wherein the bacterium is selected from Clostridium difficile, Escherichia coli, Clostridium tetani, Helicobacter pylori, Fusobacterium nucleatum, Gardnerella vaginitis, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Listeria monocytogenes, Staphylococcus aureus, Campylobacter jejuni, Vibrio vulnificus, Salmonella typhi, Clostridium botulinum, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium leprae, Corynebacterium diphtheriae, Klebsiella pneumoniae, Bacteria including: Burkholderia, Acinetobacter baumannii, Streptococcus mutans, Group B Streptococcus, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae, Enterococcus spp., Enterococcus faecalis, Listeria, Yersinia, Rickettsia, Shigella, Salmonella spp., Legionella, Chlamydia, Brucella, Neisseria, Burkholderia, Bordetella, Borrelia, Coxiella, Mycobacterium, Helicobacter, Staphylococcus, Streptococcus, Porphyromonas, Vibrio, Treponema, Lactobacillus, and Bifidobacterium.

18. The bacterium expressing a chimeric invasin polypeptide according to claim 16, wherein the bacterium is an Escherichia coli bacterium.

19. A method for treating or preventing a disease in a subject, comprising the step of administering to the subject a bacterium according to any one of claims 16 to 18, wherein the bacterium further comprises a therapeutic nucleic acid produced by the bacterium.

20. A method for treating or preventing a disease in a subject, comprising the step of administering to the subject a bacterium according to any one of claims 16-18, wherein the bacterium has been modified to express a therapeutic nucleic acid, protein, antibody, antibody derivative, polypeptide, gene editing system (CRISPR and other gene editing nucleases), eukaryotic translatable mRNA or a combination thereof from a sequence on a bacterial chromosome or plasmid.

21. A chimeric invadin polypeptide, comprising an Inv polypeptide and a linker polypeptide, wherein the linker polypeptide has a first end (N-terminus) and a second end (C-terminus), wherein the first end (N-terminus) of the linker polypeptide is attached to the C-terminus of the Inv polypeptide.

22. The chimeric invasin polypeptide of claim 21, further comprising a peptidase or protease cleavage site.

23. The chimeric invadin polypeptide of claim 21, wherein the chimeric invadin polypeptide further comprises a binding domain of a heterologous protein or a synthetic binding domain attached to the second end (C-terminus) of the linker polypeptide.

24. The chimeric invasin polypeptide of claim 21, wherein the binding domain is a binding domain from a protein listed in Tables 1-3.

25. The chimeric invadin polypeptide of claim 21, wherein the amino acid sequence of the chimeric invadin Inv polypeptide is 90% identical to amino acids 1-986 of SEQ ID. NO. 1, or 90% identical to amino acids 1-985 of SEQ ID. NO.

39.

26. The chimeric invadin polypeptide of claim 19, wherein the amino acid sequence of the chimeric invadin Inv polypeptide is 95% identical to amino acids 1-986 of SEQ ID. NO. 1, or 95% identical to amino acids 1-985 of SEQ ID. NO.

39.

27. The chimeric invadin polypeptide of claim 19, wherein the chimeric invadin Inv polypeptide amino acid sequence has 99% identity with amino acids 1-794 of SEQ ID. NO. 1 or has 99% identity with amino acids 1-794 of SEQ ID. NO.

39.

28. The chimeric invasin polypeptide of claim 19, wherein the chimeric invasin Inv polypeptide amino acid sequence has 90% identity to amino acids 1-794 of SEQ ID. NO.

1.

29. The chimeric invasin polypeptide of claim 19, wherein the chimeric invasin Inv polypeptide amino acid sequence has 95% identity to amino acids 1-794 of SEQ ID. NO.

1.

30. The chimeric invasin polypeptide of claim 19, wherein the chimeric invasin Inv polypeptide amino acid sequence is 99% identical to amino acids 1-986 of SEQ ID. NO.

1.

31. A composition for selectively binding a matrix to a target molecule, comprising a chimeric invadin polypeptide according to any one of claims 21-30, wherein the chimeric invadin polypeptide is bound to a biological and synthetic matrix surface at the amino terminus of the Inv polypeptide, wherein the matrix is ​​selected from beads, viruses, exosomes, rigid matrices (e.g., for producing lateral flow test strips), paper-based biosensors, plastic matrices (e.g., for producing plastic-based biosensors), graphene-based substrates, or nanomaterials (e.g., lipid nanoparticles, metal nanoparticles, mesoporous silica nanoparticles, nanowires, ITO, organic polymers).

32. A chimeric invasin polypeptide comprising the D1-D3 domains of an Inv polypeptide attached to a binding domain of a heterologous protein or a synthetic binding domain.

33. The chimeric invasin polypeptide of claim 32, wherein the binding domain is a binding domain from a protein listed in Tables 1-3.

34. The chimeric invadin polypeptide of claim 32, further comprising a linker polypeptide, wherein the linker polypeptide has a first end (N-terminus) and a second end (C-terminus), wherein the first end of the linker polypeptide is linked to the C-terminal amino acid of the Inv polypeptide and the second end of the linker polypeptide is attached to the amino terminus of the binding domain of the heterologous protein or the amino terminus of the synthetic binding protein.

35. The chimeric invasin polypeptide of claim 32, further comprising a peptidase or protease cleavage site located between the Inv polypeptide region and the binding domain.

36. The chimeric invasin polypeptide of claim 32, wherein the chimeric invasin Inv polypeptide amino acid sequence is 99% identical to amino acids 1-794 of SEQ ID. NO.

1.

37. The chimeric invasin polypeptide of claim 32, wherein the chimeric invasin Inv polypeptide amino acid sequence has 90% identity to amino acids 1-794 of SEQ ID. NO.

1.

38. The chimeric invasin polypeptide of claim 30, wherein the chimeric invasin Inv polypeptide amino acid sequence has 95% identity to amino acids 1-794 of SEQ ID. NO.

1.

39. A composition for selectively binding a matrix to a target molecule, comprising a chimeric invadin polypeptide according to any one of claims 32-38, wherein the chimeric invadin polypeptide is bound to the surface of a biological and synthetic matrix at the amino terminus of the Inv polypeptide region of the chimeric polypeptide, wherein the matrix is ​​selected from beads, viruses, exosomes, rigid matrices (e.g., for producing lateral flow test strips), paper-based biosensors, plastic matrices (e.g., for producing plastic-based biosensors), graphene-based matrices, or nanomaterials (e.g., lipid nanoparticles, metal nanoparticles, mesoporous silica nanoparticles, nanowires, ITO, organic polymers).

40. A chimeric invadin polypeptide comprising a non-binding domain of an Inv protein, a binding domain from a heterologous protein, and a linker, wherein the linker sequence has a first end and a second end, wherein the first end is attached to the non-binding domain of the Inv protein, the second end is attached to the binding domain from the heterologous protein, and wherein the heterologous protein is heterologous to the Inv polypeptide.

41. A non-pathogenic bacterium engineered to express a chimeric invadin polypeptide (i.e., a chimeric Inv with a targeting ligand), wherein the bacterium comprises a nucleic acid sequence encoding a non-binding domain of the Inv protein or a complete Inv protein fused to a sequence encoding a binding domain from a heterologous protein, thereby producing a chimeric Inv protein having an altered binding domain relative to native Inv when the chimeric sequence is expressed.

42. The non-pathogenic bacterium engineered to express a chimeric Inv targeting ligand according to claim 41, wherein the nucleic acid sequence comprises a linker sequence to connect the non-binding domain of the Inv protein fused to a sequence encoding a binding domain from a heterologous protein or synthetic polypeptide or protein that is bound to a chemical moiety capable of cell targeting.

43. The non-pathogenic bacterium engineered to express a chimeric Inv targeting ligand according to claim 42, wherein the linker sequence is a sequence selected from SEQ ID NOS. 2-20.

44. The non-pathogenic bacterium engineered to express a chimeric Inv targeting ligand according to claim 41, wherein the linker sequence comprises a sequence selected from SEQ ID NOS. 2-20 and one or more protease or peptidase cleavage sequences selected from Table 4.

45. The non-pathogenic bacterium engineered to express a chimeric Inv targeting ligand according to claim 44, wherein the protease or peptidase cleavage sequence is recognized by the peptidases and proteases given in Table 4.

46. ​​The non-pathogenic bacterium engineered to express a chimeric Inv targeting ligand of claim 41, wherein the bacterium is engineered to express the hlyA gene from a sequence on the chromosome of the bacterium.

47. The non-pathogenic bacterium engineered to express a chimeric Inv targeting ligand according to claim 41, wherein the binding domain sequence is a sequence encoding a binding domain of any one of the polypeptides mentioned in Tables 1-3.

48. The non-pathogenic bacterium engineered to express a chimeric Inv targeting ligand according to claim 41, wherein the binding domain sequence is a synthetic (ie, non-natural) polypeptide that interacts with a specific binding moiety on the surface of the target cell.

49. The non-pathogenic bacterium engineered to express a chimeric Inv targeting ligand according to claim 41, wherein the sequence encoding the non-binding domain of the Inv protein encodes a polypeptide having 90% (or 95%) identity with amino acids 1-790 of SEQ ID NO.

1.

50. A non-pathogenic bacterium engineered to express a chimeric Inv targeting ligand, wherein the expressed chimeric Inv targeting ligand comprises an Inv protein or a non-binding domain of an intact Inv protein fused to a binding domain from a heterologous protein to produce a chimeric Inv protein.

51. Bacteria for delivering nucleic acids to eukaryotic cells, comprising non-pathogenic bacteria, wherein the bacteria have been modified to express at least one invasion factor from a sequence on the chromosome of the bacteria, and wherein the invasion factor comprises an Inv protein or a non-binding domain of an intact Inv protein fused to a binding domain from a heterologous protein or a synthetic binding protein to produce a chimeric Inv protein targeting portion on the surface of the modified bacteria.

52. The bacterium for delivering a nucleic acid to a eukaryotic cell according to claim 51, wherein the heterologous protein binds to a cell surface protein or a cell surface chemical moiety on the target eukaryotic cell.

53. The bacterium for delivering a nucleic acid to a eukaryotic cell according to claim 51, wherein the binding domain of the heterologous protein is translated from a sequence encoded in a bacterial, fungal, viral or animal genome.

54. The bacterium for delivering a nucleic acid to a eukaryotic cell according to claim 51, wherein the chimeric Inv targeting ligand is expressed from a sequence on the chromosome of the bacterium.

55. The bacterium for delivering nucleic acids to eukaryotic cells according to claim 51, wherein the chimeric Inv targeting ligand comprises a peptide linker connecting the Inv protein or the non-binding domain of the complete Inv protein and the binding domain from a heterologous protein, and wherein the peptide linker comprises one or more amino acids fused in frame to the non-binding domain of the Inv protein or the complete Inv protein and the binding domain from a heterologous protein.

56. The bacterium for delivering a nucleic acid to a eukaryotic cell according to claim 51, wherein the non-binding domain of the Inv protein comprises the D1, D2 and D3 domains of Inv or a combination or subset thereof.

57. The bacterium for delivering a nucleic acid to a eukaryotic cell according to claim 51, wherein the bacterium has been further modified to express at least one therapeutic nucleic acid from a sequence on the chromosome of the bacterium or from a plasmid in the bacterium.

58. The non-pathogenic bacteria engineered to express a chimeric Inv targeting ligand of claim 51, wherein the bacteria has been further engineered to express a therapeutic nucleic acid, protein, antibody, antibody derivative, polypeptide, gene editing system (CRISPR and other gene editing nucleases), eukaryotic translatable mRNA, or a combination thereof.

59. The non-pathogenic bacterium engineered to express a chimeric Inv targeting ligand according to claim 58, wherein the bacterium expresses a therapeutic nucleic acid, protein, antibody, antibody derivative, polypeptide, gene editing system (CRISPR and other gene editing nucleases), eukaryotic translatable mRNA or a combination thereof from a sequence on the chromosome of the bacterium or from a plasmid in the bacterium.

60. The non-pathogenic bacterium engineered to express a chimeric Inv targeting ligand according to claim 51, wherein the bacterium is selected from the group consisting of Clostridium difficile, Escherichia coli, Clostridium tetani, Helicobacter pylori, Fusobacterium nucleatum, Gardnerella vaginitis, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Listeria monocytogenes, Staphylococcus aureus, Campylobacter jejuni, Vibrio vulnificus, Salmonella typhi, Salmonella enterica subspecies, Salmonella typhi, Clostridium botulinum, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium leprae, Mycobacterium tumefaciens, Corynebacterium diphtheriae, Klebsiella pneumoniae, Acinetobacter baumannii, Streptococcus mutans, Group B Streptococcus, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae, Enterococcus spp., Enterococcus faecalis, Listeria monocytogenes, Yersinia, Rickettsia, Shigella, Salmonella spp., Legionella, Chlamydia, Brucella, Neisseria, Burkholderia, Bordetella, Borrelia, Coxiella, Mycobacterium, Helicobacter pylori, Staphylococcus, Streptococcus, Porphyromonas, Vibrio, Treponema, Lactobacillus, and Bifidobacterium.

61. The non-pathogenic bacterium of claim 51 engineered to express a chimeric Inv targeting ligand, wherein the bacterium is an Escherichia coli bacterium.

62. The non-pathogenic bacterium engineered to express a chimeric Inv targeting ligand of claim 51, wherein the bacterium is further engineered to include a marker, wherein contact with a target cell can be detected by visualizing or otherwise detecting the marker in the engineered bacterium.

63. A method for detecting a target cell, comprising the step of contacting the target cell with a bacterium comprising an expression cassette for producing a chimeric invadin polypeptide, wherein the expression cassette is controlled by a prokaryotic promoter that expresses a nucleic acid sequence or a sequence having 99%, 95% or 90% homology to the nucleic acid sequence of the D1, D2 and / or D3 regions and a binding domain from a heterologous protein or a synthetic binding domain (see, e.g., SEQ ID NO. 38), wherein the bacterium further comprises a marker or detectable element (e.g., a fluorescent protein).

64. A method for detecting a target cell, comprising the step of contacting the target cell with a bacterium comprising an expression cassette for producing a chimeric invasin polypeptide, wherein the expression cassette is controlled by a prokaryotic promoter, which expresses a nucleic acid sequence or a sequence having 99%, 95% or 90% homology to the nucleic acid sequence of the D1, D2 and / or D3 regions of invasin (see, e.g., SEQ ID NO. 38) and a sequence encoding a binding domain from a heterologous protein or a sequence encoding a synthetic binding domain, wherein the bacterium further comprises a marker or detectable element (e.g., a fluorescent protein).

65. A method for detecting a target cell, comprising the step of contacting the target cell with a bacterium comprising an expression cassette for producing a chimeric invasin polypeptide, wherein the expression cassette is controlled by a prokaryotic promoter, which expresses a nucleic acid sequence or a sequence having 99%, 95% or 90% homology to the nucleic acid sequence of the D1-D5 region of invasin (see, for example, SEQ ID NO. 37) and a sequence encoding a binding domain from a heterologous protein or a sequence encoding a synthetic binding domain, wherein the bacterium further comprises a marker or detectable element (e.g., a fluorescent protein).

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