A bacterial platform for the delivery of gene editing systems into eukaryotic cells

Abstract

A bacterial-mediated gene editing delivery platform that uses invasive, non-pathogenic bacteria to deliver gene editing cargo containing the CRISPR / Cas system into eukaryotic cells. The bacteria contain a prokaryotic expression cassette encoding the gene editing cargo.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 856,055, filed June 1, 2019.

[0002] The present invention relates to the delivery of gene editing systems into eukaryotic cells using a non-pathogenic bacterial delivery platform. [Background technology]

[0003] Gene editing is a form of genetic engineering that inserts, deletes, alters, or replaces DNA in the genome of an organism. Editing specific sequences in a genome can be used for research purposes as well as gene therapy by targeting mutations to specific genes or inserting functional genes into an organism and targeting it to replace defective genes associated with pathogenic phenotypes or genetic diseases. A common approach to gene editing is to use engineered or synthetic nucleases. One example is the CRISPR (clustered regularly interspaced short palindromic repeats) / Cas or CRISPR / Cas system, which is rapidly advancing the field of genome editing. Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention provides a bacteria-mediated gene editing delivery platform that uses invasive, non-pathogenic bacteria to deliver gene editing cargo, including but not limited to, a CRISPR / Cas system, to eukaryotic cells, wherein the bacteria comprises a prokaryotic promoter associated with a prokaryotic expression cassette encoding the gene editing cargo. [Means for solving the problem]

[0005] According to one aspect, the present invention provides a method for generating and delivering a gene editing system, including a nuclease-based gene editing system, to a eukaryotic cell. The method includes invading an invasive non-pathogenic bacterium into a eukaryotic cell. The bacterium includes a plasmid having a prokaryotic expression cassette encoding the gene editing system. The prokaryotic expression cassette can operate under the control of one or more prokaryotic promoters, such as T7, lacUV5, gapA, T5, recA, Ptac, Patac, pA1, lac, Sp6, araBad, trp, and hybrid promoters. These prokaryotic promoters can be inducible, constitutive, or combinations thereof. If multiple promoters are present on a plasmid or multiple plasmids are present in a bacterium, it is contemplated that different promoter types can be used based on the relative activity of the promoters in the system. By using different types of prokaryotic promoters, transcription can be tailored to control the level of the transcript. By using prokaryotic promoters in a bacterium, transcription of the components of the gene editing system occurs in the bacterium and not in the target eukaryotic cell.

[0006] According to a second aspect, the present invention provides a composition comprising a bacterium for modifying the genome of a eukaryotic cell. The bacterium has a prokaryotic vector. The prokaryotic vector (or vectors) can be a plasmid or a plurality of plasmids that use a prokaryotic promoter to control transcription from the plasmid. The one or more plasmids can encode a CRISPR / Cas system having (a) at least one CRISPR-associated (Cas) enzyme, (b) at least one tracr sequence, and (c) at least one CRISPR RNA (crRNA). The crRNA can have (i) at least one CRISPR direct repeat region, and (ii) at least one spacer sequence capable of hybridizing to a target sequence in a eukaryotic cell. The plasmid encoding the CRISPR / Cas system can optionally include a nuclear localization signaling sequence to facilitate nuclear translocation of the gene editing system to the nucleus of the eukaryotic cell. The bacterium is engineered to enter a eukaryotic cell. According to this second aspect of the present invention, the bacterium produces a programmable nuclease system to direct CRISPR complex activity in the nucleus of the eukaryotic cell.

[0007] According to a third aspect, the present invention provides a composition comprising the use of a bacterial delivery system to produce and deliver a gene editing system (gene editing cargo) capable of entering a eukaryotic cell using the entry factors, invasin protein (encoded by the inv gene) and listeriolysin O (LLO) protein (encoded by the hlyA gene). The bacterial system is engineered to produce and / or deliver the gene editing system to a eukaryotic cell. The composition comprising the use of a bacterial cell to produce and / or deliver the gene editing system may comprise one or more prokaryotic plasmids with one or more prokaryotic promoters, one or more eukaryotic plasmids with one or more eukaryotic promoters, or any combination thereof. According to this third aspect of the present invention, the bacterial system utilized is capable of attaching to a eukaryotic cell by expressing an invasin protein, and escaping the eukaryotic endosome by expressing an LLO protein, thereby allowing the gene editing cargo to be released into the cytoplasm of the eukaryotic cell, and, if appropriate, translocating to the nucleus of the eukaryotic cell.

[0008] According to a third aspect, the present invention provides compositions for in vitro, in vivo, or ex vivo engineering of eukaryotic cells to insert, remove, or replace genes. When used in vitro, engineered bacterial systems to generate and deliver gene editing systems can be used to insert, remove, or replace regions of interest on eukaryotic cell genomes and modify target DNA in vitro. For example, CRISPR motifs or PAM sequences present on target DNA in vitro (in living cells outside a living system) can be recognized by a CRISPR / Cas gene editing system generated from a prokaryotic plasmid and delivered to a eukaryotic cell by a bacterial cell. According to another embodiment, CRISPR motifs or PAM sequences on target DNA in vitro are recognized by a CRISPR / Cas gene editing system transcribed and translated from a eukaryotic plasmid in a target eukaryotic cell following delivery by a bacterial cell. These genome modification experiments can be performed in vitro, including but not limited to use with primary cells, immortalized cells, dividing cells, non-dividing cells, and hematopoietic stem / progenitor cells. These in vitro gene editing applications include, but are not limited to, for use in studying disease, etiology of specific genetic modifications, infectious agents, genetic disorders, functional genomic screening, chromosome painting, building disease models (including cancer models), in vitro selection libraries, mismatch detection nuclease assays, high throughput profiling assays, diagnostics, and related next generation sequencing activities. For ex vivo genome editing and modification, eukaryotic cells are isolated from a living system, and bacterial systems engineered to generate and deliver gene editing systems are used to insert, remove, or replace regions of interest on the isolated eukaryotic cell genome and modify the target DNA ex vivo. For example, CRISPR motifs or PAM sequences present ex vivo (in cells isolated from a living system) can be recognized by a CRISPR / Cas gene editing system generated from a prokaryotic plasmid and delivered to eukaryotic cells ex vivo by bacterial cells.According to another embodiment, the CRISPR motif or PAM sequence present on the DNA ex vivo is recognized by a CRISPR / Cas gene editing system that is transcribed and translated from a eukaryotic plasmid in a target eukaryotic cell following delivery by a bacterial cell. In some cases, the present invention is used to achieve therapeutic success in which isolated cells are treated and returned to the target tissue for transplantation back into the biological system from which the eukaryotic cells originated. An example of an ex vivo (or in vitro) application is chimeric antigen receptor (CAR) T cell therapy, where T cells from a patient's blood are extracted and the genome of the isolated T cells is edited to express an artificial receptor targeted to a specific tumor antigen using the present invention. For in vivo genome editing and modification, a bacterial system engineered to generate and deliver a gene editing system can be used to insert, remove, or replace regions of interest on the eukaryotic cell genome to perform specific genome modifications of DNA in a biological system. In some cases, the present invention is used to deliver gene editing systems to target tissues (eyes, muscles, lungs, brain, nasal cavity, gastrointestinal tract, heart, skin, oral cavity, reproductive tissues, or any combination thereof). For example, CRISPR motifs or PAM sequences present in vivo can be recognized by CRISPR / Cas gene editing systems generated from prokaryotic plasmids and delivered to target eukaryotic cells in vivo by bacterial cells. According to another embodiment, CRISPR motifs or PAM sequences present on DNA in vivo are recognized by CRISPR / Cas gene editing systems transcribed and translated from eukaryotic plasmids in target eukaryotic cells following delivery to specific tissues by bacterial cells. These in vivo gene editing applications include, but are not limited to, studies of disease, etiology of specific genetic modifications, infectious agents, genetic disorders, development of gene knock-in and knock-out animals (including generation of transgenic embryos), disease treatment, disease prevention, and treatment of rare genetic disorders.

[0009] According to a fifth aspect, the present invention provides a composition comprising the use of a bacterial delivery system to produce and deliver a gene editing system (gene editing cargo), wherein the elements comprising the gene editing system are expressed from a recombinant plasmid, expressed from a bacterial chromosome (integrated into the bacterial chromosome), or expressed using any combination thereof. Similarly, the entry factors (inv and hlyA) required for entering and exiting eukaryotic cells from endosomes of eukaryotic cells are expressed from a recombinant plasmid, expressed from a bacterial chromosome, or expressed using any combination thereof. Any components of the gene editing system and any factors required for bacterial entry may be expressed in combination from one or more recombinant plasmids, bacterial chromosomes, or any combination thereof.

[0010] According to a sixth aspect, the present invention provides a plasmid comprising one or more of the following: a Cas gene, an NLS, a CRISPR direct repeat region, a spacer sequence (or an insertion site for a spacer sequence) and a tracr sequence. These elements can be expressed from the plasmid upon transformation of a bacterial cell, such as by one or more prokaryotic promoters. According to an advantageous embodiment, the plasmid comprises a spacer sequence (or an insertion site for a spacer sequence) and a prokaryotic or eukaryotic promoter.

[0011] 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: [Brief description of the drawings]

[0012] [Figure 1] Figure 1 provides an illustration of a bacteria-mediated gene editing delivery system that uses invasive, non-pathogenic bacteria to deliver a gene editing cargo, such as a CRISPR / Cas system, to a eukaryotic cell. The bacteria can contain a prokaryotic expression cassette that encodes the gene editing cargo.

[0013] [Figure 2A]Figure 2 is a series of diagrams (Figures 2A, 2B, and 2C) of the plasmid system used to engineer bacteria to generate and deliver a gene editing system in one or more embodiments of the present invention. Figure 2A is a schematic diagram with the estimated sequence length of the CRISPR-mediated gene editing plasmid (pSiCRISP) containing at least one prokaryotic promoter, an origin of replication, a selection marker, a crRNA leader, at least one spacer / crRNA homologous to a target DNA gene of interest, a tracr / tracrRNA, a CRISPR direct repeat sequence, a nuclear localization signal (NLS), and a Cas gene.

[0014] [Figure 2B] Figure 2 is a series of diagrams (Figures 2A, 2B, and 2C) of the plasmid system used to engineer bacteria to generate and deliver a gene editing system in one or more embodiments of the present invention. Figure 2B is a schematic with estimated sequence length of an invasion plasmid (pSiVEC) that contains at least one prokaryotic promoter, an origin of replication, a selection marker, at least one entry factor for receptor-mediated endocytosis (Entry Factor A), and at least one entry factor for endosomal escape (Entry Factor B).

[0015] [Figure 2C] Figure 2 is a series of diagrams (Figures 2A, 2B, and 2C) of the plasmid system used to engineer bacteria to generate and deliver the gene editing system in one or more embodiments of the present invention. Figure 2C is a schematic diagram showing the estimated sequence length of the CRISPR-mediated gene editing plasmid (pSiCRISP) containing at least one eukaryotic promoter, an origin of replication, a selection marker, a crRNA leader, a target of interest, at least one spacer / crRNA homologous to the target DNA of interest, tracr / tracrRNA, CRISPR direct repeats, a nuclear localization signal (NLS), and a Cas gene.

[0016] [Diagram 3] Figure 3 is a set of three images (Figure 3A, Figure 3B, and Figure 3C) of FEC19 E. coli co-transformed with pSiCRISP_GFP and pSiVEC and PCR screened to verify the presence of pSiCRISP_GFP and pSiVEC components via PCR amplification of (Figure 3A) the 424 bp NLS, (Figure 3B) the 727 bp Cas9, and (Figure 3C) the 201 bp inv gene expression cassette using specific primers for PCR.

[0017] [Figure 4] FIG. 4 is a graph showing the percentage of A549 cells expressing GFP. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Gene editing is a form of genetic engineering that inserts, deletes, alters, or replaces DNA in the genome of an organism. Editing specific sequences in a genome can be used for research purposes as well as gene therapy by targeting mutations to specific genes or inserting functional genes into an organism and targeting them to replace defective genes associated with pathogenic phenotypes or genetic diseases. A common approach to gene editing is to use engineered or synthetic nucleases. One example is the CRISPR (clustered regularly interspaced short palindromic repeats) system, which is rapidly advancing the field of genome editing.

[0019] CRISPR / CRISPR-associated proteins (Cas) have been used to edit the genomes of various model organisms, such as bacteria, yeast, Caenorhabditis elegans, Drosophila melanogaster, plants, zebrafish, mouse and human cells (Mali et al. "Cas9 as a versatile tool for engineering biology," Nat. Methods. 10:957-963 (2013)). The CRISPR / Cas system has been delivered to these model organisms using a variety of delivery mechanisms and vectors. More specifically, the CRISPR / Cas system has been delivered to eukaryotic cells using a number of delivery methods, including viral and non-viral vectors.

[0020] Despite advances in the field, efficient in vitro and in vivo delivery of gene editing nuclease systems, including CRISPR / Cas systems, to target cells remains a challenge. There are many drawbacks associated with current CRISPR / Cas system delivery strategies that impede clinical translation of gene editing applications and therapeutics in human and animal medicine. We developed a novel bacterial delivery platform to efficiently deliver gene editing nuclease systems, including CRISPR / Cas systems, to target eukaryotic cells, providing cellular uptake and, when necessary, nuclear translocation of the gene editing nuclease systems without the host genome for transient gene editing applications.

[0021] The present invention provides a novel bacterial delivery platform for gene editing systems, including CRISPR systems, to provide tissue-specific delivery and intracellularization of genome editing components in any eukaryotic cell (dividing and non-dividing) without genomic integration in the eukaryotic cell.

[0022] Gene editing approaches are generally characterized by the use of nuclease-based systems (i.e., CRISPR / Cas) to effectively address a wide range of gene editing applications, including the prevention of infectious diseases, chronic non-infectious diseases, and other genetic diseases. Generally, these nucleases create site-specific double-strand breaks at desired locations in the genome. The induced double-strand breaks are repaired by non-homologous end joining (NHEJ) or homologous recombination (HR), resulting in targeted mutations in the genome. An example of a nuclease-based gene editing system is the CRISPR system. Other examples include, but are not limited to: transcription activator-like effector-based nucleases (TALENs), meganucleases, zinc finger nucleases (ZFNs), ARC nucleases, nucleases based on the Argonaute system, the TtAgo nuclease system, and other homing endonucleases.

[0023] The CRISPR system consists of two key components. The first component is a single guide RNA oligonucleotide (sgRNA) or guide RNA (gRNA) that represents a fusion of CRISPR RNA (crRNA) and tracrRNA. The second component is a Cas nuclease. These two components combined represent the SGRNA / gRNA and the CRISPR-associated protease / nuclease. If a protospacer adjacent motif (PAM) is present at a genomic locus of a target DNA molecule, the Cas nuclease is directed to that PAM locus by the gRNA, and a site-specific double-stranded break in the DNA occurs, inducing a stable gene editing event. This gene editing event can result in a permanent gene knockout (gene removal) or knock-in (gene insertion).

[0024] CRISPR / Cas systems are classified into three main types: I, II, and III. Although the mechanism of gene editing differs between the three types, all three are believed to be applicable to eukaryotic cells. The most commonly used CRISPR / Cas system in eukaryotes is type II, which requires a single Cas protein, namely, Cas9. Similar to CRISPR / Cas, other gene editing systems provide the desired genetic mutation or genome modification event.

[0025] These gene editing nuclease proteins and gene editing systems have been difficult to deliver for clinical and research discovery applications. For example, due to the large size of the CAS9 protein, the positive charge of Cas9, and the strong negative charge of the sgRNA, effective co-delivery of the Cas9 / sgRNA complex into the host eukaryotic cell and across the cell membrane has been difficult with previous systems. Cas9, sgRNA, and the Cas9 / sgRNA complex cannot cross the lipid bilayer of the cell membrane without a delivery vehicle. One way to achieve CRISPR / Cas gene editing is to deliver the Cas9 protein along with the sgRNA using a plasmid. The sgRNA along with the sgRNA scaffold is incorporated into the plasmid along with the Cas9 cassette. The plasmid can also contain a nuclear localization signal (NLS) for the gene editing components to be delivered to the nucleus. For example, the sgRNA and Cas9 protein may require an NLS to allow both to move from the cytoplasm to the nucleus for transcription. The present invention uses a plasmid with a prokaryotic promoter to drive expression of the gene editing system in the bacterial cell used for delivery to the eukaryotic cell. In the present invention, a plasmid (i.e., direct repeats, tracr / tracrRNA, spacer, Cas9, and NLS) using a prokaryotic promoter to drive expression of the gene editing system is transformed into a non-pathogenic bacterial cell. Furthermore, the bacterial cell is engineered to be invasive and can enter eukaryotic cells via receptor-mediated endocytosis. The combined construct of bacteria and plasmid, with the gene editing cargo expressed under the control of a prokaryotic promoter according to certain embodiments, constitutes a bacteria-mediated gene delivery system that is delivered to eukaryotic cells.

[0026] The bacterial-mediated gene editing delivery platform taught herein offers many advantages when compared to other gene editing delivery methods. First, the system facilitates transient delivery. The bacterial delivery platform rapidly achieves the desired editing event, minimizes off-target editing, enhances DNA specificity, and reduces the undesirable consequences of prolonged CRISPR / Cas9 or other nuclease editing systems in cells.

[0027] Second, the system is non-immunogenic. The bacterial cells are engineered to avoid recognition by antigen-presenting cells, and the system does not induce immune or other cytokine responses in the host. In contrast, antibodies can be formed against other delivery vehicles, such as nanoparticles, and liposomes and viral vectors can induce immune responses.

[0028] Third, the system is not integrated. According to certain embodiments, expression of the gene editing components is solely from prokaryotic promoters. This inhibits integration in the host genome and provides controlled delivery of CRISPR / Cas9 or other nuclease editing systems, eliminating the risk of oncogenic and other undesirable side effects to the host. However, the system uses a plasmid with a eukaryotic promoter and uses non-pathogenic bacteria as a delivery vehicle for the plasmid, which may then be transcribed in the target cell. Fourth, the system is highly stable. The delivery platform is not inhibited by exposure to serum, proteases, and nucleases, allowing the bacterial vehicles to remain stable until they reach their target site. Unlike other non-viral vectors, our bacterial delivery platform is not eliminated by phagocytic clearance, further contributing to its stability.

[0029] Fifth, the system allows for large payload delivery capacity. The bacterial delivery platform can effectively deliver Cas enzymes, such as Cas9, despite the large size of the enzyme. While other non-viral vectors struggle to deliver sufficient Cas9 concentrations to target tissues, the system taught herein functions via receptor-mediated endocytosis, resulting in effective intracellularization and endosomal release of the bacterial vehicle containing the delivered gene editing system.

[0030] Sixth, the system offers a large therapeutic window: the bacterial delivery platform can be administered at a range of dosages to achieve gene editing effects without inducing toxicity.

[0031] Seventh, the system enables efficient delivery: the delivery platform can efficiently enter eukaryotic cells via receptor-mediated endocytosis and efficiently escape host endosomes to facilitate delivery of the gene editing system.

[0032] To date, efficient in vivo delivery of gene editing and CRISPR / Cas systems to target cells has been a challenge. Current strategies for CRISPR / Cas delivery are mainly based on mechanical approaches (electroporation, hydrodynamic injection, microinjection) and viral vector delivery (lentivirus, adenovirus, adeno-associated virus). Many of these methods cannot be easily translated for clinical applications in animals or human patients. Non-viral delivery methods such as liposomes and nanoparticles have also been used. However, to achieve therapeutic efficacy, CRISPR / Cas systems need to be delivered more efficiently and specifically to avoid unwanted side effects and safety concerns due to systemic administration, trafficking to unwanted tissues, and host integration.

[0033] Viral vectors have been used to deliver the CRISPR / Cas9 system in vitro and in vivo, but the risk of oncogenesis due to host genome integration, limited insert size, stimulation of undesirable immune responses, and difficulty in large-scale production severely limit their further applications. The difficulty in large-scale production significantly limits future applications. Host genome integration using viral vector delivery may result in high-level and long-term expression of Cas9 protein or other nucleases, contributing to higher off-target effects and stronger immune responses in the host. Alternative non-viral delivery systems for CRISPR / Cas are urgently needed. Non-viral vectors are another potential option for CRISPR / Cas9 delivery. These include, but are not limited to, liposomes, nanoparticles, exosomes, microvesicles, cell-penetrating peptides (CPPs), and other lipid, polymer, and lipid nanoparticles. Despite their utility in vitro, complex physiological conditions create barriers to using these non-viral delivery approaches in vivo.

[0034] The present invention provides systems and methods for delivering gene editing or CRISPR / Cas systems to eukaryotic cells using invasive, non-pathogenic bacteria that contain prokaryotic expression cassettes to drive expression of those genes prior to delivery. In other words, the present invention utilizes bacteria that are both invasive and non-pathogenic and contain plasmids driven by prokaryotic promoters that confer invasiveness to the bacteria, thereby allowing them to enter eukaryotic cells.

[0035] The advantage of using bacteria containing prokaryotic expression cassettes is that the necessary gene editing cargo (oligonucleotides and nuclease proteins) are expressed only by the bacteria. They are produced by recombination prior to delivery. Once the bacterial cells are uptaken and incorporated into the cytoplasm of the host eukaryotic cell, the plasmid containing the gene editing genes and proteins cannot be expressed in the eukaryotic cell due to polymerase limitations. This prevents these gene editing genes and proteins, as well as other plasmid DNA, from being integrated into the host genome. This is an advantage, as genome integration is associated with adverse side effects including, among others, the inability to control the expression of these genes, carcinogenesis, and toxicity (Kim S et al. 2014. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Research 24:1012-1019). The gene editing event (targeted DNA modification) needs to occur only once per copy of DNA to drive a permanent gene modification through all cell populations. Therefore, in some cases, it is advantageous to use a delivery system for transiently introducing a gene editing system in which the gene editing components (Cas9 and sgRNA) are not integrated into the genome of the host cell. In the integrated gene editing approach (i.e., viral vector delivery), the gene editing components (Cas9 and sgRNA) are integrated into the genome of the target cell, so that DNA mutations continue to occur in all copies of DNA. By avoiding integration into the genome of the target cell, the controlled expression of the gene editing components is provided, further limiting toxicity and off-target mutagenesis in the target cell.

[0036] The present invention uses bacteria that contain entry factors (e.g., inv and hlyA) that allow the bacteria to enter eukaryotic cells and deliver nuclease-based gene editing cargo (e.g., CRISPR, Talens, zinc finger are examples) to eukaryotic cells. Using bacteria that contain a plasmid with a eukaryotic promoter provides a system in which the plasmid with the gene editing system is delivered and the gene editing cargo is created in the eukaryotic cell. Systems that use prokaryotic promoters include the bacteria as a site of transcription and expression of the gene editing system. Although both delivery routes (e.g., using prokaryotic and eukaryotic promoters) are contemplated herein, systems that use prokaryotic promoters offer several important advantages, including ensuring that the system does not integrate into the target cell.

[0037] The present invention will find a wide range of applications. For example, in diagnostics, to detect genomic mutations associated with genetic diseases, or to detect rejection markers before organ transplantation. The present invention can be used for genome-wide genetic screening, where foreign DNA from infectious agents present in eukaryotic cells causes mutations, CAR-T applications for cancer therapy, where DNA modifications are associated with reduced infectious phenotypes, disease prevention, treatment of genetic diseases (i.e., cystic fibrosis, Huntington's disease, fragile X syndrome, sickle cell anemia, familial hypercholesterolemia) where DNA mutations are associated with undesirable genetic phenotypes, and where it is important to identify functional genes or gene sequences that affect physiological effects.

[0038] The invention further provides a method for chromosome painting in live cells. This system can be used to perform CRISPR imaging as a method to detect chromatin dynamics in live cells. Using a catalytically inactive dCas9 (nuclease-dead Cas9 binds to DNA but cannot cleave the bound DNA) fused to a fluorescent marker such as green fluorescent protein (GFP), researchers have turned dCas9 into a customizable DNA labeler compatible with live-cell fluorescence microscopy. Alternatively, CRISPR gRNAs can be fused to protein-interacting RNA aptamers, which recruit specific RNA-binding proteins (RBPs) tagged with fluorescent proteins to visualize target genomic loci. Further applications include the ability to generate transgenic animals, where animals are genetically modified to produce a targeted genotype or phenotype, which is useful for scientific research applications such as, for example, genetics and disease studies, or for generating, for example, agricultural animals resistant to a particular disease or agricultural species (plants) resistant to a particular disease or disorder.

[0039] FIG. 1 shows a graphical representation of a bacteria-mediated gene editing delivery system using invasive non-pathogenic bacteria to deliver gene editing cargo, such as a CRISPR / Cas system, to eukaryotic cells. In the exemplary system, the bacteria contain a prokaryotic expression cassette encoding the gene editing cargo. Provided as a non-limiting example, a representation of the system's ability to generate and deliver CRISPR / Cas9 gene editing cargo to mammalian cells is depicted. The diagram shows the operation of the bacteria-mediated gene editing delivery system proceeding through multiple steps of the process of delivering and executing gene editing in eukaryotic cells.

[0040] First, all parts of the gene-editing plasmid, including the CRISPR-associated protein 9 (Cas9) sequence, the trans-activating CRISPR RNA (tracrRNA), at least one CRISPR direct repeat sequence, a nuclear localization signal (NLS), and at least one spacer sequence, are transcribed in the bacterium (the invasive bacterial delivery vehicle). Such transcription can occur prior to entry into the target eukaryotic cell. In the bacterium, the tracrRNA hybridizes to the direct repeats of the crRNA and is processed into a mature single guide RNA (sgRNA) containing an individual spacer sequence, and the Cas9RNA transcript is translated into an active enzyme. These components (Cas9 enzyme and mature sgRNA) constitute the CRISPR / Cas9 gene editing system. The bacterium can optionally express the rnc gene of RNaseIII from either a recombinant plasmid or the bacterial chromosome. Second, the bacterial vehicle that generates the CRISPR / Cas9 gene editing system is targeted to mammalian cells by an entry factor such as an invasin protein and taken up into the cell by receptor-mediated endocytosis (RME). Third, the endosome is disassembled by a second entry factor for endosomal release such as LLO, releasing the sgRNA (mature crRNA:tracrRNA complex) and Cas9 enzyme for translocation into the eukaryotic cell nucleus, guided by the NLS. Fourth, once inside the nucleus, the sgRNA guides Cas9 to the DNA target consisting of a protospacer and a downstream protospacer adjacent motif (PAM) via heteroduplex formation between the crRNA and the protospacer DNA. Fifth, Cas9 creates a double-strand break in the protospacer via cleavage of the target DNA upstream of the PAM. In the sixth and final step, the target DNA is permanently altered. This diagram describes an example process for adapting this bacterial platform to generate, deliver, and direct CRISPR complex activity within the nucleus of a eukaryotic cell to perform targeted gene editing.

[0041] 2A and 2B provide diagrams of plasmid systems used to engineer bacteria to generate and deliver gene editing systems in one or more embodiments of the present invention. FIG. 2A is a schematic diagram including the estimated sequence length of a CRISPR-mediated gene editing plasmid (pSiCRISP), including at least one prokaryotic promoter, an origin of replication, a selection marker, a crRNA leader, at least one spacer / crRNA homologous to a target DNA gene of interest, a CRISPR direct repeat sequence, an NLS, and a Cas gene. In addition to the pSiCRISP plasmid providing the gene editing system, this system requires components that allow the bacteria to enter eukaryotic cells. FIG. 2B is a schematic diagram showing the estimated sequence length of an invasion plasmid (pSiVEC), including at least one prokaryotic promoter, an origin of replication, a selection marker, at least one entry factor for receptor-mediated endocytosis (entry factor A), and at least one entry factor for endosomal escape (entry factor B). These two plasmid systems can be co-transformed into bacteria. According to another embodiment of the present invention, the components of these two plasmids can be combined into a single recombinant plasmid for bacterial transformation. According to another embodiment of the invention, the components of these two plasmids may be expressed from the bacterial chromosome.

[0042] Glossary: All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless a technical or scientific term is defined differently herein.

[0043] The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit any embodiment. As used herein, the singular forms "a," "an," and "the" are intended to include the plural unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," as used herein, specify the presence of the stated features, integers, steps, operations, elements, and / or components, but may not exclude the presence or addition of one or more other features, integers, steps, operations, elements, ingredients, and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. When used to define products, compositions, and methods, "consisting essentially of" shall mean excluding essentially significant amounts of other components or steps. Thus, a composition consisting essentially of the recited components would not exclude trace amounts of contaminants and pharma- ceutically acceptable carriers. "Consisting of" shall mean excluding trace amounts or more of other components or steps.

[0044] The terms "polynucleotide" and "nucleic acid", used interchangeably herein, refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the terms include, but are not limited to: single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. An "oligonucleotide" generally refers to a polynucleotide of about 5 to about 100 nucleotides, either single-stranded or double-stranded DNA. However, for purposes of this disclosure, there is no upper limit to the length of an oligonucleotide. Oligonucleotides are also known as "oligomers" or "oligos" and can be isolated from genes or chemically synthesized by methods known in the art. The terms "polynucleotide" and "nucleic acid" should be understood to include single-stranded (such as sense or antisense) and double-stranded polynucleotides, as applicable to the embodiments being described. The terms "nucleic acid", "polynucleotide" or "oligonucleotide" refer to DNA molecules, RNA molecules, or analogs thereof. As used herein, the terms "nucleic acid", "polynucleotide" and "oligonucleotide" include, but are not limited to, DNA molecules such as cDNA, genomic DNA or synthetic DNA, and RNA molecules such as guide RNA, messenger RNA or synthetic RNA. Furthermore, as used herein, the terms "nucleic acid" and "polynucleotide" include single-stranded and double-stranded forms.

[0045] "Genomic DNA" refers to the DNA of an organism's genome, including, but not limited to, the DNA of a bacterium, virus, fungus, archaea, plant or animal genome.

[0046] "Manipulation" of DNA includes binding of DNA, nicking of one strand or cleaving (i.e., cutting) both strands of DNA, or modifying (e.g., amidation, methylation, etc.) the DNA or polypeptides associated with the DNA. Manipulation of DNA can suppress, activate, or modulate (increase or decrease) the expression of an RNA or polypeptide encoded by the DNA.

[0047] "Gene editing" refers to the process of changing the genetic information present in the genome of a cell. This gene editing is performed by manipulating genomic DNA, resulting in a change in the genetic information. Such gene editing may or may not affect the expression of the edited DNA.

[0048] As used herein, the term "guide RNA" generally refers to an RNA molecule (or collectively a group of RNA molecules) that can bind to a Cas protein and help target the Cas protein to a specific location within a target polynucleotide (e.g., DNA). A guide RNA can include a crRNA segment and a tracrRNA segment. As used herein, the term "crRNA" or "crRNA segment" refers to an RNA molecule or a portion thereof that includes a polynucleotide-targeting guide sequence, a stem sequence, and optionally, a 5' overhang sequence. As used herein, the term "tracrRNA" or "tracrRNA segment" refers to an RNA molecule or a portion thereof that includes a protein-binding segment (e.g., a protein-binding segment can interact with, for example, a CRISPR-associated protein, such as Cas9). The term "guide RNA" encompasses a single guide RNA (sgRNA) in which the crRNA segment and the tracrRNA segment are located within the same RNA molecule. The term "guide RNA" also collectively encompasses a group of two or more RNA molecules, in which the crRNA segment and the tracrRNA segment are located on separate RNA molecules. The term "modification" in the context of an oligonucleotide or polynucleotide includes, but is not limited to, (a) terminal modifications, such as a 5' terminal modification or a 3' terminal modification, (b) nucleobase (or "base") modifications, including base substitution, insertion, or removal, (c) sugar modifications, including modifications at the 2', 3', and / or 4' positions, and (d) backbone modifications, including modifications or replacements of phosphodiester bonds. The term "modified nucleotide" generally refers to a nucleotide having modifications in the chemical structure of one or more of the base, sugar, and phosphodiester bond or backbone moieties, including the nucleotide phosphate.

[0049] As used more generally herein, "modification" refers to a chemical moiety or part of a chemical structure that is different from that found in unmodified ribonucleotides, i.e., adenosine, guanosine, cytidine, and uridine ribonucleotides. The term "modification" may refer to the type of alteration. For example, "same modification" means the same type of modification, and "same modified nucleotide" means that the modified nucleotides have the same type of modification, but the base (A, G, C, U, etc.) may be different. Similarly, an adapter polynucleotide with "two modifications" is a guide RNA with two types of modifications, which may or may not be at the same nucleotide, and each type may appear at multiple nucleotides in the adapter. Similarly, an adapter polynucleotide with "three modifications" is an adapter with three types of modifications, which may or may not be at the same nucleotide, and each type may appear at multiple nucleotides.

[0050] A "donor polynucleotide" is a nucleotide polymer or oligomer intended for insertion into a target polynucleotide. A donor polynucleotide can be a natural or modified polynucleotide, an RNA-DNA chimera, or a DNA fragment, either single-stranded or double-stranded, or a PGR-amplified ssDNA or dsDNA fragment. A fully double-stranded donor DNA is advantageous due to the potential for increased stability, since dsDNA fragments are generally more resistant to nuclease degradation than ssDNA. The terms "xA", "xG", "xC", "xT", or "x(A, G, C, T)" and "yA", "yG", "yC", "yT", or "y(A, G, C, T)" refer to a nucleotide, nucleobase, or nucleobase analog (Krueger et al., "Synthesis and Properties of Size-Expanded DNAs: Toward Designed, Functional Genetic Systems", Acc. Chem. Res. 2007, 40, 141-150 (2007); the contents of which are incorporated herein by reference in their entirety).

[0051] As used herein, the term "target polynucleotide" or "target" refers to a polynucleotide that includes a target nucleic acid sequence: the target polynucleotide can be single-stranded or double-stranded, and according to certain embodiments, is double-stranded DNA. According to certain embodiments, the target polynucleotide is single-stranded RNA. As used herein, "target nucleic acid sequence" or "target sequence" refers to a specific sequence or its complement that you want to bind, nick, or cleave using a CRISPR / Cas system or other gene editing system.

[0052] The term "hybridization" or "hybridization" refers to the process in which fully or partially complementary polynucleotide strands come together under suitable hybridization conditions to form a double-stranded structure or region in which the two constituent strands are linked by hydrogen bonds. As used herein, the term "partial hybridization" includes cases in which the double-stranded structure or region contains one or more bulges or mismatches. Hydrogen bonds are usually formed between adenine and thymine, or adenine and uracil (A and T or A and U), or cytosine and guanine (C and G), although other non-standard base pairs may be formed (see, for example, Adams el 21 5 al., The Biochemistry of the Nucleic Acids, 11th ed., 1992). It is contemplated that modified nucleotides may form hydrogen bonds that allow or facilitate hybridization. "Hybridizable" or "complementary" or "substantially complementary" means that a nucleic acid (e.g., RNA) contains a sequence of nucleotides that allows non-covalent binding, i.e., forming Watson-Crick base pairs and / or G / U base pairs, to "anneal" or "hybridize" to another nucleic acid in a sequence-specific, antiparallel manner (i.e., the nucleic acid specifically binds to a complementary nucleic acid) under suitable in vitro and / or in vivo conditions of temperature and ionic strength of the solution. As known in the art, standard Watson-Crick base pairing includes adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C). In addition, for hybridization between two RNA molecules (e.g., dsRNA), hybridization of guanine (G) base pairs with uracil (U) is also known in the art. For example, G / U base pairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anticodon base pairing with mRNA codons. In the context of the present disclosure, guanine (G) of the protein-binding segment (dsRNA duplex) of a guide RNA molecule is considered to be complementary to uracil (U), and vice versa.Therefore, if the protein-binding segment of a guide RNA molecule (the dsRNA duplex) can be G / U base-paired at a particular nucleotide position, that position is not considered non-complementary, but instead is considered complementary.

[0053] Hybridization and washing conditions are well known and are exemplified in: Sambrook, J., Fritsch, EF and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein; and Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001). The conditions of temperature and ionic strength determine the "stringency" of hybridization.

[0054] Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases may occur. Suitable conditions for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, which are variables well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the melting temperature (Tm) value of the hybrid of nucleic acids having those sequences. For hybridization between nucleic acids with short stretches of complementarity (e.g., 35 or less, 30 or less, 25 or less, 22 or less, 20 or less, or 18 or less nucleotides of complementarity), the position of the mismatch becomes important (see Sambrook et al., supra, 11.7-11.8). Typically, the length of a hybridizable nucleic acid is at least about 10 nucleotides. Exemplary minimum lengths of hybridizable nucleic acids are: at least about 15 nucleotides; at least about 20 nucleotides; at least about 22 nucleotides; at least about 25 nucleotides; and at least about 30 nucleotides. Moreover, one of skill in the art will recognize that the temperature and salt concentration of the wash solutions can be adjusted as necessary depending on factors such as the length of the complementary regions and the degree of complementation.

[0055] It is understood in the art that the sequence of a polynucleotide need not be 100% complementary to the sequence of its target nucleic acid to be specifically hybridizable or hybridizable. Furthermore, a polynucleotide can hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., loop or hairpin structures). Polynucleotides can contain at least 55%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence complementarity to a target region within the target nucleic acid sequence to which they are targeted. For example, an antisense nucleic acid in which 18 of 20 nucleotides of an antisense compound are complementary to a target region and thus would specifically hybridize would represent 90 percent complementarity. In this example, the remaining non-complementary nucleotides may be clustered or interspersed with complementary nucleotides and do not need to be adjacent to each other or to complementary nucleotides. The percent complementarity between particular stretches of nucleic acid sequences within a nucleic acid can be routinely determined using the BLAST program (basic local alignment search tool) and the PowerBLAST program known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403- 410; Zhang and Madden, Genome Res., 1997, 7, 649-656), or using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for 35 Unix, Genetics Computer Group, University Research Park, Madison Wis.) using default settings using the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).

[0056] The term "cleavage" or "cleaving" refers to the cleavage of a covalent phosphodiester bond in the ribosylphosphodiester backbone of a polynucleotide. The term "cleavage" or "cleaving" encompasses both single-stranded and double-stranded breaks. Double-stranded breaks can occur as a result of two different single-stranded cleavage events. Cleavage can generate either blunt ends or sticky ends.

[0057] The term "CRISPR-associated protein" or "Cas protein" refers to a wild-type Cas protein, a fragment thereof, or a mutant or variant thereof. The term "Cas mutant" or "Cas variant" refers to a protein or polypeptide derivative of a wild-type Cas protein, such as a protein having one or more point mutations, insertions, deletions, truncations, fusion proteins, or combinations thereof. According to certain embodiments, the "Cas mutant" or "Cas variant" substantially retains the nuclease activity of the Cas protein. According to certain embodiments, the "Cas mutant" or "Cas variant" is mutated such that one or both nuclease domains are inactive. According to certain embodiments, the "Cas mutant" or "Cas variant" has nuclease activity. According to certain embodiments, a "Cas mutant" or "Cas variant" lacks some or all of the nuclease activity of its wild-type counterpart.

[0058] A synthetic guide RNA with "gRNA functionality" is one that has one or more of the functions of a naturally occurring guide RNA, such as association with a Cas protein, or the functions performed by a guide RNA in association with a Cas protein. According to certain embodiments, the functionality includes binding a target polynucleotide. According to certain embodiments, the functionality includes targeting a Cas protein or a gRNA:Cas protein complex to a target polynucleotide. According to certain embodiments, the functionality includes nicking a target polynucleotide. According to certain embodiments, the functionality includes acting in a gRNA:Cas system to cleave a target polynucleotide. According to certain embodiments, the functionality includes association or binding with a Cas protein. According to certain embodiments, the functionality is any other known function of a guide RNA in a CRISPR / Cas system with a Cas protein, including an artificial CRISPR / Cas system with an engineered Cas protein. According to certain embodiments, the functionality is any other function of a naturally occurring guide RNA. A synthetic guide RNA may have more or less gRNA functionality than a naturally occurring guide RNA. According to certain embodiments, a synthetic guide RNA may have greater functionality with respect to one property and less functionality with respect to another property.

[0059] As used herein, the term "segment" of a sequence refers to any portion of a sequence that is less than the complete sequence (e.g., a nucleotide subsequence or an amino acid subsequence). A segment of a polynucleotide can be of any length, e.g., at least 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 300, or 500 nucleotides in length or more. A portion of a guide sequence can be about 50%, 40%, 30%, 20%, 10% of the guide sequence, e.g., one-third of the guide sequence, or less, e.g., 7, 6, 5, 4, 3, or 2 nucleotides in length.

[0060] The term "derived from" in the context of a molecule refers to a molecule that is isolated or created using a parent molecule or information from that parent molecule. For example, Cas9 single mutant nickase and Cas9 double mutant null nuclease are derived from the wild-type Cas9 protein.

[0061] The term "substantially identical" in the context of two or more polynucleotides (or two or more polypeptides) refers to sequences or subsequences that have at least about 60%, at least about 70%, at least about 80%, at least about 90%, about 90-95%, at least about 95%, at least about 98%, at least about 99% or more nucleotide (or amino acid) sequence identity when compared and aligned for maximum correspondence using a sequence comparison algorithm or by visual inspection. Preferably, "substantial identity" between polynucleotides exists over a region of the polynucleotide that is at least about 50 nucleotides in length, at least about 100 nucleotides in length, at least about 200 nucleotides in length, at least about 300 nucleotides in length, at least about 500 nucleotides in length, or the entire length of the polynucleotide. Preferably, "substantial identity" between polypeptides exists over a region of the polypeptide that is at least about 50 amino acid residues in length, at least about 100 amino acid residues in length, or the entire length of the polypeptide.

[0062] As disclosed herein, several ranges of values ​​are provided. It is understood that each intervening value between the upper and lower limits of the range, to one-tenth of the unit of the lower limit, is also specifically contemplated. Each smaller range or intervening value that falls within a stated range is also specifically contemplated. The term "about" generally refers to plus or minus 10% of the indicated numerical value. For example, "about 10%" indicates a range of 9% to 11%, and "about 20" means 18 to 22. Other meanings of "about" may be apparent from the context, such as rounding, so for example, "about 1" may also mean 0.5 to 1.4.

[0063] A DNA sequence that "encodes" a particular RNA is a DNA nucleic acid sequence that is transcribed into RNA. A DNA polynucleotide may encode an RNA that is translated into a protein (mRNA), or a DNA polynucleotide may encode an RNA that is not translated into a protein (e.g., tRNA, rRNA, or guide RNA; also called "non-coding" RNA or "ncRNA"). A "protein coding sequence" or a sequence that encodes a particular protein or polypeptide is a nucleic acid sequence that is transcribed into mRNA (in the case of DNA) and translated into a polypeptide in vitro or in vivo (in the case of mRNA) when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5'-terminus (N-terminus) and a translation termination nonsense codon at the 3'-terminus (C-terminus). Coding sequences can include, but are not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic nucleic acids. A transcription termination sequence is usually located 3' to the coding sequence.

[0064] As used herein, a "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding or non-coding sequence. For purposes of defining the present invention, a promoter sequence is bounded at its 3' end by a transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at a level detectable above background. Within the promoter sequence is the transcription initiation site and protein binding domains responsible for binding RNA polymerase. As described in this disclosure, a variety of promoters can be used to drive vectors, including inducible promoters.

[0065] The promoter can be a constitutively active promoter (i.e., a promoter that is in a constitutively active ("on") state), or it can be an inducible promoter (i.e., a promoter whose state, activity ("on") or inactivity ("off"), is controlled by an external stimulus (e.g., temperature, the presence of a particular compound, or protein).

[0066] In the prior art, when a promoter suitable for the expression of Cas proteins is derived from a virus (and therefore can be referred to as a viral promoter), the promoter used to express Cas9 proteins in bacteria is also preferred. A promoter known to promote the expression of Cas9 in one bacterium can also be used to promote the expression of Cas9 proteins from a different (species) bacterium. In such cases, the promoter is said to be heterologous with respect to the Cas9 protein. The term "prokaryotic promoter" refers to a promoter that is active in bacteria for transcription of sequences. The use of phage promoters is facilitated because bacteria can be further engineered to express phage RNA polymerase. According to certain aspects, the use of phage promoters can be beneficial to drive bacteria to express toxic genes. For example, the use of a phage promoter and its placement upstream of a toxic gene can, for example, allow or force the bacterium to make large and possibly toxic amounts of gene editing nuclease.

[0067] Invasive microorganisms: 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 may be a microorganism that is capable of passing through a cell membrane, 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 delivery of at least one molecule to a target cell preferably does not significantly modify the invasion apparatus.

[0068] As used herein, the term "transkingdom" refers to a delivery system that uses bacteria (or another invasive microorganism) to produce nucleic acids and deliver them intracellularly within a target tissue for processing without host genome integration (i.e., across kingdoms: prokaryotes to eukaryotes, or across phyla: invertebrates to vertebrates).

[0069] Invasive microorganisms include microorganisms that can naturally deliver at least one molecule to a target cell, such as by crossing a cell membrane, e.g., a eukaryotic cell membrane, and entering the cytoplasm, as well as microorganisms that are not naturally invasive and have been modified, e.g., genetically, to be invasive. According to another preferred embodiment, a microorganism that is not naturally invasive can be modified to be invasive by linking the bacterium or BTP to an "invasion factor," also called 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 have generally been described in the art, for example, in U.S. Patent Publication Nos. US 20100189691 A1 and US20100092438 A1, and Xiang, S. et al., Nature Biotechnology 24, 697 - 702 (2006), each of which is incorporated by reference in its entirety for all purposes.

[0070] According to a preferred embodiment, as taught in the examples of this application, the invasive microorganism is Escherichia coli. However, it is contemplated that additional microorganisms can potentially be adapted to function as transkingdom delivery vehicles for the delivery of gene editing cargo. These non-toxic invasive bacteria and BTPs can be invasive or modified to be invasive and invade host cells via various mechanisms. In contrast to the uptake of bacteria or BTPs by professional phagocytes, which usually results in the destruction of 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 are Yersinia, Rickettsia, Legionella, Brucella, Mycobacterium, Helicobacter, Coxiella, Chlamydia, Neisseria, Burkolderia, 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 E. coli, Lactobacillus, Lactococcus or Bifidobacteriae, including probiotics, by the transfer of invasion-related genes (P. Courvalin, S. Goussard, C. Grillot-Courvalin, CR Acad. Sci. Paris 318, 1207 (1995)). Factors to consider or address when evaluating additional bacterial species as candidates for use as transkingdom delivery vehicles include: the candidate's pathogenicity or lack thereof, the tropism of the candidate bacterium for target cells or alternatively the extent to which the bacterium can be engineered to deliver gene editing cargo to the interior of target cells, and any synergistic value the candidate bacterium may provide by triggering the host's innate immunity.

[0071] A colony is defined as a visible cluster of microorganisms that are all derived from a single mother cell, and thus constitute a clone of bacteria that are all genetically similar.

[0072] promoter: Among gene expression regulatory elements, promoters play a central role. Along the promoter molecule, the transcription machinery is assembled and transcription is initiated. This initial step is often rate-limiting compared to the subsequent steps of protein production. Transcription initiation at a promoter can be regulated in several ways. For example, a promoter can be induced by the presence of a particular compound or external stimulus, express a gene during a particular developmental stage, or express a gene constitutively. Thus, transcription of a transgene can be regulated by operably linking the coding sequence to a promoter with different regulatory properties. Thus, regulatory elements such as promoters play a vital role in enhancing the value of transgenic organisms.

[0073] As used herein, the term "promoter" refers to a polynucleotide molecule that is involved in the recognition and binding of RNA polymerase and other proteins, such as associated sigma factors and activator proteins, to initiate transcription of an operably linked gene. A promoter can be isolated from the 5' untranslated region (5'UTR) of a genomic copy of a gene. Alternatively, a promoter molecule can be artificially and synthetically generated or can comprise a modified DNA sequence. A promoter can be used as a regulatory element to regulate the expression of an operably linked transcribable polynucleotide molecule. The promoter itself can include subelements, such as cis elements or enhancer domains, that affect the transcription of an operably linked gene.

[0074] In prokaryotes, promoters consist of two short sequences located at positions -10 and -35 upstream from the transcription start site. Sigma factors not only help to strengthen RNAP binding to the promoter, but also help target which genes RNAP will transcribe. The Sigma element at -10 is called the Pribnow box or -10 element and is usually composed of the six nucleotides TATAAT. The Pribnow box is absolutely essential for initiating transcription in prokaryotes. The other sequence at -35 (the -35 element) is usually composed of the six nucleotides TTGACA. Its presence allows for very high transcription rates. Both of the above consensus sequences are conserved on average, but are not found intact in most promoters. On average, only three out of six base pairs of each consensus sequence are found in any given promoter. Note that the above promoter sequences are only recognized by the Sigma-70 protein, which interacts with prokaryotic RNA polymerase. Complexes of prokaryotic RNA polymerase with other Sigma factors recognize entirely different core promoter sequences.

[0075] Many regulatory elements act in cis ("cis elements") and are believed to affect DNA topology, generating local conformations that selectively allow or restrict access of RNA polymerase to the DNA template or promote selective opening of the double helix at the transcription start site. Cis elements reside within the 5'UTR associated with a particular coding sequence and are often found within promoters and promoter regulatory sequences (inducible elements). Cis elements can be identified using known cis elements as target sequences or target motifs using the BLAST program. Examples of cis-acting elements in the 5'UTR associated with a polynucleotide coding sequence include, but are not limited to, promoters and enhancers.

[0076] In prokaryotes, translation of mRNA begins with UTG, GTG, or rarely UUG, usually preceded by a sequence characteristic of a ribosome binding site (RBS; Shine and Dalgarno, PNAS 71:1342-1346, 1974). RBSs are AG-rich and are usually found 6-12 bp before the start codon. RBSs are thought to be required for efficient mRNA translation in bacteria.

[0077] At least two types of information are useful in predicting promoter regions within a genomic DNA sequence. First, promoters can be identified based on their sequence "content," such as transcription factor binding sites and various known promoter motifs (Stormo, Genome Research 10: 394-397, 2000). Such signals may be identified by computer programs that identify promoter-associated sites, such as TATA boxes and transcription factor (TF) binding sites. Second, promoters can be identified based on their "location," i.e., their proximity to known or suspected coding sequences (Stormo). Prokaryotic promoters are typically found within a region of DNA that spans approximately 1-500 base pairs in the 5' direction from the transcription or translation start codon of the coding sequence. Thus, a promoter region can be identified by identifying the translation start codon or transcription start site of the coding sequence and then moving in the 5' direction beyond the start codon to identify the promoter region.

[0078] Promoter sequences can be analyzed for the presence of common promoter sequence characteristics, such as TATA boxes and other known transcription factor binding site motifs. These motifs are not always found in all known promoters, and are not required for all promoters to function, but when present, they indicate that a segment of DNA is a promoter sequence.

[0079] The activity or strength of a promoter can be measured in terms of the amount of mRNA, tRNA, dsRNA, miRNA, rRNA, or protein that accumulates specifically during a particular period of growth of a cell containing a transgene. Enhanced levels of mRNA production may be required to produce commercially important proteins or proteins for catalyzing reactions to produce commercially important compounds. The coding sequences of commercially important proteins can be identified by those skilled in the art and used with the promoters of the present invention to result in the production of mRNA and protein levels. The activity or strength of a promoter can also be measured in terms of enhanced cell growth when the cells are grown under selective pressure, for example, after being transformed with a gene that allows for the detoxification of a compound under the control of the promoter.

[0080] A hybrid promoter is defined as a promoter created by engineering a bacterium or expression cassette on a plasmid with the -35 region of one promoter and the -10 region of another promoter.

[0081] Selection marker: As used herein, the term "marker" refers to any transcribable polynucleotide molecule whose expression or lack thereof can be screened or scored in some manner. Marker genes for use in the practice of the present invention include, but are not limited to, transcribable polynucleotide molecules encoding β-glucuronidase (GUS, described in U.S. Pat. No. 5,599,670, which is incorporated herein by reference), green fluorescent protein and variants thereof (GFP, RFP, etc., described in U.S. Pat. Nos. 5,491,084 and 6,146,826, all of which are incorporated herein by reference), or proteins that confer antibiotic resistance.

[0082] Useful antibiotic resistance markers are known in the art, including those that encode proteins that confer resistance to kanamycin (nptII), hygromycin B (aph IV), streptomycin or spectinomycin (aad, spec / strep), and gentamicin (aac3 and aacC4). An advantageous positive selection marker is hisD (histidinol dehydrogenase). The hisD gene of Salmonella typhimurium encodes histidinol dehydrogenase, which catalyzes the two-step NAD'-dependent oxidation of L-histidine from L-histidine. On medium lacking histidine and containing histidinol, only cells expressing the hisD product survive.

[0083] The term "selectable marker" also includes genes that encode secretable markers whose secretion can be detected as a means of identifying or selecting for transformed cells. Examples include markers that encode secretable enzymes that can be detected catalytically. Other possible selectable marker genes will be apparent to those of skill in the art.

[0084] According to certain embodiments, the use of selectable markers can be hisD, green fluorescent protein (GFP), neomycin phosphotransferase II (nptII), luciferase (LUX), or antibiotic resistance coding sequences (aadA). According to certain embodiments, the selectable marker is a kanamycin, streptomycin, and / or spectinomycin resistance marker. Examples of coding sequences that provide resistance to antibiotics and herbicides can be found, for example, in U.S. Patent Application Publication Nos. 20080305952 and 20080280361, which are incorporated herein by reference.

[0085] Cell transformation: The term "transformation" refers to the introduction of a nucleic acid into a recipient host. The term "host" refers to a prokaryotic cell, particularly a bacterial cell. As used herein, the term "transformed" refers to a cell or organism into which an exogenous polynucleotide molecule, such as a construct, has been introduced. The introduced polynucleotide molecule may be integrated into the genomic DNA of the recipient cell or organism such that the introduced polynucleotide molecule is inherited by subsequent progeny or may remain in the cytoplasm as a self-replicating unit. A "transgenic" or "transformed" cell or organism also includes the progeny of the cell or organism. The term "transgenic" refers to a cell or other organism that contains one or more heterologous polynucleic acid molecules.

[0086] There are many methods for introducing heterologous polynucleic acid molecules into cells. The methods generally include the steps of selecting a suitable host cell, transforming the host cell with a recombinant vector, and obtaining the transformed host cell. Suitable methods for introducing heterologous polynucleic acid molecules into prokaryotes include, inter alia, freeze-thaw (heat shock), triparental mating, and electroporation. These methods are known to those skilled in the art of prokaryotic transformation.

[0087] kit: Further provided are kits for carrying out the methods of the invention. By "kit" is meant any manufacture (e.g., package or container) that includes at least one reagent, e.g., the gene-edited bacteria of the invention. The kit can be promoted, distributed, or sold as a unit for carrying out the methods of the invention. Additionally, the kit may include a package insert that describes the kit and how to use it. Any or all of the kit reagents may be provided in a container that protects them from the external environment, such as a sealed container or pouch.

[0088] The term "administration" and variations thereof with respect to a compound of the invention (e.g., "administration" of a compound) refers to the introduction of the compound into the system of a subject in need of treatment. When a compound of the invention is provided in combination with one or more other active agents (e.g., an AIV vaccine, etc.), "administration" and variations thereof are understood to include simultaneous and sequential introduction of the compound and the other agent, respectively.

[0089] As used herein, the term "composition" is intended to include a product containing the specified ingredients in the specified amounts, as well as any product that results directly or indirectly from combining the specified ingredients in the specified amounts.

[0090] The term "therapeutically effective amount" as used herein refers to an amount of an active compound or agent that induces a biological or medical response in a tissue, system, animal or human that is desired by a researcher, veterinarian, physician or other clinician. With respect to viral infection, an effective amount includes an amount sufficient to prevent the onset of disease or reduce the severity of disease as evidenced by clinical disease, clinical symptoms, viral titer or viral shedding from a subject, or from a subject's ability to prevent or reduce infection between animals. According to some embodiments, an effective amount is an amount sufficient to delay the onset of clinical disease and / or symptoms or to prevent disease. According to 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.

[0091] Beneficial or desirable clinical outcomes include, but are not limited to, any one or more of the following: alleviation of one or more symptoms, reduction in the extent of viral infection, a stable (i.e., not worsening) state of viral infection, prevention or slowing of spread (e.g., shedding of viral infection), prevention, slowing or deceleration 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.

[0092] A "pharmaceutical acceptable" ingredient is one that is suitable for use in humans and / or animals without undue adverse side effects (such as toxicity, irritation, and allergic reactions) commensurate with a reasonable benefit / risk ratio.

[0093] 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 a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit / risk ratio.

[0094] As used throughout the application, the terms "a" and "an" are used in the sense of meaning "at least one," "at least a 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. The term "and / or" as used herein includes the meanings of "and," "or," and "all or any other combination of the elements connected by said term."

[0095] The above advantages, and those made apparent from the preceding description, are efficiently attained. Since certain modifications can be made to the above structure without departing from the scope of the invention, it is intended that all matter contained in the above description or shown in the accompanying drawings be interpreted as illustrative and not in a limiting sense.

[0096] All references cited in this application are incorporated herein by reference in their entirety to the extent not inconsistent herewith.

[0097] The advantages set forth above, and those made apparent from the foregoing description, are efficiently attained, and certain modifications can be made to the structure described above without departing from the scope of the invention, It is intended that all matter contained in the foregoing description or shown in the accompanying drawings be interpreted as illustrative and not in a limiting sense. It is also to be understood that the following claims are intended to cover all of the general and specific features of the invention described herein, and all statements of the scope of the invention that may be found therebetween as a matter of language. The invention now being described.

[0098] The following examples are given for the purpose of illustrating various embodiments of the present invention and are not meant to limit the present invention in any manner. The following examples, together with the methods described herein, are representative of preferred embodiments, are illustrative, and are not intended as limiting the scope of the present invention. Modifications therein and other uses that fall within the spirit of the invention as defined by the claims will occur to those skilled in the art. EXAMPLES

[0099] Example 1 - Generation of invasive bacteria containing pSiC RISP plasmids capable of directing gene editing activities: The pSiCRISP_GFP plasmid, which represents a CRISPR complex-directed plasmid expressing the crRNA for green fluorescent protein (GFP), was synthesized and transformed into Escherichia coli (FEC19). The EC19 bacteria were further engineered to invade eukaryotic cells via co-transformation with pSiVEC, which contains the inv and hlyA genes for invasin-mediated receptor-mediated endocytosis and LLO-mediated endosomal release, respectively. The resulting FEC19 colonies containing pSiCRISP-GFP and pSiVEC were plated on brain heart infusion (BHI) agar medium containing the appropriate antibiotic for selection. The resulting FEC19 bacterial colonies (identified as A, B, C, D, E, F, and G) were screened by PCR to demonstrate successful transformation of the pSiCRISP and pSiVEC plasmids in FEC19 bacteria as a way to validate the construction of aspects of the present invention. The pSiCRISP_GFP sequences specific for the Cas9 gene and NLS, and the pSiVEC sequences specific for the inv gene were amplified using specific primers in a PCR assay. Primers amplifying a 424 bp product (Figure 3A) were used to confirm the presence of the NLS (screened in colonies A, D, E, F, and G), primers amplifying a 727 bp product (Figure 3B) were used to confirm the presence of the Cas9 gene (screened in colonies A, B, C, D, E, and F), and primers amplifying a 201 bp product (Figure 3C) were used to confirm the presence of the inv gene (screened in colonies A, D, E, and F). In the figure, a 1 kb ladder is used as a reference. The NLS sequences of Cas9 and FEC19 + pSiCRISP_GFP (FEC19 transformed with pSiCRISP_GFP) were both present in colonies D, E, and F. The Inv sequence was present in all colonies tested. For each colony that was positive by PCR for both inv and Cas9 + NLS (colonies D, E, and F), a single positive clone was sequence verified to confirm the presence of pSiCRISP_GFP and pSiVEC and was expanded.This experiment demonstrates the generation of E. coli (FEC19) containing a gene editing plasmid (pSiCRISP) and additionally containing a plasmid (pSiVEC) containing an invasion factor for target cell invasion. Bacterial stocks D, E, and F were generated in BHI medium containing the appropriate antibiotics for selection and grown until the optical density measured at wavelength 600 nm (OD600) was equal to 1.0. These FEC19 + pSiCRISP_GFP + pSiVEC stocks (D, E, F) were frozen at -80 °C in 20% glycerol. A single frozen aliquot from each stock was thawed for plate enumeration. Briefly, 1 mL aliquots were centrifuged at 5000 x g for 5 min and the cells were resuspended in 1 mL of BHI. The resulting bacterial suspensions were serially diluted and plated in triplicate on BHI agar containing antibiotics. The number of colonies at each dilution was averaged to calculate overall colony forming units (CFU) / mL, representing the viable concentration of FEC19 + pSiCRISP_GFP + pSiVEC stock. This system allowed the quantified live inoculum to be used directly in all future assays.

[0100] Example 2: CRISPR-mediated gene editing with FEC19 + pSiCRISP + pSiVEC in mammalian cell lines: Human alveolar basal epithelial (A549) cells stably expressing GFP were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM GlutaMAX, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37 °C under incubation with 5% CO2. Invasive (pSiVEC-transformed) FEC19 bacteria containing the pSiCRISP_GFP plasmid can invade A549 cells via the RME and deliver the CRISPR-mediated gene editing cargo required to knock out the gene of interest, GFP. The experiment includes a positive control treatment (FEC19 + pSiVEC_shGFP) in which the pSiVEC plasmid expresses a short hairpin RNA (shRNA) targeting GFP. This positive control treatment is included because knockdown of GFP using shRNA targeting GFP in A549 cells is expected and has been previously demonstrated. This invasion assay involves the following steps: First, seed A549 cells into black 24-well plates at a fixed concentration. On the day of bacterial invasion, thaw the following four FEC19 stocks: 1) FEC19 + pSiCRISP_GFP + pSiVEC, 2) FEC19 + pSiCRISP_GFP (non-invasive), 3) FEC19 + pSiCRISP_scramble (expressing scrambled / nonsense crRNA) + pSiVEC, and 4) FEC19 + pSiVEC_shGFP (expressing shRNA for GFP). Dissolve the FEC19 bacterial stocks at 1x10 in DMEM. 7 CFU / mL ~ 1X10 5 Serially dilute 1:10 to CFU / mL. Incubate A549 cells with 1 mL of each FEC19 treatment for 2 hours, followed by rinsing with DMEM to remove unbound FEC19 bacteria. Quantify GFP expression over a 72 hour time course using a Nexcelom Celigo, with a T0 reading just prior to invasion. Figure 4 shows the relative increase in GFP expression in A549 cells with 1x10 cells infected with one of the four FEC19 treatments at 0, 24, 48, and 72 hours post-treatment compared to untreated A549 cells. 7 The percentage of A549 cells expressing GFP after incubation with CFU / mL is shown.

Claims

1. A composition for delivery of a gene editing system to a eukaryotic cell, comprising Escherichia coli engineered to express an entry factor encoded by the inv or hlyA gene, a gene editing nuclease, and a guide RNA to facilitate entry of E. coli into a eukaryotic cell.

2. The composition for delivery of a gene editing system to a eukaryotic cell of claim 1, wherein the gene editing nuclease is a CRISPR-associated (Cas) enzyme, an effector nuclease, a Tal-effector nuclease (TALEN), a transcription activator-like effector (TALE), an ARC nuclease, a zinc finger nuclease, a nuclease based on the Argonaute system, a TtAgo nuclease system, or a combination thereof.

3. The composition for delivery of a gene editing system to a eukaryotic cell according to claim 2, wherein the gene editing nuclease is a CRISPR-associated (Cas) enzyme, and the CRISPR-associated (Cas) enzyme is encoded by the Cas9 gene.

4. The composition for delivery of a gene editing system to a eukaryotic cell of claim 1, wherein the guide RNA is a single guide RNA (sgRNA) comprising a CRISPR direct repeat region, a spacer sequence, a tracr sequence, a crRNA, or any part or combination thereof.

5. The composition for delivery of a gene editing system to a eukaryotic cell of claim 1, wherein the invasion factor, gene editing nuclease or guide RNA is expressed from the chromosome of E. coli.

6. The composition for delivery of a gene editing system to a eukaryotic cell of claim 1, wherein two or more of the invasion factors, gene editing nucleases or guide RNAs are expressed from the chromosome of E. coli.

7. The composition for delivery of a gene editing system to a eukaryotic cell according to claim 1, wherein the gene editing nuclease has a nuclear localization signal (NLS).

8. The composition for delivery of a gene editing system to a eukaryotic cell of claim 1, wherein the E. coli comprises a plasmid encoding a gene editing nuclease and a guide RNA, the transcription of which is under the control of a prokaryotic promoter.

9. 9. The composition for delivery of a gene editing system to a eukaryotic cell of claim 8, wherein the plasmid encodes a nuclear localization signal.

10. 9. The composition for delivery of a gene editing system to a eukaryotic cell of claim 8, wherein the plasmid encodes a gene editing nuclease with a nuclear localization signal.

11. 9. The composition for delivery of a gene editing system to a eukaryotic cell of claim 8, wherein the prokaryotic promoter is a hybrid or synthetic prokaryotic promoter.

12. 2. The composition for delivery of a gene editing system to a eukaryotic cell of claim 1, wherein the E. coli comprises a plasmid encoding a gene editing nuclease and a guide RNA, the plasmid comprising a eukaryotic promoter that controls transcription of the gene editing nuclease and the guide RNA.

13. The composition for delivery of a gene editing system to a eukaryotic cell of claim 12, wherein the gene editing nuclease is a CRISPR-associated (Cas) enzyme, and the CRISPR-associated (Cas) enzyme has a nuclear localization signal (NLS).

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