HK1160488A

HK1160488A - E. coli mediated gene silencing of beta-catenin

Info

Publication number: HK1160488A
Application number: HK12100927.4A
Authority: HK
Inventors: 约翰内斯．海因里希．弗吕奥夫; 莫雷斯沃尔．巴努达斯．瓦泽; 弗洛伊德．斯蒂芬．拉鲁; 杰西卡．安．塞克斯顿; 吉勒．雷默．博尔达克
Original assignee: 玛瑞纳生物技术有限公司
Priority date: 2008-11-14
Filing date: 2009-11-13
Publication date: 2012-08-17

Description

Escherichia coli mediated gene interference of beta-catenin

Cross Reference to Related Applications

This application claims priority from U.S. patent application No.61/114,610, filed on 14/11/2008, the contents of which are incorporated herein by reference in their entirety.

Background

Gene silencing by RNAi (RNA interference) using small interfering RNAs (sirnas) has become a powerful tool in molecular biology and has potential for therapeutic gene silencing. Short hairpin rnas (shrnas) transcribed from small DNA plasmids in target cells have also been shown to mediate stable gene silencing and achieve gene knockdown comparable to levels obtained by transfection of chemically synthesized sirnas (t.r. brummelkamp, r. bernards, r. agami, Science 296, 550(2002), p.j.paddison, a.a.caudiy, g.j.hannon, PNAS 99, 1443 (2002)).

The possible applications of RNAi for therapeutic purposes are broad and include silencing and knocking down disease genes such as oncogenes or viral genes. One major obstacle to the therapeutic use of RNAi is the delivery of siRNA into target cells (Zamore PD, Aronin N.Nature Medicine 9, (3): 266-8 (2003)). Indeed, delivery has been identified as the major obstacle to current RNAi (Phillip Sharp, citdby Nature news feature, Vol 425, 2003, 10-12).

Thus, there is a need for new methods of administering interfering RNA to mammals safely and predictably.

Summary of The Invention

The present invention provides at least one invasive bacterium or at least one invasive Bacterial Therapeutic Particle (BTP) comprising a prokaryotic vector comprising one or more DNA molecules encoding one or more sirnas, a modified P_lacUV5A promoter, at least one Inv locus, and at least one HlyA gene, wherein the siRNA interferes with mRNA of a target gene target, and the RNase III activity of the invasive bacterium is reduced compared to a wild-type bacterium. Preferably, the invasive bacterium is an invasive escherichia coli (e. Preferably, the siRNA interferes with the mRNA of beta-catenin. The invention also provides modified P comprising one or more DNA molecules encoding one or more siRNAs_lacUV5At least one prokaryotic vector of a promoter, at least one Inv locus, and at least one HlyA gene, wherein the siRNA interferes with the mRNA of the target gene target. Preferably, the siRNA interferes with the mRNA of beta-catenin.

The invention also provides methods of using the various invasive bacteria, BTPs and vectors provided herein. For example, the invention provides methods of delivering one or more siRNAs to a mammalian cell. The method comprises introducing at least one invasive bacterium or at least one invasive Bacterial Therapeutic Particle (BTP) comprising a prokaryotic vector comprising one or more DNA molecules encoding one or more siRNAs, a modified P_lacUV5A promoter, at least one Inv locus, and at least one HlyA gene, wherein the siRNA interferes with mRNA of a target gene target, and the RNase III activity of the invasive bacterium is reduced compared to a wild-type bacterium. Preferably, the invasive bacterium is an invasive escherichia coli bacterium.

The invention also relates toMethods of modulating gene expression in mammalian cells are provided. The method comprises introducing at least one invasive bacterium or at least one invasive Bacterial Therapeutic Particle (BTP) comprising a prokaryotic vector comprising one or more DNAs encoding one or more siRNAs, a modified P_lacUV5A promoter, at least one Inv locus and at least one HlyA gene, wherein the siRNA interferes with mRNA of a target gene target, and the invasive bacterium has reduced RNase III activity compared to a wild-type bacterium, wherein the expressed siRNA interferes with at least one mRNA of a target gene and thereby modulates gene expression. Preferably, the invasive bacterium is an invasive escherichia coli bacterium. Preferably, the siRNA interferes with the mRNA of beta-catenin.

The invention also provides methods of treating or preventing a disease or condition in a mammal. The method comprises modulating the expression of at least one gene in a mammalian cell known to cause a disease or disorder, e.g., known to increase proliferation, growth or dysplasia, by introducing into the cell at least one invasive bacterium or at least one invasive Bacterium Therapeutic Particle (BTP) comprising a prokaryotic vector, wherein the vector comprises one or more DNA molecules encoding one or more sirnas, a modified P_lacUV5A promoter, at least one Inv locus and at least one HlyA gene, wherein the siRNA interferes with mRNA of a target gene target, and the invasive bacterium has reduced RNase III activity compared to a wild-type bacterium, wherein the expressed siRNA interferes with mRNA of a gene known to cause a disease or disorder. Preferably, the invasive bacterium is an invasive escherichia coli bacterium. Preferably, the siRNA interferes with the mRNA of beta-catenin.

The expressed siRNA is capable of directing the interaction of the multiple enzyme complex RNA-induced silencing complex of the cell with the mRNA (e.g., beta-catenin) of one or more target genes. Preferably, the expression of β -catenin is reduced compared to the expression of wild-type β -catenin or compared to the expression of β -catenin prior to administration or treatment with invasive bacteria or BTP comprising one or more DNA molecules encoding one or more sirnas. The reduced expression of β -catenin may be reduced expression of β -catenin mRNA or reduced expression of β -catenin. Preferably, the expression of β -catenin is reduced by at least 50% compared to the expression of wild-type β -catenin (when compared to normal healthy cells) or compared to the expression of β -catenin prior to administration or treatment with invasive bacteria or BTP comprising one or more DNA molecules encoding one or more sirnas; more preferably, the expression of β -catenin is reduced by at least 75% compared to the expression of wild-type β -catenin (when compared to normal healthy cells) or compared to the expression of β -catenin prior to administration or treatment with invasive bacteria or BTP comprising one or more DNA molecules encoding one or more sirnas; most preferably, the expression of β -catenin is reduced by at least 90% compared to the expression of wild-type β -catenin (when compared to normal healthy cells) or compared to the expression of β -catenin prior to administration or treatment with invasive bacteria or BTP comprising one or more DNA molecules encoding one or more sirnas.

Preferably, the disease or disorder may be, but is not limited to, a disease or disorder associated with overexpression of β -catenin. That is, the disease or condition is characterized by increased expression of β -catenin (DNA, RNA, or protein) in a cell or mammal in need of such treatment when compared to a normal (non-diseased) or wild type cell or mammal. Preferably, the disease or disorder to be treated is selected from the group consisting of: colon cancer, rectal cancer, colorectal cancer, Crohn's disease, ulcerative colitis, Familial Adenomatous Polyposis (FAP), Gardner syndrome, hepatocellular carcinoma (HCC), basal cell carcinoma, pilomas, medulloblastomas, and ovarian cancer.

The invention also provides a composition comprising at least one invasive bacterium or BTP and a pharmaceutically acceptable carrier.

The invasive bacterium or BTP of the invention can be an attenuated, nonpathogenic or avirulent bacterium.

The mammalian cell may be ex vivo, in vivo or in vitro. The mammalian cells can be, but are not limited to, human, bovine, ovine, porcine, feline, bison, canine, caprine, equine, donkey, deer, avian, chicken, and primate cells. Preferably, the mammalian cell is a human cell. In some preferred embodiments, the mammalian cell can be, but is not limited to, a colonic epithelial cell, a rectal epithelial cell, an intestinal epithelial cell, a liver cell, a skin epithelial cell, a hair cell, a nerve cell, and an ovarian cell.

Can use about 10³-10¹¹(or any integer within the range) of a viable invasive bacterium or BTP to infect the mammalian cell. Preferably, about 10 may be used⁵-10⁹(or any integer within the range) of a viable invasive bacterium or BTP to infect the mammalian cell. May be used in the range of about 0.1-10⁶(or any integer within the range) infects the mammal. Preferably, a useful range is about 10²-10⁴(or any integer within the range) infects the mammal.

The mammal may be, but is not limited to, a human, a cow, a sheep, a pig, a cat, a bison, a dog, a goat, a horse, a donkey, a deer, a bird, a chicken, and a primate. Preferably, the mammal is a human.

The invasive bacterium has a deletion of the gene encoding RNaseIII. Preferably, the invasive bacterium has a deletion in the rnc gene encoding RNaseIII. Preferably, the RNase III activity of the invasive bacterium is reduced by at least 90% compared to the wild-type bacterium; more preferably, the RNase III activity of the invasive bacterium is reduced by at least 95% compared to the wild-type bacterium; most preferably, the RNase III activity of the invasive bacterium is reduced by at least 99% compared to the wild type bacterium. Preferably, the invasive bacterium is an invasive escherichia coli bacterium.

The one or more DNA molecules may be transcribed into one or more shRNA within the invasive bacterium. Preferably, the one or more shRNA comprise a 3' overhang or blunt end. Preferably, the one or more shrnas do not comprise or include a 5' overhang (with blunt ends). Preferably, the one or more shRNA comprises a 3' overhang of 2-5 base pairs; more preferably, the one or more shRNA comprises a 3' overhang of no more than 2 base pairs (overhang of one or two base pairs); most preferably, the one or more shrnas do not comprise or include a 3' overhang (with blunt ends). The one or more shrnas are processed into one or more sirnas. Preferably, the one or more shrnas are processed into one or more sirnas within the mammalian cell.

Prokaryotic vectors comprising one or more DNAs encoding the one or more sirnas may comprise one or more promoter sequences, enhancer sequences, terminator sequences, invasion factor sequences, or lytic regulatory sequences. The promoter may be a prokaryotic promoter. Preferably, the prokaryotic promoter is the T7 promoter, P_gapAPromoter, P_araBADPromoter, P_tacPromoter, P_lacUV5A promoter or a recA promoter. Preferably, the promoter is a prokaryotic promoter. Preferably, the prokaryotic promoter is a modified P_lacUV5A promoter. The modified P_lacUV5The promoter may comprise SEQ ID NO: 573. Preferably, the modified P_lacUV5The promoter may comprise an UP element. The UP element may comprise SEQ ID NO: 573 nucleotides 7-26. Preferably, the prokaryotic vector further comprises at least one terminator sequence. Preferably, the terminator sequence comprises a series of consecutive thymidine base pairs. More preferably, the terminator sequence may comprise at least 5 contiguous thymidine base pairs. The terminator sequence preferably comprises less than 20 contiguous thymidine base pairs. The prokaryotic vector may further comprise a second terminator sequence. Preferably, the second terminator sequence may be an rrnC terminator sequence. Preferably, the rrnC terminator sequence may comprise SEQ ID NO: 30-31 or SEQ ID NO: 574. Preferably, the two terminator sequences are adjacent (which are contiguous sequences) in the prokaryotic vector. More preferably, the two terminator sequencesThe columns are separate. Preferably, the prokaryotic vector comprises the validated sequence of pMBV 43. Preferably, the validated pMBV43 sequence is SEQ id no: 564.

unless defined otherwise, 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. In this specification, the singular forms may also include the plural forms unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Drawings

FIG. 1 is a graph showing a comparison of three doses of CEQ200 and CEQ221 in COS7 cells.

FIG. 2 is a schematic diagram showing the structure of hairpin RNA with functional annotation of RNase III substrate.

FIG. 3 is a schematic diagram showing the cleavage of hairpin precursors by bacterial type I RNase III.

Fig. 4 is a schematic view showing the second step of ripening (the first Dicer cleavage step).

FIG. 5 is a schematic diagram showing a second Dicer cleavage step and maturation into active siRNA.

FIG. 6, part A is a schematic showing that CEQ505 is capable of silencing mammalian β -catenin in a dose-dependent manner by 90% in Cos-7 cells. Part B of FIG. 6 is a schematic showing that CEQ221pNJSzc lamin (an equivalent strain targeting the lamin gene) is able to silence mammalian lamin up to 65% in a dose-dependent manner in SW480 cells

FIG. 7 is a schematic diagram showing H3-shRNA having strand wobble (strand wobbble).

FIG. 8, part A, is a schematic diagram showing the invasive potential of an opa-expressing E.coli strain at MOI 1. FIG. 8, part B, is a diagram showing the invasive potential of an opa-expressing E.coli strain at MOI 10

FIG. 9, part A, is a schematic showing silencing of β -catenin mRNA in human SW480 cells using CEQ 508. Figure 9, part B, is a schematic showing silencing of β -catenin using CEQ508 in human SW480 cells.

FIG. 10 is a photograph of an immunoblot showing silencing of beta-catenin using CEQ508 in human SW480 cells.

FIG. 11 is a schematic diagram showing silencing of β -catenin mRNA in COS-7 cells using CEQ509 BTP.

Detailed Description

The present invention relates to compositions and methods for the delivery of small interfering RNA (siRNA) to eukaryotic cells using non-pathogenic or therapeutic strains of bacteria or Bacterial Therapeutic Particles (BTP). The bacterium or BTP delivers DNA encoding siRNA or siRNA itself to exert RNA interference (RNAi) effects by invasion into eukaryotic host cells. Generally, to trigger RNA interference within a target cell, siRNA needs to be introduced into the cell. The siRNA is introduced directly or by transfection into the target cell or may be transcribed into hairpin-structured dsrna (shrna) within the target cell by a specific plasmid with an RNA polymerase III compatible promoter (e.g. U6, H1) (p.j.paddison, a.a.caudiy, g.j.hannon, PNAS 99, 1443(2002), t.r.brummelkamp, r.bernards, r.agami, Science 296, 550 (2002)).

The interfering RNA of the invention modulates gene expression in a eukaryotic cell, which silences or knockdown a gene of interest (e.g., reduces gene activity) within the target cell. The interfering RNA targets the multienzyme complex RISC (RNA-induced silencing complex) to which the cell belongs to the mRNA of the gene to be silenced. The interaction of RISC and mRNA causes degradation or sequestration of the mRNA (sequestration). This results in efficient post-transcriptional silencing of the target gene. This approach is known as bacteria-mediated gene silencing (BMGS).

In the case of BMGS by delivery of DNA plasmids expressing siRNA, upon release of eukaryotic transcriptional plasmids, shRNA or siRNA is produced within the target cell and triggers a highly specific process of mRNA degradation that leads to silencing of the target gene. In addition, one or more cell-specific eukaryotic promoters that restrict expression of the siRNA or shRNA to a particular target cell or tissue for a particular metabolic state may be used. In one embodiment of the method, the cell-specific promoter is albumin and the target cell or tissue is liver. In another embodiment of the method, the cell-specific promoter is keratin and the target cell or tissue is skin.

The non-toxic bacteria and BTP of the present invention have invasive properties (or are modified to have invasive properties) and can enter mammalian host cells by a variety of mechanisms. Invading bacteria or BTP strains have the ability to invade non-phagocytic host cells, in contrast to the uptake of bacteria or BTP by specialized phagocytic cells, which often results in destruction of the bacteria or BTP in specialized lysosomes. Examples of such bacteria or BTPs occurring in nature are intracellular pathogens such as Yersinia (Yersinia), Rickettsia (Rickettsia), Legionella (Legionella), Brucella (Brucella), Mycobacterium (Mycobacterium), Helicobacter (Helicobacter), coxella (Coxiella), Chlamydia (Chlamydia), Neisseria (Neisseria), Burkolderia (Burkolderia), Bordetella (Bordetella), Brucella (Borrelia), Listeria (Listeria), Shigella (Shigella), Salmonella (Salmonella), Staphylococcus (Staphylococcus), Streptococcus (Streptococcus), porphyria (phyromonas), Treponema (Treponema) and Vibrio (Vibrio), but this property can also be transferred to other bacteria or BTPs, such as escherichia coli (escherichia coli)Lactobacillus (Lactobacillus), Lactococcus (Lactococcus) or bifidobacterium (bifidobacterium) (p. Courvalin, s. goussard, c.grilot-Courvalin, c.r.acad.sci.paris 318, 1207 (1995)). In another embodiment of the invention, the bacteria or BTP used to deliver interfering RNA to the host Cell include Shigella flexneri (D.R. Sizemore, A.A.Branstrom, J.C.Sadoff, Science 270, 299(1995)), invasive Escherichia coli (P.Courvalin, S.Goussard, C.Grillot-Courvalin, C.R.Acad.Sci.Paris 318, 1207(1995), C.Grillot-Courvalin, S.Goussard, F.Huetz, D.M.Ojcius, P.Courvalin, Nat technol 16, 1998)), Enterocolitica coli (Yersinia enterocolitica) (A.Hol-Maririi A, A.Tibor, P.Lestresen P.862, Cell K.K.E.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K. Ser. No.3, Cell K.K.K.K.K.K.K.K.K.K.K. Ser. No.3, Cell K.K.K.K.K.K.K. Ser. No.3, Cell K.K.K.K.K.K. Ser. No.3, Cell K.K.K. 3, Cell K.K. K. 3, Cell K. 3, Cell K. 3, Cell K. of Escherichia coliG.geginat, m.j.loessner, i.gentschev, w.goebel, Gene Therapy10, 2036 (2003)). Any invasive bacterium or BTP can be used to transfer DNA into eukaryotic cells (s.weiss, t.chakraborty, Curr Opinion Biotechnol 12, 467 (2001)).

BMGS was carried out using the naturally invasive pathogen Salmonella typhimurium. In one aspect of this embodiment, the Salmonella typhimurium strains include SL7207 and VNP20009(S.K. Hoiseth, B.A.D. Stocker, Nature 291, 238 (1981); Pawelek JM, Low KB, Bermudes D.cancer Res.57 (20): 4537-44 (Oct.151997)). In another embodiment of the present invention, BMGS is performed using an attenuated Escherichia coli bacterium. In another aspect of this embodiment, the CEQ201 strain is engineered to have cell invasive properties via an invasive plasmid. In one aspect of the invention, the plasmid is a TRIP (transckingdom RNA interference plasmid) plasmid or pNJSZ.

Dual "trojan horse" technology has also been used against invasive auxotrophic bacteria or BTP carrying plasmids transcribed from eukaryotic. The plasmid is in turn transcribed by the target cell to form one or more hairpin RNA structures that trigger the intracellular processes of RNAi. The method of the invention induces significant gene silencing of multiple genes. In certain aspects of this embodiment, the genes include transgenes in vitro (GFP), mutated oncogenes (k-Ras), and cancer-associated genes (β -catenin).

Another aspect of the BMGS of the invention is referred to as Transkingdom RNAi (tkRNAi). In this aspect of the invention, the siRNA is produced directly by the invasive bacterium, or accumulates in BTP after bacterial production, as opposed to the target cell. The transcription plasmid controlled by a prokaryotic promoter (e.g., T7) is inserted into a vector bacterium by standard transformation techniques. siRNA is produced in the bacteria and released into mammalian target cells following lysis of the bacteria triggered by auxotrophy or timed addition of antibiotics.

Most bacteria contain a large number of RNA degrading enzymes RNAse which can degrade siRNA resulting in a reduction in tkRNAi activity. In the case where RNAse from a particular bacterium exhibits this siRNA degrading activity, targeted deletion of the gene encoding the RNAse of interest (e.g., the rnc gene encoding RNAse III) is performed to allow higher levels of siRNA to be produced per tkRNAi bacterium, thereby allowing more siRNA to be delivered to the target cell and more effective gene silencing of the target gene in the target cell.

In contrast to genetically engineered knockdown models for discovering gene function, the RNAi methods of the invention, including BMGS and tkRNAi, are used to generate transient "knockdown" genetic animal models. The method is also useful as an in vitro transfection tool for research and drug development.

These methods use bacteria with desirable properties (invasive, attenuated, easy to handle) for BMGS and tkRNAi. As described in more detail herein, plasmids (e.g., TRIP) and vectors are used to render bacteria or BTPs invasive and eukaryotic or prokaryotic transcription of one or several shrnas.

1. Bacteria and/or Bacterial Therapy Particle (BTP)

The present invention provides at least one invasive bacterium or at least one Bacterial Therapeutic Particle (BTP) comprising one or more sirnas or one or more DNA molecules encoding one or more sirnas.

According to the present invention, any microorganism capable of delivering a molecule, such as an RNA molecule or a DNA molecule encoding an RNA, to the cytoplasm of a target cell, e.g.by entering the cytoplasm of the cell through a membrane, can be used for delivering the RNA to the cell. In a preferred embodiment, the microorganism is a prokaryote. In a more preferred embodiment, the prokaryote is a bacterium or BTP. In addition to bacteria, microorganisms that can be used to deliver RNA to cells are also included within the scope of the present invention. For example, the microorganism may be a fungus, such as Cryptococcus neoformans (Cryptococcus neoformans), protozoa, such as Trypanosoma cruzi (Trypanosoma cruzi), Toxoplasma gondii (Toxoplasma gondii), Leishmania donovani (Leishmania donovani) and plasmodium falciparum (plasmodia).

In a preferred embodiment, the microorganism is a bacterium or BTP. Preferred invasive bacteria or BTPs are capable of delivering at least one molecule, such as an RNA or a DNA molecule encoding an RNA, to a target cell, e.g. by entering the cytoplasm of a eukaryotic cell. Preferably, the RNA is siNRA or shRNA and the DNA molecule encoding RNA encodes siRNA or shRNA.

BTP is a fragment of a bacterium used for therapeutic or prophylactic purposes. BTP can include particles known in the art as minicells (minicells). Mini cells are small cells resulting from cell division with defects near the pole (pole) that lack a nucleoid and therefore are unable to grow and form colonies (Alder et al, (1967) Proc. Nat. Acad. Sci. U.S.A.57, 321-. Formation of minicells is the result of a mutation that results in a selective defect in the site of membrane formation for cell division. Such mutations include the null alleles of minC, minD (Davie et al, (1984) J. Bacteriol.158, 1202-1203; de Boer et al, 1988) J. Bacteriol.170, 2106-2112) and certain alleles of ftsZ (Bi and Lutkenhaus, (1992) J. Bacteriol.174, 5414-5423). Overexpression of FtsZ or MinC-MinD proteins has also been reported to cause minicell formation (Ward and Lutkenhaus, 1985; de Boeret et al, 1988). Although minicells do not contain a nucleoid, they are capable of transcription and translation (Roozenet al., (1971) J.Bacteriol.107, 21-33; Shepherd et al., (2001) J.Bacteriol.183, 2527-34).

BTP differs from bacteria in that it lacks a bacterial genome, thus reducing the risk of bacterial proliferation in patients. This is particularly beneficial for immunocompromised patients. Furthermore, the non-proliferative nature of BTP may allow its use in sensitive tissues such as the brain, and other body areas that are generally considered inaccessible to conventional siRNA. For example, intraperitoneal delivery of bacteria can involve risks of adhesions and peritonitis, which can be eliminated by the use of BTP. However, like the bacteria of the present invention, BTPs comprise a bacterial cell wall, some bacterial cytoplasmic content (bacterial plasma content) and subcellular particles, one or more therapeutic components (e.g., one or more sirnas), one or more invasion factors, one or more phagosome degradation factors, and one or more factors that target a particular tissue. BTP is produced by bacteria that have produced siRNA and accumulate siRNA within the bacteria, and bacterial fragments (BTP) are then isolated during cell division. In one embodiment of the invention, BTP is obtained by fermenting bacteria (which is formed in large quantities during the fermentation) and then separating BTP from viable bacteria using differential volume filtration (which will retain the bacteria while allowing BTP to pass and collect). In another embodiment of the invention, BTP is isolated from bacteria by centrifugation. In another embodiment of the invention, the viable bacterial cells are lysed by activating a death signal. After isolation, the BTP can be frozen and formulated for use.

The term "invasion" as used herein when referring to a microorganism such as a bacterium or BTP refers to a bacterium capable of delivering at least one molecule, such as an RNA or a DNA molecule encoding an RNA, to a target cell. An invasive microorganism may 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 (e.g., RNA or DNA encoding RNA) to the target cell. The process of delivering at least one molecule to the target cell preferably does not significantly alter the affected organ.

Invasive microorganisms include microorganisms that are naturally capable of delivering at least one molecule to a target cell, for example, by crossing a cell membrane (e.g., a eukaryotic cell membrane) and entering the cytoplasm, as well as microorganisms that are naturally non-invasive but have been modified (e.g., genetically modified) to be invasive. In another preferred embodiment, the bacterium or BTP can be modified to render the naturally non-invasive microorganism invasive by linking it to an "invasion factor" (also known as an "entry factor" or "cytoplasmic targeting factor"). As used herein, an "aggressive agent" is an agent, such as a protein or proteome, that when expressed by a non-aggressive bacterium or BTP, renders the bacterium or BTP aggressive. As used herein, an "invasion factor" is encoded by a "cytoplasmic targeting gene".

In one embodiment of the invention, the microorganism is a naturally invasive bacterium or BTP selected from the group consisting of, but not limited to, yersinia, rickettsia, legionella, brucella, mycobacterium, spirillum, coxsackiella, chlamydia, neisseria, burkholderia, bordetella, brucella, listeria, shigella, salmonella, staphylococcus, streptococcus, porphyria, spirochete, vibrio, escherichia coli and bifidobacterium. Optionally, the naturally invasive bacterium or BTP is yersinia expressing an invasive factor selected from the group consisting of invasin and YadA (yersinia enterocolitica plasmid adhesion factor). Optionally, the naturally invasive bacterium or BTP is rickettsia expressing the invasive factor RickA (actin-polymerizing protein). Optionally, the naturally invasive bacterium or BTP is legionella expressing the invasive factor RaIF (guanine exchange factor). Optionally, the naturally invasive bacterium or BTP is a neisseria expressing an invasive factor selected from the group including, but not limited to NadA (neisseria adhesion/invasive factor), OpaA, OpaC, and Opa52 (opacity related protein). Optionally, the naturally invasive bacterium or BTP is a listeria expressing an invasive factor selected from the group including, but not limited to, InlA (internalizing protein factor), InlB (internalizing protein factor), Hpt (hexose phosphate transporter), and ActA (actin polymerization protein). Optionally, the naturally invasive bacterium or BTP is shigella expressing an invasive factor selected from the group including, but not limited to, shigella secreting factors IpaA (invasive plasmid antigen), IpaB, IpaC, IpgD, IpaB-IpaC complex, VirA, and IcsA. Optionally, the naturally invasive bacterium or BTP is salmonella expressing an invasive factor selected from the group comprising, but not limited to, salmonella secretion/exchange factors SipA, SipC, SpiC, SigD, SopB, SopE2, and SptP. Optionally, the naturally invasive bacterium or BTP is a staphylococcus expressing an invasive factor selected from the group including, but not limited to, fibronectin binding proteins FnBPA and FnBPB. Optionally, the naturally invasive bacterium or BTP is a streptococcus expressing an invasive factor selected from the group including, but not limited to, fibronectin binding comparison ACP, Fba, F2, Sfb1, Sfb2, SOF, and PFBP. Optionally, the naturally invasive bacterium or BTP is Porphyromonas gingivalis (Porphyromonas gingivalis) expressing the invasion factor FimB (integrin binding protein fibriae).

In another embodiment of the invention, the microorganism is a bacterium or BTP that is not naturally invasive but has been modified (e.g., genetically modified) to be invasive. Optionally, the naturally non-invasive bacterium or BTP has been genetically modified to be invasive by expression of an invasive factor selected from the group consisting of invasin, YadA, RickA, RaIF, NadA, OpaA, OpaC, Opa52, InlA, InlB, Hpt, ActA, IpaA, IpaB, IpaC, IpgD, IpaB-IpaC complex, VirA, IcsA, SipA, SipC, SpiC, SigD, SopB, SopE2, SptP, FnBPA, FnBPB, ACP, Fba, F2, Sfb1, Sfb2, SOF, PFBP, and FimB.

In another embodiment of the invention, the microorganism is a bacterium or BTP that may be naturally invasive but has been modified (e.g., genetically modified) to express one or more additional invasive factors. Optionally, the invasion factor is selected from the group including, but not limited to: invasin, YadA, Ricka, RaIF, NadA, OpaA, OpaC, Opa52, InlA, InlB, Hpt, ActA, IpaA, IpaB, IpaC, IpgD, IpaB-IpaC complex, VirA, IcsA, SipA, SipC, SpiC, SigD, SopB, SopE2, SptP, FnBPA, FnBPB, ACP, Fba, F2, Sfb1, Sfb2, SOF, PFBP, and FimB.

Naturally invasive microorganisms such as bacteria or BTP may have a specific tropism, i.e. a preferred target cell. Alternatively, the microorganism (e.g., bacteria or BTP) may be modified (e.g., genetically modified) to mimic the tropism of a second microorganism. Optionally, the bacterium or BTP is streptococcus and the preferred target cell is selected from the group including, but not limited to: pharyngeal epithelial cells, buccal epithelial cells of the tongue, and mucosal epithelial cells. Optionally, the bacterium or BTP is a porphyromonas and the preferred target cell is selected from the group including, but not limited to, oral epithelial cells. Optionally, the bacterium or BTP is staphylococcus and the preferred target cell is a mucosal epithelial cell. Optionally, the bacterium or BTP is neisseria and the preferred target cell is selected from the group including, but not limited to, urothelial cells and cervical epithelial cells. Optionally, the bacterium or BTP is escherichia coli and the preferred target cell is selected from the group including, but not limited to: intestinal epithelial cells, urothelial cells, and cells of the upper urinary tract. Optionally, the bacterium or BTP is bordetella and the preferred target cell is an airway epithelial cell. Optionally, the bacterium or BTP is vibrio and the preferred target cell is an intestinal epithelial cell. Optionally, the bacterium or BTP is a spirochete and the preferred target cell is a mucosal epithelial cell. Optionally, the bacterium or BTP is mycoplasma and the preferred target cell is a respiratory epithelial cell. Optionally, the bacterium or BTP is a helicobacter and the preferred target cell is an intragastric endothelial cell. Optionally, the bacterium or BTP is chlamydia and the preferred target cell is selected from the group including, but not limited to, conjunctival cells and urothelial cells.

In another embodiment of the invention, the microorganism is a bacterium or BTP that has been modified (e.g., genetically modified) to have a particular tropism. Optionally, the preferred target cell is selected from the group including, but not limited to, consisting of: pharyngeal epithelial cells, buccal epithelial cells of the tongue, mucosal epithelial cells, oral epithelial cells, urethral epithelial cells, cervical epithelial cells, intestinal epithelial cells, respiratory epithelial cells, cells of the upper urinary tract, gastric epithelial cells, and conjunctival cells. Optionally, the preferred target cell is a dysplastic or carcinomic epithelial cell. Optionally, the preferred target cell is an activated or resting immune cell.

Delivery of the at least one molecule to the target cell can be determined according to methods known in the art. For example, the presence of the molecule may be determined by the reduced expression of the RNA or protein thus silenced, using hybridization or PCR methods, or using immunological methods which may include the use of antibodies.

Determining whether the microorganism is sufficiently invasive for use in the present invention can include determining whether sufficient siRNA is delivered to the host cell relative to the number of microorganisms contacted with the host cell. If the amount of siRNA is small relative to the amount of microorganism used, it may be desirable to further modify the microorganism to increase its invasive potential.

Bacterial or BTP entry into cells can be determined by a variety of methods. Intracellular bacteria or BTP are resistant to aminoglycoside antibiotic treatment, while extracellular bacteria are rapidly killed. Quantitative estimation of bacterial or BTP uptake can be achieved by treating a cell monolayer with the antibiotic gentamicin to inactivate extracellular bacteria or BTP, then removing the antibiotic before releasing viable intracellular organisms with mild detergent, and determining the number of survivors on standard bacterial media. In addition, bacteria or BTP that enter the cells can be directly observed by, for example, a thin sheet transmission electron microscope of the cell layer or by immunofluorescence techniques (Falkowet al (1992) Annual Rev. cell biol.8: 333). Thus, various techniques can be used to determine whether a particular bacterium or BTP is capable of invading a particular type of cell, or to determine bacterial invasion following modification of the bacterium or BTP (modifying the tropism of this bacterium to mimic the tropism of a second bacterium).

Preferably, the bacteria or BTP that can be used to deliver RNA are not pathogenic according to the methods of the invention. However, pathogenic bacteria or BTP may also be used, provided that their pathogenicity has been attenuated, thereby rendering the bacteria harmless to the subject to which they are administered. The term "attenuated bacteria or BTP" as used herein refers to bacteria or BTP that has been modified to significantly reduce or eliminate its deleterious effects on a subject. Pathogenic bacteria or BTP can be attenuated by a variety of methods as listed below.

Regardless of its specific mechanism of action, the bacteria or BTP that delivers RNA to eukaryotic cells can enter various compartments of the cell depending on the type of bacteria or BTP. For example, the bacteria or BTP may be in a vesicle (e.g., a phagocyte vesicle). Once inside the cell, the bacteria or BTP can be destroyed or lysed and their contents delivered into the eukaryotic cells. Bacteria or BTP can also be engineered to express phagosome degrading proteins to allow RNA leakage from the phagosome. In one embodiment of the invention, the bacteria or BTP express (either naturally or by modification such as genetic modification) a protein that facilitates pore formation, disruption or degradation of the phagosome. Optionally, the protein is cholesterol-dependent cytolysin. Optionally, the protein is selected from the group consisting of: listeriolysin (listeriolysin), ivermectin (ivanolysin), streptolysin (streptolysin), sphingomyelinase, perfringens lysin (perfringens), botulilysin (botulinysin), leukocidin (leukocidin), anthrax toxin, phospholipase, IpaB (invading plasmid antigen), IpaH, IcsB (intracellular transmission), DOT/Icm (organelle transport defect/intracellular reproduction defect), DOTU (stabilizer of DOT/Icm complex), IcmF, and PmrA (multidrug resistance efflux pump).

In some embodiments, the bacteria can remain viable in eukaryotic cells for varying lengths of time, and can continue to produce RNA. The RNA or DNA encoding the RNA may then be released from the bacteria into the cell by, for example, leakage. In certain embodiments of the invention, the bacterium may also replicate in eukaryotic cells. In a preferred embodiment, bacterial replication does not kill the host cell. The present invention is not limited to the delivery of RNA or DNA encoding RNA by a particular mechanism and is intended to include methods and compositions that can deliver RNA or DNA encoding RNA by bacteria regardless of its delivery mechanism.

In one embodiment, the bacteria or BTP for use in the present invention are non-pathogenic or non-toxic. In another aspect of this embodiment, the bacterium or BTP is therapeutic. In another aspect of this embodiment, the bacterium or BTP is an attenuated strain or derivative thereof selected from, but not limited to: yersinia, rickettsia, legionella, brucella, mycobacteria, spirillum, Haemophilus (Haemophilus), coxsackiella, chlamydia, neisseria, burkholderia, bordetella, brucella, listeria, shigella, salmonella, staphylococci, streptococci, porphyria, spirochete, vibrio, escherichia coli and bifidobacteria. Optionally, the Yersinia is an attenuated strain of the species Yersinia pseudotuberculosis (Yersinia pseudouberculosis). Optionally, the yersinia strain is an attenuated strain of yersinia enterocolitica species. Optionally, the Rickettsia strain is an attenuated strain of Rickettsia coronii. Optionally, the Legionella strain is an attenuated strain of the species Legionella pneumophila (Legionella pneumophila). Optionally, the Mycobacterium strain is an attenuated strain of the species Mycobacterium tuberculosis (Mycobacterium tuberculosis). Optionally, the Mycobacterium strain is an attenuated strain of Mycobacterium bovis (Mycobacterium bovis) BCG species. Optionally, the Helicobacter strain is an attenuated strain of the Helicobacter pylori (Helicobacter pylori) species. Optionally, the strain of coxsackie is an attenuated strain of coxsackie burnetti (Coxiella burnetti). Optionally, the Haemophilus strain is an attenuated strain of Haemophilus influenzae (haempophilus influenza) species. Optionally, the Chlamydia strain is an attenuated strain of the species Chlamydia trachomatis (Chlamydia trachomatis). Optionally, the Chlamydia strain is an attenuated strain of the species Chlamydia pneumoniae (Chlamydia pneumoniae). Optionally, the Neisseria strain is an attenuated strain of the species Neisseria gonorrhoeae (Neisseria gonorrhoeae). Optionally, the Neisseria strain is an attenuated strain of the species Neisseria meningitidis (Neisseria meningitidis). Optionally, the burkholderia strain is an attenuated strain of burkholderia cepacia (Burkolderia cepacia) species. Optionally, the Bordetella strain is an attenuated strain of the species Bordetella pertussis (Bordetella pertussis). Optionally, the brucella strain is an attenuated strain of the species Borrelia hermisii. Optionally, the listeria strain is an attenuated strain of listeria monocytogenes species. Optionally, the Listeria strain is an attenuated strain of the species Listeria ita (Listeria ivanovii). Optionally, the Salmonella strain is an attenuated strain of a Salmonella enterica (Salmonella enterica) species. Optionally, the salmonella strain is an attenuated strain of salmonella typhimurium. Optionally, the salmonella typhimurium strain is SL7207 or VNP 20009. Optionally, the Staphylococcus strain is an attenuated strain of the species Staphylococcus aureus (Staphylococcus aureus). Optionally, the Streptococcus strain is an attenuated strain of Streptococcus pyogenes (Streptococcus pyogenes) species. Optionally, the Streptococcus strain is an attenuated strain of Streptococcus mutans (Streptococcus mutans) species. Optionally, the Streptococcus strain is an attenuated strain of Streptococcus salivarius (Streptococcus salivarius) species. Optionally, the Streptococcus strain is an attenuated strain of the species Streptococcus pneumoniae (Streptococcus pneumoniae). Optionally, the porphyrin strain is an attenuated strain of a porphyromonas gingivalis species. Optionally, the pseudomonas strain is an attenuated strain of the species pseudomonas aeruginosa (pseudomonas aeruginosa). Optionally, the spirochete strain is an attenuated strain of the species treponema pallidum. Optionally, the Vibrio strain is an attenuated strain of the species Vibrio cholerae (Vibrio cholerae). Optionally, the escherichia coli strain is MM 294.

Listed below are examples of bacteria that have been described in the literature as being naturally invasive (section 1.1), bacteria that have been described in the literature as not being naturally invasive (section 1.2), and naturally nonpathogenic or attenuated bacteria. Although it has been described that some bacteria are not invasive (section 1.2), these bacteria may still be sufficiently invasive to be used in the present invention. Whether a naturally invasive bacterium or a non-invasive bacterium is generally described, any bacterial strain can be modified to modulate, especially improve, its invasive properties (e.g., as described in section 1.3).

1.1 naturally invasive bacteria

The particular naturally invasive bacteria used in the present invention is not critical to the present invention. Such naturally occurring invasive bacteria include, but are not limited to, Shigella, Salmonella, Listeria, Rickettsia, and Enterobacter coli.

The particular Shigella strain employed is not critical to the present invention. Examples of Shigella strains that can be used in the present invention include Shigella flexneri 2a (ATCC No.29903), Shigella sonnei (Shigella sonnei) (ATCC No.29930), and Shigella dysenteriae (Shigella disseriae) (ATCC No. 13313). Attenuated Shigella strains are preferably used in the present invention, for example, Shigella flexneri 2a 2457T aroA virG mutant CVD 1203(Noriega et al, supra), Shigella flexneri M90T icsA mutant (Goldberg et al Infect. Immun., 62: 5664-. Alternatively, a new attenuated shigella strain can also be constructed by introducing a single attenuating mutation or by introducing the single attenuating mutation in combination with one or more other attenuating mutations.

At least one advantage of shigella bacteria as a delivery vehicle is their tropism for lymphoid tissues at the surface of the colonic mucosa. Furthermore, the major site of Shigella replication is believed to be in dendritic cells and macrophages that are often found on the basal side of M cells in mucosal lymphoid tissues (reviewed by McGhee, J.R.et al (1994) Reproduction, Fertility, & Development 6: 369; Pascual, D.W.et al (1994) immunoassays 5: 56). Thus, shigella vectors can provide a means to target RNA interference or deliver therapeutic molecules to these specialized antigen presenting cells. Another advantage of Shigella vectors is that attenuated Shigella strains deliver nucleic acid reporters In vitro and In vivo (Sizemore, D.R.et. (1995) Science 270: 299; Courvalin, P.et. (1995) computers Rendus de l Academy des Sciences series III-Sciences de la Vie-Life Sciences 318: 1207; Powell, R.J.et. (1996) In: Molecular ap probes to the control of infectious diseases, F.Brown, E.Norrby, D.Burton and J.Melalanos, Cold.spring harbor Laboratory, New York.183; Anderson, R.J.et. (1997) extract for the biological 97). In practical terms, the strictly restricted host specificity of shigella can prevent diffusion of shigella vectors into the food chain through intermediate hosts. Furthermore, attenuated strains that are highly attenuated in rodents, primates and volunteers have been developed (Anderson et al (1997) supra; Li, A.et al (1992) Vaccine 10: 395; Li, A.et al (1993) Vaccine 11: 180; Karnell, A.et al (1995) Vaccine 13: 88; Sanonenti, P.J. and J.Aronde (1989) Vaccine 7: 443; Fontaine, A.et al (1990) Research in Microbiology 141: 907; Sanonenti, P.J.1991) Vaccine 9: 416; Noriega, F.R.et al (1994) Vaccine & Immunity 62: 5168; Norieega, F.R.ferty (1990) 64: Immunity & 64; Inmunity & F.r.r.5: 1996) have been developed. The last knowledge allows the development of well-tolerated shigella vectors for use in humans.

Have been obtained by non-specific mutagenesis using chemical methods such as N-methyl-N' -nitro-N-nitrosoguanidine reagents, or using recombinant DNA techniques; classical genetic techniques such as Tn10 mutagenesis, P22-mediated transduction, lambda phage-mediated crossover (crossover) and conjugal transfer; or site-directed mutagenesis using recombinant DNA techniques to introduce attenuating mutations into bacterial pathogens. Recombinant DNA technology is preferred because the strains constructed by recombinant DNA technology are more defined. Examples of such attenuating mutations include, but are not limited to:

(i) auxotrophic mutations, such as aro (Hoiseth et al. Nature, 291: 238-;

(ii) mutations which inactivate global regulatory function, such as cya (Curtiss et al. Impect. Immun., 55: 3035-3043(1987)), crp (Curtiss et al (1987), supra), phoP/phoQ (Grossman et al. Proc. Natl. Acad. Sci., USA, 86: 7077-7081 (1989); and Miller et al. Proc. Natl. Acad. Sci., USA, 86: 5054-5058(1989)), phoP^c(Miller et al J.Bact., 172: 2485-;

(iii) mutations that alter stress responses, such as recA (Buchmeier et al. mol. Micro., 7: 933-936(1993)), htrA (Johnson et al. mol. Micro., 5: 401-407(1991)), htpR (Neidhardt et al. biochem. Biophys. Res. Com., 100: 894-900(1981)), hsp (Neidhardt et al. Ann.Rev.Genet., 18: 295 (1984)) and groEL (Buchmeier et al. Sci., 248: 730-732(1990)) mutations;

(iv) mutations of specific virulence factors, such as IsyA (Libby et al Proc. Natl. Acad. Sci., USA, 91: 489-493(1994)), pag or prg (Miller et al (1990), supra; and Miller et al (1989), supra), iscA or virG (d' Hauteville et al. mol. Micro., 6: 833-19 (1992)), plcA (Menglad et al. mol. Microbiol., 5: 367-72 (1991); Camilli et al. J. exp. Med., 173: 751-754(1991)) and act (Brundage et al. Proc. Natl. Acad. Sci., USA, 90: 11890-11894 (1993));

(v) mutations that affect DNA topology, such as topA (Galan et al. Infect. Immun., 58: 1879-1885 (1990));

(vi) mutations that interrupt or alter the cell cycle, such as min (de Boer et al. cell, 56: 641-649 (1989));

(vii) introduction of a gene encoding a suicide system, such as sacB (Recorbet et al. App. environ. micro., 59: 1361- & 1366 (1993); Quandt et al. Gene, 127: 15-21(1993)), nuc (Ahrenholtz et al. App. environ. micro., 60: 3746- & 3751(1994)), hok, gef, kil or phlA (Molin et al. Ann. Rev. Microbiol., 47: 139- & 166 (1993));

(viii) mutations that alter biogenesis of lipopolysaccharide and/or lipid A, such as rFb (Raetz in Escherichia coli and Salmonella typhimurium, Neidhardt et al, Ed., ASM Press, Washington D.C.pp 1035 1063(1996)), galE (Hone et al.J.Infect.Dis., 156: 164-;

(ix) introduction of a phage lysis system, for example the P22-encoded lysin gene (Rennell et al Virol, 143: 280-289(1985)), lambda murein glycosyltransferase (Bienkowska-Szewczyk et al mol. Gen. Gene., 184: 111-114(1981)) or the S-gene (Reader et al Virol, 43: 623-628 (1971)); and

the attenuating mutation may be constitutively expressed or under the control of an inducible promoter, for example the promoter of the temperature-sensitive heat shock protein family (Neidhardt et al, supra), or the anaerobically inducible nirB promoter (Harborne et al. mol. Micro., 6: 2805. sub. 2813(1992)) or a repressible promoter, for example uapA (Gorfikiel et al. J. biol. chem., 268: 23376. sub. 23381(1993)) or gcv (Stauffer et al. J. Bact., 176: 6159. sub. 6164 (1994)).

The particular listeria strain employed is not critical to the present invention. Examples of listeria strains that can be used with the present invention include listeria monocytogenes (ATCC No. 15313). Attenuated Listeria strains, such as the Listeria monocytogenes actA mutant (Brundage et al, supra) or Listeria monocytogenes plcA (Camilli et al J. exp. Med., 173: 751-754(1991)), are preferably used in the present invention. Alternatively, a new attenuated listeria strain can be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described for shigella above.

The particular salmonella strain employed is not critical to the present invention. Examples of salmonella strains that can be used in the present invention include salmonella typhi (ATCC No.7251) and salmonella typhimurium (ATCC No. 13311). Attenuated salmonella strains comprising s.typhi-aroC-aroD (Hone et al vacc.9: 810(1991) and salmonella typhimurium-aroA mutant (massoeni et al micro. pathol.13: 477(1992)) are preferably used in the present invention or new attenuated salmonella strains can be constructed by introducing one or more attenuating mutations as described above for shigella species.

The particular rickettsia strain employed is not critical to the present invention. Examples of Rickettsia strains that can be used in the present invention include Rickettsia (Rickettsia Rickettsiae) (ATCCNOS.VR149 and VR891), Rickettsia prowaseckii (ATCC No. VR233), Rickettsia tsutsutsugamushi (ATCC Nos. VR312, VR150 and VR609), Rickettsia morganii (Rickettsia mooseri) (ATCC No. VR144), siberian Rickettsia (Rickettsia sibirica) (ATCC No. VR151) and Rickettsia pentaerythraea (Rockhamaeviantana) (ATCC No. VR358). Attenuated rickettsia strains are preferably used in the present invention and may be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described above for shigella species.

The particular enteroinvasive Escherichia strain employed is not critical to the present invention. Examples of enteroinvasive Escherichia strains that can be used in the present invention include Escherichia coli 4608-58, 1184-68, 53638-C-17, 13-80 and 6-81(Sansonetti et al, Ann. Microbiol. (Inst. Pasteur), 132A: 351-355 (1982)). Attenuated enteroinvasive strains of Escherichia are preferably used in the present invention and may be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described above for Shigella species.

Furthermore, since certain microorganisms other than bacteria are also capable of interacting with integrin molecules (which are receptors for certain invasion factors) for cellular uptake, these microorganisms can also be used to introduce RNA into target cells. For example, viruses such as oropodovirus, echovirus and adenovirus, and eukaryotic pathogens such as Histoplasma capsulatum (Histoplasma capsulatum) and Leishmania major (Leishmania major) that interact with integrin molecules.

1.2 less invasive bacteria

Examples of bacteria that have been described in the literature as being non-invasive or at least less invasive than the bacteria listed in section 1.1 above, which may be used in the present invention, include, but are not limited to, yersinia, Escherichia (Escherichia spp.), Klebsiella (Klebsiella spp.), bordetella, neisseria, Aeromonas (Aeromonas spp.), francisella (francisella spp.), Corynebacterium (Corynebacterium spp.), Citrobacter (Citrobacter spp.), chlamydia, haemophilus (haemophilus spp.), brucella, mycobacterium, legionella, Rhodococcus (Rhodococcus spp.), Pseudomonas (Pseudomonas spp.), helicobacter spp., helicobacter, vibrio, Bacillus (Bacillus spp.) and Erysipelothrix (Erysipelothrix). These bacteria may have to be modified to increase their invasive potential.

The particular yersinia strain employed is not critical to the present invention. Examples of yersinia strains that may be used in the present invention include yersinia enterocolitica (y.enterocolitica) (ATCC No.9610) or yersinia pestis (y.pestis) (ATCC No. 19428). Attenuated Yersinia strains, such as Yersinia enterocolitica Ye03-R2(al-Hendy et al. infection. Immun., 60: 870-875(1992)) or Yersinia enterocolitica aroA (O' Gaora et al. micro. Path., 9: 105-116(1990)), are preferred for use in the present invention. Alternatively, a new attenuated yersinia strain may be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described above for shigella species.

The particular Escherichia strain employed is not critical to the present invention. Examples of Escherichia strains which can be used in the present invention include Escherichia coli Nissle 1917, MM294, H10407(Elinghorst et. Infect. Immun., 60: 2409-. Attenuated strains of Escherichia, such as the attenuated turkey pathogen Escherichia coli 02 carAB mutant (Kwaga et al. infection. Immun., 62: 3766-3772(1994)) or CEQ201, are preferably used in the present invention. Alternatively, a novel attenuated Escherichia strain can be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described above for Shigella species.

The particular klebsiella strain employed is not critical to the present invention. Examples of Klebsiella strains that can be used in the present invention include Klebsiella pneumoniae (ATCC No. 13884). Attenuated klebsiella strains are preferably used in the present invention and may be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described above for shigella species.

The particular bordetella strain employed is not critical to the present invention. Examples of the Bordetella strain that can be used in the present invention include Bordetella bronchiseptica (ATCC No. 19395). Attenuated bordetella strains are preferably used in the present invention and may be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described above for shigella species.

The particular neisserial strain employed is not critical to the present invention. Examples of neisserial strains that may be used in the present invention include neisseria meningitidis (ATCC No.13077) and neisseria gonorrhoeae (ATCC No. 19424). The invention preferably uses attenuated strains of Neisseria, such as Neisseria gonorrhoeae MS11 aro mutants (Chamberland et al. Micro. Path., 15: 51-63 (1993)). Alternatively, a novel attenuated neisserial strain may be constructed by introducing one or more attenuating mutations in groups (i) to (vii), as described above for shigella species.

The particular aeromonas strain employed is not critical to the present invention. Examples of strains of aeromonas that can be used in the present invention include aeromonas hydrophila (a. eutrenophila) (ATCC No. 23309). Alternatively, a new attenuated aeromonas strain may be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described above for shigella species.

The particular Francis strain employed is not critical to the present invention. Examples of the Francisella strain that can be used in the present invention include Francisella tularensis (F.tularensis) (ATCC No. 15482). Attenuated Francisella strains are preferably used in the present invention, and may be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described above for Shigella species.

The particular Corynebacterium strain employed is not critical to the present invention. Examples of the coryneform bacteria strain that can be used in the present invention include pseudotuberculosis corynebacterium (c.pseudotuberculosis) (ATCC No. 19410). Attenuated corynebacteria strains are preferably used in the present invention, and may be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described above for shigella species.

The particular Citrobacter strain employed is not critical to the present invention. Examples of the Citrobacter strains that can be used in the present invention include Citrobacter freundii (C.freundii) (ATCC No. 8090). Attenuated Citrobacter strains are preferably used in the present invention and may be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described above for Shigella species.

The particular chlamydia strain employed is not critical to the present invention. Examples of chlamydia strains that can be used in the present invention include chlamydia pneumoniae (ATCC No. vr1310). Attenuated chlamydia strains are preferably used in the present invention and may be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described above for shigella species.

The particular haemophilus strain employed is not critical to the present invention. Examples of the haemophilus strain that can be used in the present invention include h.sornnus (ATCC No. 43625). Attenuated haemophilus strains are preferably used in the present invention and may be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described above for shigella species.

The particular brucella strain employed is not critical to the present invention. Examples of brucella strains that can be used in the present invention include brucella bovis (b.abortus) (ATCC No. 23448). Attenuated brucella strains are preferably used in the present invention and may be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described above for shigella species.

The particular mycobacterium strain employed is not critical to the present invention. Examples of the mycobacterium strains that can be used in the present invention include M.intracellulare (ATCC No.13950) and Mycobacterium tuberculosis (ATCC No. 27294). Attenuated mycobacterial strains are preferably used in the present invention and may be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described above for shigella species.

The particular legionella strain employed is not critical to the present invention. Examples of legionella strains which can be used in the present invention include legionella pneumophila (l.pneumophila) (ATCC No. 33156). Attenuated strains of Legionella, such as the legionella pneumophila mip mutant (Ott, FEMS micro. Rev., 14: 161-. Alternatively, a new attenuated legionella strain may be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described above for shigella species.

The particular Rhodococcus strain employed is not critical to the present invention. Examples of the Rhodococcus strain which can be used in the present invention include Rhodococcus equi (R.equi) (ATCC No. 6939). Attenuated Rhodococcus strains are preferably used in the present invention and may be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described above for Shigella species.

The particular pseudomonas strain employed is not critical to the present invention. Examples of Pseudomonas strains that can be used in the present invention include Pseudomonas aeruginosa (ATCC No. 23267). Attenuated pseudomonas strains are preferably used in the present invention and may be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described above for shigella species.

The particular helicobacter strain employed is not critical to the present invention. Examples of helicobacter strains that can be used in the present invention include h.mustelae (ATCC No. 43772). Attenuated helicobacter strains are preferably used in the present invention and may be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described above for shigella species.

The particular salmonella strain employed is not critical to the present invention. Examples of Salmonella strains that can be used in the present invention include Salmonella typhimurium (ATCC No.7251) and Salmonella typhimurium (ATCC No. 13311). Attenuated Salmonella strains are preferred for use herein and include Salmonella typhimurium aroC aroD (Hone et al. vacc., 9: 810-491 (1991)) and Salmonella typhimurium aroA mutant (Mastroeni et al. micro. Pathol, 13: 477-491 (1992)). Alternatively, a new attenuated salmonella strain may be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described above for shigella species.

The particular vibrio strain employed is not critical to the present invention. Examples of Vibrio strains that may be used in the present invention include Vibrio cholerae (Vibrio cholerae) (ATCC No.14035) and Vibrio cincinnati (Vibrio cincinnati) (ATCC No. 35912). Attenuated strains of Vibrio cholerae including virulent mutants of Vibrio cholerae RSI (Taylor et al.J.Infect.Dis., 170: 1518-. Alternatively, a new attenuated vibrio strain may be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described above for shigella species.

The particular bacillus strain employed is not critical to the present invention. Examples of Bacillus strains that can be used in the present invention include Bacillus subtilis (ATCC No. 6051). Attenuated strains of Bacillus are preferred for use herein and include the Bacillus anthracis (B.antrrachis) mutant pX01(Welkos et al. micro. Pathol, 14: 381-388(1993)) and the attenuated BCG strain (Stover et al. Nat., 351: 456-460 (1991)). Alternatively, a new attenuated bacillus strain may be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described above for shigella species.

The particular strain of erysipelothrix employed is not critical to the present invention. Examples of the strain of Erysipelothrix rhusiopathiae (ATCC No.19414) and Erysipelothrix tonsillis (ATCC No.43339) that can be used in the present invention. The present invention preferably uses attenuated strains of Erysipelothrix rhusiopathiae and the strains include the Erysipelothrix rhusiopathiae Kg-1a and Kg-2(Watarai et al.J.vet.Med.Sci., 55: 595-600(1993)) and the Ervac swine ORVAC mutant (Markowska-Daniel et al.int.J.Med.Microb.Virol.Parisit.Infect.Dis., 277: 547 553 (1992)). Alternatively, a new attenuated erysipelothrix strain may be constructed by introducing one or more attenuating mutations in groups (i) to (vii) as described above for shigella species.

1.3. Method for improving invasive Properties of bacterial strains

Whether invasive or non-invasive in the traditional sense, these organisms can be engineered to enhance their invasive properties, for example by mimicking the invasive properties of shigella, listeria, rickettsia or enteroinvasive escherichia coli. For example, one or more genes may be introduced into a microorganism that enable the microorganism to enter the cytoplasm of a cell (e.g., a cell of a native host of the non-invasive bacterium).

Examples of such genes, referred to herein as "cytoplasmic targeting genes", include the genes encoding proteins capable of Shigella invasion, or the similarly invasive genes of Enterobacter enteroinvasive Escherichia or Listeriolysin O of Listeria, and thus such techniques are known to enable a variety of invasive bacteria to invade and enter the cytoplasm of animal cells (formula et al. Infection. Immun., 46: 465 (1984); Bielecke et al Nature, 345: 175. 198176; Small et al. in: Microbiology-1986, pages 121-124; Levine et al. Eds., American Society for Microbiology, Washington, D.C. (1986); Zychlinsky et al. Molec. 619., 627: Immun (317; Isthschel. Biochem. 317; Ishleck. 1982. 4203. 1985; Ishly. 1987: Isberr. 767. 1985). Methods for transferring the above cytoplasmic targeting genes into bacterial strains are well known in the art. Another preferred gene that can be introduced into bacteria to improve their invasive properties encodes the invasin protein from Yersinia pseudotuberculosis (Leong et al. EMBO J., 9: 1979 (1990)). Invasin and listeriolysin may be introduced in combination, thereby further increasing the invasive properties of the bacteria relative to when these genes are introduced alone. The above description of genes is for illustrative purposes, however, any gene or combination of genes from one or more sources involved in delivering molecules from a microorganism (particularly RNA or DNA molecules encoding RNA) to the cytoplasm of a cell (e.g., an animal cell) may suffice for the present invention, as will be apparent to those skilled in the art. Thus, this gene is not limited to bacterial genes, and includes viral genes such as influenza virus hemagglutinin HA-2 (plant et al J. biol. chem., 269: 12918-12924(1994)) which promotes endosmosis lysis.

The above-mentioned cytoplasm-targeting gene can be obtained by, for example, PCR amplification of DNA isolated from an invading bacterium carrying the desired cytoplasm-targeting gene. The term "cell" may be used in accordance with what is available in the art, for example from the above listed literature and/or GenBank (publicly available from the internet,www.ncbi.nlm.nih.gov/) The obtained nucleotide sequence was used to design a primer for PCR. PCR primers can be designed to amplify a cytoplasmic targeted gene, a cytoplasmic targeted operon, a cluster of cytoplasmic targeted genes, or a regulator of a cytoplasmic targeted gene. The PCR strategy employed will depend on the genetic configuration of one or more cytoplasmic targeting genes in the target invading bacteria. The PCR primers are designed to contain sequences homologous to the beginning and end DNA sequences of the target DNA sequence. The cytoplasmic targeting gene can then be introduced into the target Bacterial strain, for example, by using Hfr transfer or plasmid migration (Miller, A Short coursing Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1992); Bothwell et al, supra; and Ausubel et al, supra), phage-mediated transduction (de Boer, supra; Miller, supra; and Ausubel et al, supra), chemical transformation (Bothwell et al, supra; Ausubel et al, supra), electroporation (Bothwell et al, supra; Ausubel et al, supra; Sambrook et al, Molecular Cloning: A Laboratory, Cold Spring Harbor Laboratory, d plasmid LaboratorySpring Harbor, n.y.) and physical transformation techniques (Johnston et al, supra; and Bothwell, supra). The cytoplasmic targeting gene can be introduced into lysogenic phages (de Boer et al. cell, 56: 641-649(1989)), into plasmid vectors (Curtiss et al. supra) or spliced into the chromosome of the target strain (Hone et al. supra).

In addition to genetically processing bacteria and BTP to improve their invasive properties, as described above, the invasive factors can also be modified by attaching them to the bacteria. Thus, in one embodiment, the bacteria are rendered more invasive by covalently or non-covalently coating the bacteria with an invasive factor, such as the protein invasin, invasin derivative, or a fragment thereof sufficient for invasiveness. In fact, it has been demonstrated that non-invasive bacterial cells coated with either purified invasin from Yersinia pseudotuberculosis, or 192 amino acids from the carboxy terminus of invasin, are able to enter mammalian cells (Leong et al (1990) EMBO J.9: 1979). In addition, latex beads coated with the carboxy-terminal region of invasin are efficiently internalized by mammalian cells; similarly, S.aureus strains coated with antibody-immobilized invasin are also efficiently internalized by mammalian cells (reviewed in Isberg and Tran van Nhieu (1994) Ann.Rev.Genet.27: 395). Alternatively, the bacteria may also be coated with an antibody, variant or fragment thereof that specifically binds to a surface molecule recognized by a bacterial entry factor. For example, it has been demonstrated that bacteria are internalized if they are coated with monoclonal antibodies targeting integrin molecules (e.g., α 5 β 1, a surface molecule known to interact with bacterial invasin proteins). Such antibodies can be prepared according to methods known in the art. The efficacy of the antibodies in mediating bacterial invasion can be determined, for example, by coating the bacteria with the antibodies, contacting the bacteria with eukaryotic cells having surface receptors recognized by the antibodies, and monitoring the presence of intracellular bacteria, according to the methods described above. Methods of attaching an invading agent to the surface of a bacterium are known in the art and include cross-linking.

3. Plasmids and vectors

The invention also provides at least one vector or plasmid comprising at least one DNA molecule encoding one or more sirnas and at least one promoter, wherein the expressed siRNA interferes with at least one mRNA of a target gene. In a preferred embodiment, the invention provides at least one prokaryotic vector comprising at least one DNA molecule encoding one or more sirnas and at least one promoter compatible with RNA polymerase III or at least one prokaryotic promoter, wherein the expressed siRNA interferes with at least one mRNA of a target gene.

TRIP (Transkingdom RNA interference plasmid) vectors and plasmids of the invention comprise a multiple cloning site, a promoter sequence and a terminator sequence. The TRIP vectors and plasmids also contain one or more sequences encoding an invasion factor to allow non-invasive bacteria or BTP to enter mammalian cells (e.g., the Inv locus encoding an invader that allows bacteria or BTP to enter β 1-integrin positive mammalian cells) (Young et al, J.cell biol.116, 197-. TRIP (containing vector/plasmid schematic) is further described in PCT publication WO 06/066048. In a preferred embodiment, the TRIP vectors and plasmids will incorporate a hairpin RNA expression cassette encoding a short hairpin RNA under the control of appropriate promoter and terminator sequences.

In the design of these constructs, algorithms were utilized to account for some of the known difficulties in siRNA development, namely: (1) reject the reject property (SNP, interferon motif); (2) excluding the sequence if it has homology to ref seq (19/21, > 17 apart from any other gene), and (3) excluding the sequence if it has a significant miRNA pattern match.

As described herein, one or more DNA molecules encoding one or more sirnas are transcribed in a eukaryotic target cell, or in bacteria or BTP.

In embodiments where the DNA is transcribed in a eukaryotic cell, the one or more sirnas are transcribed as shrnas in the eukaryotic cell. The eukaryotic cell may be in vivo, in vitro or ex vivo. In one aspect of this embodiment, the one or more DNA molecules encoding the one or more sirnas comprise a eukaryotic promoter. Optionally, the eukaryotic promoter is an RNA polymerase III promoter. Optionally, the RNA polymerase III promoter is a U6 promoter or an H1 promoter.

In embodiments where the DNA is transcribed in bacteria or BTP, the one or more DNA molecules comprise a prokaryotic promoter. Optionally, the prokaryotic promoter is an escherichia coli promoter. Preferably, the Escherichia coli promoter may be T7 promoter, lacUV5 promoter, modified lacUV5 promoter, RNA polymerase promoter, gapA promoter, pA1 promoter, lac-regulated promoter, araC + P_araBADPromoter, T5 promoter, P_tacPromoters (Estrem et al, 1998, Proc. Natl. Acad. Sci. USA 95, 9761-9766; Meng et al, 2001, Nucleic Acids Res.29, 4166-417; De Boer et al, 1983, Proc. NatL. Acad. Sci. USA 80, 21-25) or recA promoters.

Preferably, the promoter sequences are listed in table 1.

TABLE 1

In embodiments where the DNA is transcribed in bacteria or BTP, the Escherichia coli promoter is linked to a terminator. Preferably, the Escherichia coli terminator may be a T7 terminator, a lacUV5 terminator, a Rho-independent terminator, a Rho-dependent terminator or a RNA polymerase terminator.

Preferably, the terminator sequences are listed in table 2.

TABLE 2

In other embodiments, the vectors and plasmids of the invention further comprise one or more enhancer sequences, selectable markers, or lytic regulatory system sequences.

In one aspect of the invention, the one or more DNA molecules comprise a prokaryotic enhancer. Optionally, the prokaryotic enhancer is the T7 enhancer. Optionally, the T7 enhancer has the sequence GAGAGACAGG (SEQ ID NO: 22). In another aspect of this embodiment, the one or more DNA molecules comprise a prokaryotic terminator.

In another aspect of the invention, the one or more DNA molecules are linked to one or more selectable markers. In one aspect of this embodiment, the selectable marker is an amber suppressor containing one or more mutations or Diaminopimelic Acid (DAP) containing one or more mutations. Optionally, the bait gene is selected from, but not limited to, dapA and dapE.

Preferably, the sequence of the selectable marker is listed in table 3.

TABLE 3

Optionally, the amber suppressor is linked to a promoter or terminator. Optionally, the promoter is a lipoprotein promoter. Preferably, the promoter sequences are listed in table 4.

TABLE 4

Optionally, the terminator is an rrnC terminator. Preferably, the terminator sequences are listed in table 5.

TABLE 5

Terminator sequences of amber suppressor	Sequence of	SEQ ID NO：
			rrnC terminator	GATCCTTAGCGAAAGCTAAGGATTTTTTTTAC	30
rrnC terminator	GATCCTTAGCGAAAGCTAAGGATTTTTTTTTT	31

Bacterial and BTP delivery is more attractive than viral delivery because bacterial and BTP are more amenable to genetic manipulation to produce vector strains specifically tailored for specific applications. In one embodiment of the invention, the methods of the invention are used to produce bacteria and BTP that cause RNAi in a tissue-specific manner.

The siRNA-encoding plasmid or siNRA or sinras are released from the intracellular bacteria or BTP by an activation mechanism. One mechanism involves the type III export system in salmonella typhimurium, a specialized polyprotein complex spanning the membrane of bacterial or BTP cells whose function includes secretion of virulence factors extracellularly to allow signaling to target cells, but can also be used to deliver antigen to target cells by bacterial lysis and release of bacterial or BTP contents into the cytoplasm (russmann h. int J MedMicrobiol, 293: 107-12 (2003)). The lysis of intracellular bacteria or BTPs is triggered by a variety of mechanisms including the addition of an intracellular active antibiotic (tetracycline), naturally attenuated by bacterial metabolism (auxotrophy), or by a lytic regulatory system or bacterial suicide system comprising bacterial regulators, promoters and sensor proteins sensitive to the environment (e.g., pH, magnesium concentration, phosphate concentration, ferric ion concentration, osmolality, anaerobic conditions, nutritional deficiencies, and substantial stress of the target cell or host phagosome). When the bacterial or BTP lytic modulation system detects one or more of the above environmental conditions, bacterial or BTP lysis is triggered by one or more mechanisms, including but not limited to antimicrobial proteins, bacteriophage lysins, and autolysins naturally expressed by the bacteria or BTP or by expression of pore-forming proteins naturally expressed by the bacteria or BTP or by modification (e.g., genetic modification), which disrupt phagosomes containing bacteria or BTP and release plasmids encoding sirnas or one or more sirnas.

The regulators of the lytic regulatory system may be selected from the group including, but not limited to, OmpR, ArcA, PhoP, PhoB, Fur, RstA, EvgA and Rpos. Preferably, the sequence of the cleavage regulon is listed in table 6.

TABLE 6

The promoter of the lytic regulatory system may be selected from the group including, but not limited to, ompF, ompC, fadB, phoPQ, mgtA, mgrB, psiB, phnD, Ptrp, sodA, sodB, sltA, sltB, asr, csgD, emrKY, yhiUV, acrAB, mdfA and tolC. Preferably, the promoter sequences for the lytic regulatory system are listed in table 7.

TABLE 7

The sensor protein of the lytic regulatory system may be selected from the group including, but not limited to, EnvZ, ArcB, PhoQ, PhoR, RstB and EvgS. Preferably, the sequence of the sensor protein of the lytic regulatory system is listed in Table 8.

TABLE 8

The lytic modulation system may comprise any combination of one or more of the above modulators, promoters, and sensor proteins.

In one example of this embodiment, the lytic regulatory system comprises OmpR as a regulator, ompF as a promoter and EnvZ as a sensor protein, and the stimulus is a reduced osmolality. In another example of this embodiment, the lytic regulatory system comprises OmpR as a regulator, ompC as a promoter and EnvZ as a sensor protein, and the stimulus is a reduced osmolality.

In another example of this embodiment, the lytic regulatory system comprises ArcA as a regulator, fad as a promoter, and Arc B as a sensor protein, and the stimulus is anaerobic conditions.

In another example of this embodiment, the lytic regulatory system comprises PhoP as a regulator, phoPQ as a promoter, and PhoQ as a sensor protein, and the stimulus is a reduced magnesium concentration. In another example of this embodiment, the lytic regulatory system comprises PhoP as a regulator, mgtA as a promoter, and PhoQ as a sensor protein, and the stimulus is a reduced magnesium concentration. In another example of this embodiment, the lytic regulatory system comprises PhoP as a regulator, mgrB as a promoter, and PhoQ as a sensor protein, and the stimulus is a reduced magnesium concentration.

In another example of this embodiment, the lytic regulatory system comprises PhoB as a regulator, psiB as a promoter, and PhoR as a sensor protein, and the stimulus is a reduced phosphate concentration. In another example of this embodiment, the lytic regulatory system comprises PhoB as a regulator, phnD as a promoter, and PhoR as a sensor protein, and the stimulus is a reduced phosphate concentration. In another example of this embodiment, the lytic regulatory system comprises RstA as a regulator, asr as a promoter, and RstB as a sensor protein. In another example of this embodiment, the lytic regulatory system comprises RstA as a regulator, csgD as a promoter, and RstB as a sensor protein.

In another example of this embodiment, the lytic modulation system comprises EvgA as a regulator, emryk as a promoter, and EvgS as a sensor protein. In another example of this embodiment, the lytic regulatory system comprises EvgA as a regulator, yhiUV as a promoter, and EvgS as a sensor protein. In another example of this embodiment, the lytic regulatory system comprises EvgA as a regulator, acrAB as a promoter, and EvgS as a sensor protein. In another example of this embodiment, the lytic regulatory system comprises EvgA as a regulator, mdfA as a promoter, and EvgS as a sensor protein. In another example of this embodiment, the lytic regulatory system comprises EvgA as a regulator, tolC as a promoter, and EvgS as a sensor protein.

In another example of this embodiment, the lytic regulatory system comprises Fur as a regulator and a promoter selected from the group comprising sodA, sodB, sltA or sltB.

The antimicrobial protein may be selected from the group including, but not limited to, alpha-and beta-defensins, protegrins, cathelicidins (such as indolicidin and bactenecin), granulysin, lysozyme, lactoferrin, azuridin (azurocidin), elastase, bacterial permeability-inducing peptide (BPI), adrenomedullin, brevin, histatin (histatin) and hepcidin. Other antimicrobial proteins are disclosed in the following documents, all of which are incorporated herein by reference in their entirety: devine, d.a.et., Current pharmaceutical design, 8, 703-; jack R.W., et al, Microbiological Reviews, 59(2), 171-.

Optionally, the antimicrobial protein is alpha-defensin, beta-defensin or protegrin. Preferably, the sequence of the antimicrobial protein is listed in table 9.

TABLE 9

The phage cytolysin may be selected from the group including, but not limited to, perforin and endolysins or cytolysins (e.g. lysozyme, amidase and transglycosylase). Other cytolysins are disclosed in the following documents, all of which are incorporated herein by reference in their entirety: kloos d. -u., et al., Journal of bacteriology, 176(23), 7352-7361(December 1994); jain V, et al, Infection and simulation, 68(2), 986-989(February 2000); srividhya k.v., et al, j.biosci, 32, 979-; young R.V., Microbiological Reviews, 56(3), 430-481(September 1992).

The autolysin may be selected from the group including, but not limited to, peptidoglycan hydrolases, amidases (e.g. N-acetylmuramyl-L-alanine amidase), transglycosylases, endopeptidase and glucosaminidase. Other autolysins are disclosed in the following documents, all of which are incorporated herein by reference in their entirety: heidrich C., et al, Molecular Microbiology, 41(1), 167-; kitano K., et, Journal of Bacteriology, 167(3), 759-765(September 1986); lommatzsch J., et al, Journal of Bacteriology, 179(17), 5465-5470(September 1997); oshida t., et al, PNAS, 92, 285-; lenz l.l., et al, PNAS, 100(21), 12432-; ramadurai L., et al, Journal of Bacteriology, 179(11), 3625-; kraft A.R., et al, Journal of Bacteriology, 180(12), 3441-; dijkstra a.j., et al., FEBS Letters, 366, 115-; huard C., et al, Microbiology, 149, 695-.

In one aspect of the invention, the control exerted by the lytic regulatory system can be further enhanced by bacterial or BTP strain-specific regulation. In one aspect of this embodiment, the strain-specific modulation is attenuation by deletion of a trophic gene. The trophic genes may be selected from the group including, but not limited to, dapA, aroA, and guaBA. In one example of this embodiment, dapA attenuation causes a defect in the biosynthesis of lysine and peptidoglycan. In this particular embodiment, transcription of genes including, but not limited to, lysC may be activated by mechanisms such as transcription induction, anti-termination, and riboswitch (riboswitch). In another example of this embodiment, attenuation of aroA results in a defect in aromatic amino acids and suppression of one or more genes including, but not limited to, aroF, aroG, and aroH by regulators such as TrpR and TyrR. In another example of this embodiment, the guaBA attenuation results in the inhibition of one or more genes inhibited by PurR.

In addition to lytic regulation systems and strain-specific regulation, the bacteria or BTP may further comprise an inducible system including, but not limited to, the Tet-on expression system to facilitate lysis of the bacteria or BTP at a time desired by the clinician. Following administration of tetracycline, which activates the Tet-on promoter, the bacteria or BTP express proteins that trigger bacterial or BTP cleavage. In one example of this embodiment, the protein expressed under the Tet-on expression system is selected from the group including, but not limited to, defensins and protegrins.

The invention also provides a lytic modulation system in combination with strain-specific attenuation (e.g., nutritional attenuation). As shown in PCT publication No. WO2008/156702 in fig. 30, unlike laboratory cultures in which there is an excess of nutrients and either positive or negative regulators that respond to starvation, global regulators can sense the extracellular environment and regulate transcription and starvation of specific nutrients (e.g., amino acids) in vivo. In the schematic shown in PCT publication No. WO2008/156702, fig. 31, there are three cassettes, any of which can be placed on the bacterial chromosome or on a plasmid.

As described, the present invention provides plasmids comprising a lytic regulatory system comprising OmpR as regulator, ompF or ompC as promoter and protegrin or β -defensin as antimicrobial protein and a Tet-on expression system providing two levels of control over bacterial lysis. This embodiment is illustrated in figure 32 of PCT publication No. WO 2008/156702.

In another aspect of the invention, the DNA insert comprises one or more constructs, as shown in table 10, each comprising an HPV target sequence, a hairpin sequence, and a BamH1 and Sal1 restriction site for ease of introduction into the hairpin RNA expression cassette of the TRIP plasmid.

Watch 10

4. Cellular and gene targets

The invention also provides methods of using the various bacteria, BTPs and vectors provided herein. For example, the invention provides methods of delivering one or more siRNAs to a mammalian cell. The method comprises introducing into a mammalian cell at least one invasive bacterium or at least one Bacterial Therapeutic Particle (BTP) comprising one or more sirnas or one or more DNA molecules encoding one or more sirnas.

The invention also provides methods of modulating gene expression in mammalian cells. The method comprises introducing into a mammalian cell at least one invasive bacterium or at least one Bacterial Therapeutic Particle (BTP) comprising one or more sirnas or one or more DNA molecules encoding one or more sirnas, wherein the expressed sirnas interfere with at least one mRNA of a target gene and thereby modulate gene expression.

The present invention provides methods for delivering RNA to any type of target cell. The term "target cell" as used herein refers to a cell that can be invaded by bacteria, i.e., a cell that has a surface receptor that is necessary for bacterial recognition.

Preferred target cells are eukaryotic cells. More preferably the target cell is an animal cell. An "animal cell" is defined as a nucleated and chloroplast-free cell derived from or present in a multicellular organism that is taxonomically located within the kingdom animalia. The cells may be present in whole animals, primary cell cultures, explant cultures, or transformed cell lines. The particular tissue origin of the cells is not important to the present invention.

The recipient animal cells used in the present invention are not critical to the present invention and include cells that are present in or derived from all organisms within the kingdom animalia (e.g., those of the class mammalia, piscidae, birds, reptiles).

Preferred animal cells are mammalian cells, such as human, bovine, ovine, porcine, feline, canine, caprine, equine and primate cells. Most preferably the mammalian cell is a human cell. The cells may be in vivo, in vitro or ex vivo.

In some embodiments of the invention, the cell is a cervical, rectal or pharyngeal epithelial cell, a macrophage, a gastrointestinal epithelial cell, a skin cell, a melanocyte, a keratinocyte, a hair follicle, a colon cancer cell, an ovarian cancer cell, a bladder cancer cell, a pharyngeal cancer cell, a rectal cancer cell, a prostate cancer cell, a breast cancer cell, a lung cancer cell, a renal cancer cell, a pancreatic cancer cell, a liver cell, a hepatocellular carcinoma (HCC) cell, a neural cell, or a hematopoietic cell such as a lymphoma cell or a leukocyte. In one aspect of this embodiment, the colon cancer cells are SW480 cells. In another aspect of this embodiment, the pancreatic cancer cells are CAPAN-1 cells.

In a preferred embodiment, the target cell is within a mucosal surface. Certain enteropathogens, such as escherichia coli, shigella, listeria and salmonella, are naturally adapted for this application because of the ability of these organisms to bind to and invade host mucosal surfaces (Kreig et al, supra). Thus, in the present invention, such bacteria can deliver RNA molecules or DNA encoding RNA into cells within the mucosal compartment of the host.

While certain types of bacteria may have a certain tropism, i.e., target cells are preferred, delivery of RNA or DNA molecules encoding RNA to a particular type of cell may be achieved by selecting bacteria that are tropic for the desired cell type, or modifying bacteria to be able to invade the desired cell type. Thus, as discussed above, for example, bacteria can be genetically engineered to mimic mucosal tissue tropism and invasion properties, thereby enabling the bacteria to invade mucosal tissues and deliver RNA or DNA encoding RNA into cells at those sites.

Bacteria can also be targeted to other types of cells. For example, bacteria can be targeted to human and primate erythrocytes by modifying them to express on their surface either or both of Plasmodium vivax reticulocyte binding proteins-1 and-2 that specifically bind to human and primate erythrocytes (Galinski et al cell, 69: 1213-. In another embodiment, the bacteria are modified to have on their surface non-salivary orosomucoid (a ligand for asialoglycoprotein receptor on hepatocytes) (Wu et al.J.biol.chem., 263: 14621-14624 (1988)). In another embodiment, the bacteria are coated with insulin-poly-L-lysine, which has been shown to cause uptake of plasmids by cells with insulin receptors (Rosenkranz et al expt. cell Res., 199: 323-329 (1992)). Also included within the scope of the invention are bacteria modified to have on their surface a Listeria monocytogenes p60 that gives rise to tropism for hepatocytes (Heass et al. infection. Immun., 63: 2047-.

In another embodiment, the cell may be modified to be a target cell for a bacterium for delivering RNA. Thus, cells can be modified to express surface antigens, i.e., receptors for invasion factors, that are recognized by bacteria for entry into the cell. The cells may be modified by introducing into the cells a nucleic acid encoding an invasion factor receptor, thereby allowing expression of the surface antigen under desired conditions. Alternatively, the cells may be coated with an invasion factor receptor. The invasion factor receptors include proteins belonging to the integrin receptor superfamily. A list of integrin receptor types recognized by a variety of bacteria and other microorganisms can be found, for example, in Isberg and Tran Van Nhieu (1994) ann.rev.genet.27: 395. the nucleotide sequence of the integrin subunit can be found, for example, in GenBank, which is publicly available on the Internet.

As set forth above, other target cells include fish, avian, and reptile cells. Examples of bacteria that are naturally invasive to fish, avian and reptile cells are listed below.

Examples of bacteria that are naturally capable of entering the cytoplasm of fish cells include, but are not limited to, Aeromonas salmonicida (ATCC No.33658) and Aeromonas schuberii (ATCCNO.43700). Attenuated bacteria are preferably used in the present invention, and include Aeromonas salmonicida vapA (Gustafson et al.J.mol.biol., 237: 452-.

Examples of bacteria that naturally enter the cytoplasm of avian cells include, but are not limited to, Salmonella gallinarum (ATCC No.9184), Salmonella enteritidis (ATCC No.4931), and Salmonella typhimurium (ATCC No. 6994). Attenuated bacteria are preferred in the present invention and include attenuated Salmonella strains, such as the Salmonella gallinarum cya crp mutant (Curtiss et al (1987) supra) or the Salmonella enteritidis aroA aromatic-dependent mutant CVL30(Cooper et al Infect. Immun., 62: 4739-4746 (1994)).

Examples of bacteria that are naturally capable of entering the cytoplasm of a reptile cell include, but are not limited to, Salmonella typhimurium (ATCC No. 6994). Attenuated bacteria are preferred in the present invention and include attenuated strains, such as aromatic-dependent mutants of Salmonella typhimurium (Hormeche et al, supra).

The invention also provides for the delivery of RNA to other eukaryotic cells, such as plant cells, provided that a microorganism is present that is capable of attacking (either naturally capable of attacking or modified to be invasive) such cells. Examples of microorganisms that can attack plant cells include agrobacterium tomerificum, which uses cilium-like structures that bind to plant cells through specific receptors and then deliver at least some of their contents to the plant cells by a process similar to bacterial conjugation.

Listed below are examples of cell lines into which RNA can be delivered according to the methods of the invention.

Examples of human cell lines include, but are not limited to, ATCC nos. CCL 62, CCL 159, HTB151, HTB 22, CCL2, CRL 1634, CRL 8155, HTB 61, and HTB 104.

Examples of bovine cell lines include ATCC nos. CRL 6021, CRL 1733, CRL 6033, CRL 6023, CCL 44, and CRL 1390.

Examples of sheep cell lines include ATCC nos. CRL 6540, CRL 6538, CRL 6548 and CRL 6546.

Examples of porcine cell lines include ATCC nos. cl 184, CRL 6492 and CRL 1746.

Examples of feline cell lines include CRL 6077, CRL 6113, CRL 6140, CRL 6164, CCL 94, CCL 150, CRL 6075, and CRL 6123.

Examples of buffalo cell lines include CCL 40 and CRL 6072.

Examples of canine cell lines include ATCC nos. CRL 6213, CCL 34, CRL 6202, CRL6225, CRL 6215, CRL 6203, and CRL 6575.

Examples of goat derived cell lines include ATCC No. ccl 73 and ATCC No. crl 6270.

Examples of horse derived cell lines include ATCC nos. ccl 57 and CRL 6583.

Examples of deer cell lines include ATCC Nos. CRL 6193-6196.

Examples of primate-derived cell lines include cell lines from chimpanzees, such as ATCC nos. CRL 6312, CRL 6304, and CRL 1868; monkey cell lines, such as ATCC nos. crl 1576, CCL 26 and CCL 161; chimpanzee cell line ATCC No. CRL 1850; and gorilla cell line atccno.crl 1854.

The invention also provides methods of modulating the expression of one or more genes. Preferably, modulating the expression of one or more genes means reducing or decreasing the expression of said genes and/or reducing or decreasing the activity of said genes and their corresponding gene products.

In one embodiment, the expressed siRNA directs the cellular multienzyme complex RISC (RNA-induced silencing complex) to interact with the mRNA to be modulated. The complex degrades or sequesters the mRNA, thereby causing a reduction or inhibition of gene expression.

In some embodiments, the gene is an animal gene. Preferred animal genes are mammalian genes, such as human, bovine, ovine, porcine, feline, canine, caprine, equine and primate genes. The most preferred mammalian gene is a human cell.

The gene to be modulated may be a viral gene, an anti-inflammatory gene, an obesity gene, or a gene of an autoimmune disease or disorder. In some embodiments, more than one gene may be regulated by a single plasmid or vector.

In a preferred embodiment, the gene may be, but is not limited to, ras, β -catenin, one or more HPV oncogenes, APC, eotaxin-1(CCL11), HER-2, MCP-1(CCL2), MDR-1, MRP-2, FATP4, SGLUT-1, GLUT-2, GLUT-5, apobec-1, MTP, IL-6R, IL-7, IL-12, IL-13Ra-1, IL-18, IL-21R, IL-32 α, the p19 subunit of IL-23, LY6C, p38/JNK MAP kinase, p65/NF- κ B, CCL20(MIP-3 α), Claudin-2, chitinase 3-like 1 enzyme, apoA-IV, MHC type I and MHC type II. In one aspect of this embodiment, the Ras is k-Ras. In another aspect of this embodiment, the HPV oncogene is E6 or E7.

Preferred beta-catenin targeting gene sequences are listed in table 11. The sequences in table 11 are species-spanning targeting sequences because they are capable of silencing human, mouse, rat, canine, and monkey β -catenin genes (CTNNB 1).

TABLE 11

Beta-catenin targeting gene sequences	SEQ ID NO：
		AGCCAATGGCTTGGAATGAGA	55
ATCAGCTGGCCTGGTTTGATA	56
		CTGTGAACTTGCTCAGGACAA	57
AGCAATCAGCTGGCCTGGTTT	58
		CCTCTGTGAACTTGCTCAGGA	59
TTCCGAATGTCTGAGGACAAG	60
		CCAATGGCTTGGAATGAGACT	61
GGTGCTGACTATCCAGTTGAT	62
		CAATCAGCTGGCCTGGTTTGA	63
CACCCTGGTGCTGACTATCCA	64
		CACCACCCTGGTGCTGACTAT	65
TGCTTTATTCTCCCATTGAAA	66
		CTGGTGCTGACTATCCAGTTG	67
TCTGTGCTCTTCGTCATCTGA	68
		TGCCATCTGTGCTCTTCGTCA	69
TGGTGCTGACTATCCAGTTGA	70
		CCTGGTGCTGACTATCCAGTT	71
ACCCTGGTGCTGACTATCCAG	72
		GAGCCTGCCATCTGTGCTCTT	73
CTGGTTTGATACTGACCTGTA	74
		TGGTTTGATACTGACCTGTAA	75
TCGAGGAGTAACAATACAAAT	76
		ACCATGCAGAATACAAATGAT	77
AGGAGTAACAATACAAATGGA	78
		GTCGAGGAGTAACAATACAAA	79
TTGTTGTAACCTGCTGTGATA	80
		GAGTAATGGTGTAGAACACTA	81
AGTAATGGTGTAGAACACTAA	82
		CACACTAACCAAGCTGAGTTT	83
TTTGGTCGAGGAGTAACAATA	84
		TACCATTCCATTGTTTGTGCA	85
TAGGGTAAATCAGTAAGAGGT	86
		CTAACCAAGCTGAGTTTCCTA	87
TGGTCGAGGAGTAACAATACA	88
		CTGGCCTGGTTTGATACTGAC	89
TAACCTCACTTGCAATAATTA	90
		ATCCCACTGGCCTCTGATAAA	91
GACCACAAGCAGAGTGCTGAA	92
		CACAAGCAGAGTGCTGAAGGT	93
CTAACCTCACTTGCAATAATT	94
		AGCTGATATTGATGGACAG	95

Preferred HPV target gene sequences are listed in table 12. The sequences of table 12 are targeting sequences because they are capable of silencing the HPV E6 oncogene.

TABLE 12

HPV targeted gene sequencesColumn(s) of	SEQ ID NO：
		CGGTGCCAGAAACCGTTGAATCC	96
CACTGCAAGACATAGAAATAACC	97
		AGGTGCCTGCGGTGCCAGAAACC	98
GCGGTGCCAGAAACCGTTGAATC	99
		TCACTGCAAGACATAGAAATAAC	100
CCCATGCTGCATGCCATAAATGT	101
		ATGCTGCATGCCATAAATGTATA	102
GTGGTGTATAGAGACAGTATACC	103
		GCGCGCTTTGAGGATCCAACACG	104
CTGCGGTGCCAGAAACCGTTGAA	105
		CCCCATGCTGCATGCCATAAATG	106
ACCCCATGCTGCATGCCATAAAT	107
		AACACTGGGTTATACAATTTATT	108
ACGACGCAGAGAAACACAAGTAT	109
		AAGGTGCCTGCGGTGCCAGAAAC	110
GGTGCCTGCGGTGCCAGAAACCG	111
		CATGCTGCATGCCATAAATGTAT	112
GACGCAGAGAAACACAAGTATAA	113
		TTCACTGCAAGACATAGAAATAA	114
GGTGCCAGAAACCGTTGAATCCA	115
		TGGCGCGCTTTGAGGATCCAACA	116
TGTGGTGTATAGAGACAGTATAC	117
		GTGCCTGCGGTGCCAGAAACCGT	118
CTGCATGCCATAAATGTATAGAT	119
		GACTCCAACGACGCAGAGAAACA	120
CTGGGCACTATAGAGGCCAGTGC	121
		TGCTGCATGCCATAAATGTATAG	122
GTGCCAGAAACCGTTGAATCCAG	123
		TTACAGAGGTATTTGAATTTGCA	124
GAGGCCAGTGCCATTCGTGCTGC	125

Other preferred HPV target gene sequences are listed in table 13. The sequences of table 13 are targeting sequences because they are capable of silencing the HPV E7 oncogene.

Watch 13

HPV targeted gene sequences	SEQ ID NO：
		ATTCCGGTTGACCTTCTATGTCA	126
GATGGAGTTAATCATCAACATTT	127
		AAGCCAGAATTGAGCTAGTAGTA	128
CATGGACCTAAGGCAACATTGCA	129
		AACCACAACGTCACACAATGTTG	130
ATGGACCTAAGGCAACATTGCAA	131
		TAAGCGACTCAGAGGAAGAAAAC	132
GAAGCCAGAATTGAGCTAGTAGT	133
		GAGCCGAACCACAACGTCACACA	134
ACGTCACACAATGTTGTGTATGT	135
		GAACCACAACGTCACACAATGTT	136
AGGCAACATTGCAAGACATTGTA	137
		AAGACATTGTATTGCATTTAGAG	138
TAAGGCAACATTGCAAGACATTG	139
		CCAGCCCGACGAGCCGAACCACA	140
AAGCTCAGCAGACGACCTTCGAG	141
		GCCCGACGAGCCGAACCACAACG	142
TTCCGGTTGACCTTCTATGTCAC	143
		TGCATGGACCTAAGGCAACATTG	144
TTCCAGCAGCTGTTTCTGAACAC	145
		AACACCCTGTCCTTTGTGTGTCC	146
CTTCTATGTCACGAGCAATTAAG	147
		ACGAGCCGAACCACAACGTCACA	148
TTGAGCTAGTAGTAGAAAGCTCA	149
		CAGCAGACGACCTTCGAGCATTC	150
AGCCAGAATTGAGCTAGTAGTAG	151
		GTCACACAATGTTGTGTATGTGT	152
CCGACGAGCCGAACCACAACGTC	153
		AATTCCGGTTGACCTTCTATGTC	154
ATTCCAGCAGCTGTTTCTGAACA	155

Other preferred HPV-targeting gene sequences are listed in table 14. The sequences of table 14 are targeting sequences common to HPV E6 and E6.

TABLE 14

HPV targeted gene sequences	SEQ ID NO：
		TAGGTATTTGAATTTGCAT	156
GAGGTATTTGAATTTGCAT	157

Preferred MDR-1 targeting gene sequences are listed in Table 15. The sequences of Table 15 are capable of silencing the human MDR-1 gene.

Watch 15

MDR-1 targeting gene sequence	SEQ ID NO：
		ATGTTGTCTGGACAAGCACT	158

Preferred k-Ras-targeting gene sequences are listed in Table 16. The sequences of Table 16 are capable of silencing the human k-Ras gene.

TABLE 16

k-Ras targeted gene sequence	SEQ ID NO：
		GTTGGAGCTGTTGGCGTAG	159

Preferably, the IL-6R targeting gene sequences are listed in Table 17. The sequences of Table 17 are capable of silencing the human IL-6R gene.

TABLE 17

IL-6R targeting gene sequences	SEQ ID NO：
		CTCCTGGAACTCATCTTTCTA	160
GCTCTCCTGCTTCCGGAAGAG	161
		CTCCACGACTCTGGAAACTAT	162
CAGAAGTTCTCCTGCCAGTTA	163
		CCGGAAGACAATGCCACTGTT	164
CTGAACGGTCAAAGACATTCA	165
		CACAACATGGATGGTCAAGGA	166
ATGCAGGCACTTACTACTAAT	167
		ATCGGGCTGAACGGTCAAAGA	168
AGCTCTCCTGCTTCCGGAAGA	169
		CAGCTCTCCTGCTTCCGGAAG	170
CAGGCACTTACTACTAATAAA	171
		CACTTGCTGGTGGATGTTCCC	172
AACGGTCAAAGACATTCACAA	173
		TGCACAAGCTGCACCCTCAGG	174

Other reference IL-6R targeting gene sequences are listed in Table 18. The sequences of Table 18 are capable of silencing the IL-6R gene of mice.

Watch 18

IL-6R targeting gene sequences	SEQ ID NO：
		ATCCTGGAGGGTGACAAAGTA	175
TGGGTCTGACAATACCGTAAA	176
		AACGAAGCGTTTCACAGCTTA	177
CCGCTGTTTCCTATAACAGAA	178
		ACGAAGCGTTTCACAGCTTAA	179
CTGCTGTGAAAGGGAAATTTA	180
		AACCTTGTGGTATCAGCCATA	181
CACAGTGTGGTGCTTAGATTA	182
		CAGCTTCGATACCGACCTGTA	183
CAGTGTGGTGCTTAGATTAAA	184
		CCCGGCAGGAATCCTCTGGAA	185
CCCGCTGTTTCCTATAACAGA	186
		AACCACGAGGATCAGTACGAA	187
ACCTGCCGTCTTACTGAACTA	188
		ACCACGAGGATCAGTACGAAA	189
ACAGCTTGTGATGACTGAATA	190
		AGGATCAGTACGAAAGTTCTA	191
AACCCGCTGTTTCCTATAACA	192
		CAGTACGAAAGTTCTACAGAA	193
TACGCGAGTGACAATTTCTCA	194
		ACGAAAGTTCTACAGAAGCAA	195
CAGGCACTTACTACTAATAAA	196
		CACTTGCTGGTGGATGTTCCC	197
AACGGTCAAAGACATTCACAA	198
		TGCACAAGCTGCACCCTCAGG	199

Preferred IL-7 targeting gene sequences are listed in Table 19. The sequences of Table 19 are capable of silencing the human IL-7 gene.

Watch 19

IL-7 targeting gene sequences	SEQ ID NO：
		TAAGAGAGTCATAAACCTTAA	200
AACAAGGTCCAAGATACCTAA	201
		AAGATTGAACCTGCAGACCAA	202
AAGAGATTTCAAGAGATTTAA	203
		AAGCGCAAAGTAGAAACTGAA	204
TAGCATCATCTGATTGTGATA	205
		TAAGATAATAATATATGTTTA	206
ATGGTCAGCATCGATCAATTA	207
		TTGCCTGAATAATGAATTTAA	208
ATCTGTGATGCTAATAAGGAA	209
		AACAAACTATTTCTTATATAT	210
AACATTTATCAATCAGTATAA	211
		ATCAATCAGTATAATTCTGTA	212
AAGGTATCAGTTGCAATAATA	213

Other preferred IL-7 targeting gene sequences are listed in Table 20. The sequences of Table 20 are capable of silencing the IL-7 gene of mice.

Watch 20

IL-7 targeting gene sequences	SEQ ID NO：
		CGGATCCTACGGAAGTTATGG	214
GACCATGTTCCATGTTTCTTT	215
		AACCTAAATGACCTTTATTAA	216
CAGGAGACTAGGACCCTATAA	217
		TAGGGTCTTATTCGTATCTAA	218
ATGAGCCAATATGCTTAATTA	219
		GCCAATATGCTTAATTAGAAA	220
CAGCATCGATGAATTGGACAA	221
		TTGCCTGAATAATGAATTTAA	222
CTGATAGTAATTGCCCGAATA	223
		AAGGGTTTGCTTGTACTGAAT	224
AACATGTATGTGATGATACAA	225
		TTGCAACATGTAATAATTTAA	226
AAGAGACTACTGAGAGAAATA	227
		AAGAATCTACTGGTTCATATA	228
TGCCGTCAGCATATACATATA	229
		AGGGCTCACGGTGATGGATAA	230

Other preferred IL-7 targeting gene sequences are listed in Table 21. The sequences of table 21 are cross-species sequences because they are capable of silencing human and mouse IL-7 genes.

TABLE 21

IL-7 targeting gene sequences	SEQ ID NO：
		CGCCTCCCGCAGACCATGTTC	231
TCCGTGCTGCTCGCAAGTTGA	232
		GCCTCCCGCAGACCATGTTCC	233
CCTCCCGCAGACCATGTTCCA	234
		CTCCCGCAGACCATGTTCCAT	235
TCCCGCAGACCATGTTCCATG	236
		CCCGCAGACCATGTTCCATGT	237
CCGCAGACCATGTTCCATGTT	238
		CGCAGACCATGTTCCATGTTT	239
GCAGACCATGTTCCATGTTTC	240
		CAGACCATGTTCCATGTTTCT	241
AGACCATGTTCCATGTTTCTT	242

Preferred IL-13Ra-1 targeting gene sequences are listed in Table 22. The sequences of Table 22 are capable of silencing the human IL-13Ra-1 gene.

TABLE 22

IL-13Ra-1 targeting gene sequences	SEQ ID NO：
		AACCTGATCCTCCACATATTA	243
CCTGATCCTCCACATATTAAA	244
		AGAAATGTTTGGAGACCAGAA	245
CAAATAATGGTCAAGGATAAT	246
		TTCCTGATCCTGGCAAGATTT	247
TAAAGAAATGTTTGGAGACCA	248
		ATGTTTGGAGACCAGAATGAT	249
CTCCAATTCCTGATCCTGGCA	250

Other preferred IL-13Ra-1 targeting gene sequences are listed in Table 23. The sequences of Table 23 are capable of silencing the IL-13Ra-1 gene of mice.

TABLE 23

IL-13Ra-1 targeting gene sequences	SEQ ID NO：
		CAAGAAGACTCTAATGATGTA	251
CACAGTCAGAGTAAGAGTCAA	252
		ACCCAGGGTATCATAGTTCTA	253
CTGCTTTGAAATTTCCAGAAA	254
		ATCATAGTTCTAAGAATGAAA	255
AAGGCTTAAGATCATTATATT	256
		AACTACTTATAAGAAAGTAAA	257
CACAGAACATCTAGCAAACAA	258
		CTCGTTCTTGTTCAATCCTAA	259
AACTTGTAGGTTCACATATTA	260
		AACCATTTCTGCAAATTTAAA	261
CTCAGTGTAGTGCCAATGAAA	262
		CAGGCCTTAGGGACTCATAAA	263
AAGTATGACATCTATGAGAAA	264
		GTGGAGGTCAATAATACTCAA	265
CAGAGTATAGGTAAGGAGCAA	266

Preferred IL-18 targeting gene sequences are listed in Table 24. The sequences of Table 24 are capable of silencing the human IL-18 gene.

Watch 24

IL-18 targeting gene sequences	SEQ ID NO：
		TTGAATGACCAAGTTCTCTTC	267

Other preferred IL-18 targeting gene sequences are listed in Table 25. The sequences of Table 25 are capable of silencing the IL-18 gene of mice.

TABLE 25

IL-18 targeting gene sequences	SEQ ID NO：
		CTCTCTGTGAAGGATAGTAAA	268
CCGCAGTAATACGGAATATAA	269
		CAAGGAAATGATGTTTATTGA	270
CAGACTGATAATATACATGTA	271
		TTGGCCGACTTCACTGTACAA	272
CCAGACCAGACTGATAATATA	273
		AAGATGGAGTTTGAATCTTCA	274
ACGCTTTACTTTATACCTGAA	275
		TACAACCGCAGTAATACGGAA	276
CTGCATGATTTATAGAGTAAA	277
		CCCGAGGCTGCATGATTTATA	278
CACGCTTTACTTTATACCTGA	279
		CGCCTGTATTTCCATAACAGA	280
CGCAGTAATACGGAATATAAA	281
		TACATGTACAAAGACAGTGAA	282
CAGGCCTGACATCTTCTGCAA	283
		TTCGAGGATATGACTGATATT	284
CTGTATTTCCATAACAGAATA	285
		GAGGATATGACTGATATTGAT	286
CAAGTTCTCTTCGTTGACAAA	287
		CACTAACTTACATCAAAGTTA	288
ACCGCAGTAATACGGAATATA	289
		CTCTCACTAACTTACATCAAA	290

Preferred CCL20 targeting gene sequences are listed in table 26. The sequences of table 26 are capable of silencing the human CCL20 gene.

Watch 26

CCL20 targeting gene sequence	SEQ ID NO：
		ATCATCTTTCACACAAAGAAA	291
AACAGACTTGGGTGAAATATA	292
		ATGGAATTGGACATAGCCCAA	293
GAGGGTTTAGTGCTTATCTAA	294
		CTCACTGGACTTGTCCAATTA	295
ATCATAGTTTGCTTTGTTTAA	296
		TTGTTTAAGCATCACATTAAA	297
AAGCATCACATTAAAGTTAAA	298
		CCCAAAGAACTGGGTACTCAA	299
CACATTAAAGTTAAACTGTAT	300
		CAGATCTGTTCTTTGAGCTAA	301
TTGGTTTAGTGCAAAGTATAA	302
		CAGACCGTATTCTTCATCCTA	303
AACATTAATAAGACAAATATT	304
		GACCGTATTCTTCATCCTAAA	305

Other reference CCL20 targeting gene sequences are listed in table 27. The sequences of table 27 were able to silence the CCL20 gene in mice.

Watch 27

CCL20 targeting gene sequence	SEQ ID NO：
		AAGCTTGTGACATTAATGCTA	306
CAATAAGCTATTGTAAAGATA	307
		ATCATCTTTCACACGAAGAAA	308
AGCTATTGTAAAGATATTTAA	309
		CAGCCTAAGAGTCAAGAAGAT	310
CCCAGTGGACTTGTCAATGGA	311
		ATGAAGTTGATTCATATTGCA	312
AAGTTGATTCATATTGCATCA	313
		TCACATTAGAGTTAAGTTGTA	314
CACATTAGAGTTAAGTTGTAT	315
		TATGTTATTTATAGATCTGAA	316
ATGTTTAGCTATTTAATGTTA	317
		TTAGTGGAAGGATTAATATTA	318
ACCCAGCACTGAGTACATCAA	319
		TATGTTTAAGGGAATAGTTTA	320

Other preferred CCL20 targeting gene sequences are listed in table 28. The sequences in table 28 are species-spanning targeting sequences because they are capable of silencing the human and mouse CCL20 genes.

Watch 28

CCL20 targeting gene sequence	SEQ ID NO：
		ATGAAGTTGATTCATATTGCA	321
TGAAGTTGATTCATATTGCAT	322
		GAAGTTGATTCATATTGCATC	323
AAGTTGATTCATATTGCATCA	324
		AGTTGATTCATATTGCATCAT	325
GTTGATTCATATTGCATCATA	326
		TTGATTCATATTGCATCATAG	327
TGATTCATATTGCATCATAGT	328
		TCAATGCTATCATCTTTCACA	329
CAATGCTATCATCTTTCACAC	330
		TAATGAAGTTGATTCATATTG	331
AATGAAGTTGATTCATATTGC	332

Preferred CCL20 targeting gene sequences are listed in table 29. The sequences of table 29 are capable of silencing the human CCL20 gene.

Watch 29

Claudin-2 targeting gene sequence	SEQ ID NO：
		AGCATGAAATTTGAGATTGGA	333
TACAGAGCCTCTGAAAGACCA	334
		CACTACAGAGCCTCTGAAAGA	335
CTGACAGCATGAAATTTGAGA	336
		ATCTCTGTGGTGGGCATGAGA	337
CATGAAATTTGAGATTGGAGA	338
		TCTGGCTGAGGTTGGCTCTTA	339
GTGGGCTACATCCTAGGCCTT	340

Other preferred CCL20 targeting gene sequences are listed in table 30. The sequences of table 30 were able to silence the CCL20 gene in mice.

Watch 30

Claudin-2 targeting gene sequence	SEQ ID NO：
		CAGCTTCCTGCTAAACCACAA	341
CAAGAGTGAGTTCAACTCATA	342
		CTGGTTCCTGACAGCATGAAA	343
TGGCTGGGACTATATATATAA	344
		GAGGGCAATTGCTATATCTTA	345
CAGCAGCCAAACGACAAGCAA	346
		CAAGGGTTTCCTTAAGGACAA	347
CAGATACTTGTAAGGAGGAAA	348
		AAGAAATGGATTAGTCAGTAA	349
AAGGAAAGCACAAGAAGCCAA	350
		CTGGCTGAGGTTGGCTCTTAA	351
AACCTGGGATCTAAAGAAACA	352
		AAGGGCTTGGGTATCAAAGAA	353
CAGGCTCCGAAGATACTTCTA	354
		CCCAATATATAAATTGCCTAA	355
CTGACCCAGCTTCCTGCTAAA	356

Preferred chitinase-3 targeting gene sequences are listed in Table 31. The sequences of table 31 are capable of silencing the human chitinase-3 gene.

Watch 31

Chitinase-3 targeted gene sequences	SEQ ID NO：
		ACCCACATCATCTACAGCTTT	357
CATCATCTACAGCTTTGCCAA	358
		CAGCTGGTCCCAGTACCGGGA	359
CACCAAGGAGGCAGGGACCCT	360
		CCGGTTCACCAAGGAGGCAGG	361
AGCTGGTCCCAGTACCGGGAA	362
		CAGGCCGGTTCACCAAGGAGG	363
GGCCGGTTCACCAAGGAGGCA	364

Other preferred chitinase-3 targeted gene sequences are listed in Table 32. The sequences of table 32 are capable of silencing the chitinase-3 gene of mice.

Watch 32

Chitinase-3 targeted gene sequences	SEQ ID NO：
		TAGGTTTGACAGATACAGCAA	365
AACCCTGTTAAGGAATGCAAA	366
		ATCAAGTAGGCAAATATCTTA	367
CGCAGCTTTGTCAGCAGGAAA	368
		TTGGATCAAGTAGGCAAATAT	369
TTGAGGGACCATACTAATTAT	370
		GAGGACAAGGAGAGTGTCAAA	371
TGCGTACAAGCTGGTCTGCTA	372
		CAGGAGTTTAATCTCTTGCAA	373
ATCAAGGAACTGAATGCGGAA	374
		CACCCTGATCAAGGAACTGAA	375
CACTTGGATCAAGTAGGCAAA	376
		CAGGATTGAGGGACCATACTA	377
AACTATGACAAGCTGAATAAA	378
		ATGCAAATTCTCAGACTCTAA	379
ATCCTTCCCTTAGGAACTTAA	380

5. Treatment of diseases and disorders

The invention also provides methods of treating or preventing a disease or condition in a mammal. The method comprises modulating the expression of at least one gene known to cause a disease or disorder in a cell of a mammal by introducing into the cell at least one invasive bacterium or at least one Bacterial Therapeutic Particle (BTP) comprising one or more sirnas or one or more DNA molecules encoding one or more sirnas, wherein the expressed siRNA interferes with mRNA of a gene known to cause the disease or disorder of interest.

The RNAi methods of the invention, including BMGS and tkRNAi, are useful for treating any disease or disorder for which modulation of gene expression is beneficial. The methods function by silencing or knocking down (lowering) genes involved in one or more diseases and pathologies.

Genes that need to be modulated to treat or prevent a disease or disorder of interest may be, but are not limited to, ras, β -catenin, one or more HPV oncogenes, APC, eotaxin-1(CCL11), HER-2, MCP-1(CCL2), MDR-1, MRP-2, FATP4, SGLUT-1, GLUT-2, GLUT-5, apobec-1, MTP, IL-6R, IL-7, IL-12, IL-13Ra-1, IL-18, IL-21R, IL-32 α, p19 subunit of IL-23, LY6C, p38/JNK MAP kinase, p65/NF- κ B, CCL20(MIP-3 α), Claudin-2, chitinase 3-like 1 enzyme, apoA-IV, MHC type I, and MHC type II. In one aspect of this embodiment, the Ras is k-Ras. In another aspect of this embodiment, the HPV oncogene is E6 or E7.

The present invention provides methods of treating or preventing diseases or disorders associated with overexpression of genes, including, but not limited to, ras, β -catenin, one or more HPV oncogenes, APC, eotaxin-1(CCL11), HER-2, MCP-1(CCL2), MDR-1, MRP-2, FATP4, SGLUT-1, GLUT-2, GLUT-5, apobec-1, MTP, IL-6R, IL-7, IL-12, IL-13Ra-1, IL-18, IL-21R, IL-32 α, p19 subunit of IL-23, LY6C, p38/JNK kinase, p 65/NF-B, CCL20 (3 α), Claudin-2, chitinase 3-like 1 enzyme, apoA-IV, Class I MHC and class II MHC. Preferably, the gene is β -catenin and the disease or disorder to be treated is associated with overexpression of β -catenin. The term "overexpression" as used herein refers to increased expression (DNA, RNA or protein) when compared to normal or wild type expression. Preferably, the disease or condition to be treated is selected from the group consisting of: colon cancer, rectal cancer, colorectal cancer, Crohn's disease, ulcerative colitis, Familial Adenomatous Polyposis (FAP), Gardner syndrome, hepatocellular carcinoma (HCC), basal cell carcinoma, pilomas, medulloblastomas, and ovarian cancer.

Preferably, the present invention provides methods for treating or preventing cancer or cell proliferative disorders, viral diseases, inflammatory diseases or disorders, metabolic diseases or disorders, autoimmune diseases or disorders, or skin or hair related diseases, disorders or cosmetic events in a mammal by introducing bacteria or BTP into the cells to modulate the expression of one or several genes known to be associated with the onset, spread or delay of the disease or disorder. The bacterium or BTP contains one or more sirnas or one or more DNA molecules encoding one or more sirnas, wherein the expressed sirnas interfere with mRNA of a gene known to cause, propagate, or prolong a target disease or disorder.

In some preferred embodiments, the viral disease may be, but is not limited to, hepatitis b, hepatitis c, Human Papilloma Virus (HPV) infection or epithelial dysplasia or cancer caused by HPV infection or HPV-induced transformation, including cervical, rectal and pharyngeal cancer.

In some preferred embodiments, the inflammatory disease or disorder may be, but is not limited to, inflammatory bowel disease, Crohn's disease, ulcerative colitis, allergic reactions, rheumatoid arthritis, or respiratory disease.

In some preferred embodiments, the autoimmune disease or disorder can be, but is not limited to, celiac disease, rheumatoid arthritis, systemic lupus erythematosus, or encephalomyelitis.

In some preferred embodiments, the disease, disorder or cosmetic event may be, but is not limited to, psoriasis, eczema, albinism, alopecia or graying hair.

The mammal may be any mammal including, but not limited to, a human, a bovine, an ovine, a porcine, a feline, a canine, a caprine, a equine, or a primate. Preferably, the mammal is a human.

The term "treating" as used herein refers to administering an agent or formulation (e.g., a bacterium comprising an siRNA or DNA encoding an siRNA and/or BTP) to a clinically symptomatic individual afflicted with an adverse condition, pathology, or disease, thereby reducing the severity and/or frequency of the symptoms, eliminating the symptoms and/or their underlying cause, and/or promoting amelioration or remediation of the injury.

The term "prevention" refers to the administration of an agent or composition to an individual who is predisposed to a particular adverse condition, disorder or disease without clinical symptoms, and thereby prevents the appearance of symptoms and/or their underlying causes.

6. Pharmaceutical compositions and modes of administration

In a preferred embodiment of the invention, the invasive bacteria or BTP comprising an RNA molecule and/or DNA encoding such RNA molecule is introduced into the animal by intravenous, intramuscular, intradermal, intraperitoneal, oral, intranasal, intraocular, intrarectal, intravaginal, intraosseous, oral, maceration and urethral inoculation.

The amount of invasive bacteria or BTP of the invention administered to a subject may vary depending on the species of the subject and the disease or condition being treated. Typically, the dosage employed is about 10 per subject³-10¹¹Living organisms, preferably about 10⁵-10⁹Living organisms.

The invasive bacterium or BTP of the invention is typically administered with a pharmaceutically acceptable carrier and/or diluent. The particular pharmaceutically acceptable carrier and/or diluent employed is not critical to the invention. Examples of diluents include phosphate buffers, buffers which buffer gastric acid in the stomach, such as citrate buffers containing sucrose (pH 7.0), sodium bicarbonate buffers alone (pH 7.0) (Levine et al.J.Clin. Invest., 79: 888-902 (1987); and Black et al J.Infect. Dis., 155: 1260-1265(1987)) or bicarbonate buffers containing ascorbic acid, lactose and optionally aspartic acid (pH 7.0) (Levine et al.Lancet, II: 467-470 (1988)). Examples of carriers include proteins, such as those found in skim milk, sugars, such as sucrose, or polyvinylpyrrolidone. Generally, these carriers are used at a concentration of about 0.1-30% (w/v), but preferably in the range of 1-10% (w/v).

Listed below are other pharmaceutically acceptable carriers or diluents that may be used for a particular delivery route. Any such carrier or diluent can be used to administer the bacteria of the present invention, provided that the bacteria or BTP are still capable of invading the target cells. Invasive in vitro or in vivo tests can be performed to determine the appropriate diluents and carriers. The compositions of the present invention are formulated for a variety of administration types, including systemic administration and topical or local administration. Lyophilized forms may also be included, provided that the bacteria are invasive after contact with the target cells or after administration to the subject. Related techniques and formulations are commonly found in Remmington's Pharmaceutical Sciences, Meade Publishing co., Easton, Pa. For systemic administration, injections are preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous injections. For injection, the compositions of the invention (e.g., bacteria or BTP) can be formulated as liquid solutions, preferably in physiologically compatible buffers (e.g., Hank's solution or Ringer's solution).

For oral administration, the pharmaceutical compositions may be in the form of tablets or capsules prepared, for example, by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethyl cellulose), fillers (e.g., lactose, microcrystalline cellulose or dibasic calcium phosphate), lubricants (e.g., magnesium stearate, talc or silica), disintegrants (e.g., potato starch or sodium starch glycolate), or wetting agents (e.g., sodium lauryl sulfate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared in conventional manner using pharmaceutically acceptable additives such as suspending agents (e.g. sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifiers (e.g., lecithin or gum arabic); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g. methyl or propyl-p-hydroxybenzoate or sorbic acid). The formulations may also contain suitable buffer salts, flavouring agents, colouring agents and sweetening agents.

Formulations for oral administration may be suitably formulated to provide controlled release of the active compound. For oral administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the pharmaceutical compositions used in the present invention may be delivered in aerosol spray packs, from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, metered delivery can be achieved by providing a valve to determine the dosage unit. Powder mixtures comprising the components (e.g. bacteria) and a suitable powder base such as lactose or starch may be formulated for use in gelatin capsules and cartridges in inhalers or inhalers.

The pharmaceutical composition may be formulated for parenteral administration by injection, for example by bolus injection (bolus) or continuous infusion. Formulations for injection may be presented in unit dosage form, for example in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The pharmaceutical compositions may also be formulated as rectal, intravaginal or intraurethral compositions, for example as suppositories or retention enemas containing conventional suppository bases such as cocoa butter or other glycerides.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration can be by nasal spray or by use of suppositories. For topical administration, the bacteria of the present invention may be formulated as an ointment, salve, gel, or cream, as is generally known in the art, provided that the bacteria remain invasive after contact with the target cells.

If desired, the compositions may be packaged in the form of a packaging or dispensing device and/or kit which may contain one or more unit dosage forms containing the active ingredient. The packaging may comprise, for example, a metal or plastic sheet, such as a blister pack (bib). The packaging or dispensing device may be accompanied by instructions for use.

Invasive bacteria or BTPs containing the RNA or DNA encoding the RNA to be introduced can be used to infect animal cells cultured in vitro, e.g., cells obtained from a subject. These in vitro infected cells can then be introduced into an animal, e.g., the subject from which the cells were originally obtained, intravenously, intramuscularly, intradermally, or intracranially, or by any route of inoculation that allows the cells to enter the host tissue. When delivering RNA to a single cell, the dose of live organism administered will have a multiplicity of infection of about 0.1-10 per cell⁶Bacteria, preferably about 10 per cell²-10⁴Bacteria.

In another embodiment of the invention, the bacterium can also deliver an RNA molecule encoding a protein to a cell (e.g., an animal cell), from which the protein can subsequently be harvested or purified. For example, proteins can be produced in tissue culture cells.

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

The invention is further illustrated below by means of examples, which, however, do not limit the invention in any way. The contents of all references, including citations, issued patents, published patent applications, cited throughout this application are expressly incorporated herein by reference. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are fully described in the literature. See, e.g., Molecular Cloning A Laboratory Manual, 2nd Ed., ed.by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (d.n. glover ed., 1985); oligonucletoideosynthesis (m.j.gait ed., 1984); mullis et al.u.s.pat.no: 4,683,195; nucleic Hybridization (B.D. Hames & S.J. Higgins eds. 1984); transcription AndTranslation (b.d. hames & s.j.higgins eds.1984); culture Of Animal Cells (r.i. freshney, Alan r.loss, inc., 1987); immobilized Cells And Enzymes (IRLPress, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the enzyme, Methods In Enzymology (Academic Press, Inc., N.Y.); gene transfer vectors For Mammalian Cells (J.H.Miller and M.P.Calos eds., 1987, Cold spring Harbor Laboratory); methods In Enzymology, Vols.154 And 155(Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer And Walker, eds., Academic Press, London, 1987); handbook Of Experimental immunology, Volumes I-IV (D.M.Weir and C.C.Blackwell, eds., 1986); manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

The following non-limiting examples are intended to illustrate preferred embodiments of the invention only and are not intended to limit the invention.

Examples

Example 1: knock-down of beta-catenin and k-Ras

Previous studies have demonstrated the effectiveness of the siRNA knockdown techniques disclosed herein. For example, PCT publication No. WO 06/066048, which is incorporated herein by reference in its entirety, describes in vitro and in vivo knockdown of β -catenin and k-ras using bacterial delivery.

Example 2: TRIP with multiple shRNA expression cassettes

The TRIPs described herein and described in detail in PCT publication No. WO 06/066048 can all be modified to produce plasmids that can target multiple genes simultaneously or multiple sequences within a gene simultaneously. For example, a TRIP with multiple hairpin expression cassettes that generate shRNA can target different sequences within a given gene or target multiple genes by simultaneous bacterial treatment.

The TRIP plasmid can introduce multiple (up to 10) cloning sites to express different shRNA constructs (as shown in figure 1 of PCT publication No. WO 2008/156702). The purpose of this plasmid is to silence multiple genes by a single therapeutic bacterium, which can be achieved by multiple expression cassette-TRIP (mec-TRIP) to synthesize short hairpin RNAs directed to multiple targets simultaneously.

These different hairpins can compete for expression at high levels by using the same high level promoter (e.g., the T7 promoter or different high level bacterial promoters), or can be expressed at different levels by using promoters with different levels of activity, depending on the intended use of the plasmid and the desired relative level of silencing of the target gene.

This mec-TRIP can be used to treat a complex disease (e.g., inflammatory disease or cancer) as described herein by simultaneously silencing (targeting) multiple targets (e.g., multiple oncogenes, such as k-ras and β -catenin in the case of colon cancer, or HER-2 and MDR-1 in breast cancer, or other combinations).

Example 3: operator-inhibitor titration system

The TRIP system (bacteria and plasmids) has been modified to include the ORT (operon suppressor titration) system from CobraBiomanufacturing (Keele, UK). This modification helps to maintain the plasmid in the appropriate strain in the absence of the selective antibiotic. Thus, the bacterial vector strain has been modified to allow the ORT system to function (deletion of the DAP gene and exchange for the ORT-controlled DAP gene expression system). The plasmid has been modified to remove antibiotic selection sequences to support the ORT system. Other changes have also been introduced into the bacterial genome, including for example (a) deletion of the aroA gene (in some CEQ strains) to make the bacteria more susceptible to auxotrophy, especially within intracellular compartments where it dies due to lack of nutrition; (b) inserting a T7RNA polymerase gene into the chromosome, and/or (c) integrating an shRNA expression cassette under the control of a T7 promoter into the chromosome.

PCT publication No. WO2008/156702 shows a development example of a bacterial strain in fig. 2. Further developed strains include, but are not limited to, CEQ922 (CEQ 919 without aroA deletion), CEQ923 (CEQ 920 without aroA deletion), CEQ924 (CEQ 921 without aroA deletion).

Example 4: intestinal gene delivery

Salmonella typhimurium has been studied to determine whether it can be used as a vector for delivering RNAi to epithelial cells in the gut. By means of 10⁸SL7207 mice were treated orally with a single dose and mice were sacrificed at different time points after dosing. SL7207 was then stained with salmonella specific antibodies. After 2 hours of treatment, many SL7207 intrusions into the intestinal epithelial layer (salmonella stained red) were observed, suggesting that oral SL7207 may be a useful tool for delivering a payload (payload) to the intestinal and colonic mucosa. In subsequent experiments, mice were treated with SL7207 with a GFP expression plasmid (pEGFPC1, Invitrogen). A small proportion (about 1%) of the cells were clearly found to express GFP 24 hours after a single treatment.

PCT publication No. WO2008/156702 shows in fig. 3 an efficient invasion of salmonella typhimurium and plasmid delivery to the intestinal mucosa. SL7207 was stained with red fluorescent antibody 6 hours after oral administration. Intact SL7207 and fragments of SL7207 were observed in epithelial cells as well as in the lower cells of the lamina propria (upper left/right). SL7207 successfully delivered the expressed DNA into the intestinal mucosa: intestinal mucosal cells express GFP after treatment with SL7207 carrying a GFP eukaryotic expression plasmid (pEGFP-C1) (bottom left). For fluorescence microscopy, SL7207 was stained with red fluorescent antibody and nuclei were counterstained with Hoechst 37111.

To test whether SL7207 was able to deliver RNAi targeting a target gene to the gut, shRNA with a targeted GFP was usedGFP transgenic mice (4 per group) were treated with Salmonella typhimurium either expression plasmid (SL-siGFP) or with shRNA expression plasmid targeting k-RAS (SL-siRAS). By oral gavage, 10 were administered three times a week⁸U., 2 weeks total. Subsequently, specific antibodies (LivingColors) are usedInvitrogen) for immunohistochemical staining of GFP expression, colon tissue was observed using a fluorescence microscope (data not shown) and analyzed for staining. Overall GFP expression levels were significantly reduced in SL-siGFP treated animals and the number of GFP-expressing vesicles was also significantly reduced (33.9% vs 50%, p < 0.05) compared to animals treated with SL-siRAS, indicating that this approach can be used to deliver therapeutic RNAi to colonic epithelial cells.

PCT publication No. WO2008/156702 in fig. 4 shows that bacteria-mediated RNA interference reduces expression of target genes in gastrointestinal epithelial cells. Colon tissues show lower levels of GFP expression and fewer colon vesicles staining positive for GFP after treatment with SL7207 carrying a GFP-targeting expression plasmid (SL-siGFP, bottom right) compared to animals treated with SL-siRAS (bottom left). Slides were stained with GFP-specific antibodies.

Example 5: construction of CEQ503 bacterial strains

Derivation and description of CEQ503 (Strain CEQ201(pNJSZ))

CEQ503 consists of a combination of an attenuated escherichia coli strain (CEQ201) and a specially engineered TRIP plasmid (pNJSZ). The plasmid confers the ability required to induce tkRNAi (in this case: invasiveness, escape from entering vesicles, expression of short hairpin RNAs). Strain description of CEQ503 (pNJSZ):

1. genotype: escherichia coli CEQ201[ glnV44(AS), LAM^-，rfbC1，endA1，spoT1，thi-1，hsdR17，(r_k ^-m_k ⁺)，creC510ΔdapA，ΔrecA]。

Derivatization of CEQ201

MM294(Meselson and Yuan, Nature 217, 1110, 1968; from CGSC)

↓ transformed with plasmid pKD46

MM294(pKD46)

↓.

MM294ΔdapA::kan(pKD46)

↓.

MM294ΔdapA::kanΔrecA::cat(pKD46)

↓ remaining (cured) plasmid pKD46 by culturing cells at 43 ℃

MM294ΔdapA::kanΔrecA::cat

Plasmid pCP20 for ↓¹Transformation of

MM294ΔdapA::kanΔrecA::cat(pCP20)

I Retention plasmid pCP20 and treatment by inducing FLP recombinase at 43 ℃

↓ and deleting kan gene from chromosome

MM294ΔdapAΔrecA(CEQ201)

3. Plasmid: pNJSZ, illustrated in figure 5 of PCT publication No. WO2008/156702, is a 10.4kb plasmid that confers resistance to kanamycin on the bacterial strain of the present invention (CEQ 503). This plasmid contains two genes hly and inv and the hairpin sequence of H3 is:

ggatccAGGAGTAACAATACAAATGGATTCAAGAGATCCATTTGTATTGTTACTCCTTTgtcgac (SEQ ID NO: 383) that contains BamHI and SalI restriction sites. To demonstrate the presence of this plasmid, PCR was performed to demonstrate chromosomal deletion of dapA, and miniprep and/or PCR was performed to confirm inv, hly, and 341-H3 on the plasmid.

4. The nutrient conditions are as follows: altha Media Broth or LB, Miller (Luria-Bertani) Broth (Amresco; cat. No.: J106-2KG) and 50. mu.g/ml DL-. DELTA.; ε -Diaminopimelic Acid (DAP) (SIGMA; cat. No.: D1377-10G).

5. The culture conditions are as follows: 37 ℃ is carried out.

Example 6: production of BTP

BTP or minicells containing suitable plasmids such as TRIP have been engineered for delivery of tkRNAi. These cells may express invasin or Opa to enable entry into mammalian cells, and listeriolysin may lyse the phagosome after degradation/lysis of the minicells. In addition, methods of making minicells have been developed that utilize suicide constructs to kill intact cells to aid in the purification of minicells. Such suicide plasmids have been described in the literature (Kloos et al, (1994) J.bacteriol.176, 7352-61; Jain and Mekalanos, (2000) infection. Immun.68, 986-. In summary, the lambda S and R genes encoding pore forming and lysozyme were placed under the regulation of inducible promoters on bacterial chromosomes. When induced, the minicells will lyse intact cells rather than minicells due to their lack of chromosomes. Many different types of regulators may be used, such as lacI, araC,. lambda.cI 857 and rhaS-rhaR, for the development of inducible suicide gene constructs. Similarly, a number of different types of suicide genes, including escherichia coli self-cleaving gene and antimicrobial small peptides, can be used in a similar protocol. Purification is enhanced by treatment or mutation that induces filamentation (filamentation) (see, e.g., Ward and Lutkenhaus, (1985) Cell 42, 941-949; Bi and Lutkenhaus, 1992). Initial purification included low speed centrifugation to separate intact cells and retain the mini-cells in the supernatant. This may be followed by density gradient purification or filtration (e.g., Shull et al, (1971) J.Bacteriol.106, 626-.

Any cell death trigger gene (also known as a suicide gene, including but not limited to genes encoding an antimicrobial protein, a bacteriophage lysin, or an autolysin) can be used in the present methods for obtaining BTP from a mixture comprising BTP and bacteria. Suicide genes can kill living bacteria by mechanisms including, but not limited to, cell lysis or by destroying, degrading, or poisoning cellular components (e.g., chromosomal DNA or silk components). Any inducible promoter can be used in conjunction with the system. In one embodiment of the invention, the suicide genes are integrated into the chromosome, thereby limiting their presence only in intact bacteria, whereas BTP or minicells would not introduce these genes due to the absence of chromosomal DNA.

As shown in figure 6 of PCT publication No. WO2008/156702, induction of the suicide gene will lyse the intact bacterial cells. Place the S and R genes (suicide genes) in P_lacUV5(inducible promoter) under control. Missing basal activity quilt P_gapAlacI on (Strong promoter)^qGene-encoded "super-repressor" repression. The cassette is placed at the minCD locus.

Example 7: siRNA inhibition of Human Papilloma Virus (HPV) oncogene

Cell culture: hela cells were cultured in Minimum Essential Medium (MEM, ATCCNO.30-2003) supplemented with antibiotic 100U/ml penicillin G, 10. mu.g/ml streptomycin (Sigma) and containing 10% FBS.

And (3) bacterial culture: the plasmid was transformed into BL21(DE3) strain (Invitrogen). The bacteria were cultured at 37 ℃ in LB medium containing 100. mu.g/ml ampicillin. Using OD₆₀₀The assay calculates bacterial cell density (CFU/ml). For cell infection, overnight cultures were inoculated into fresh medium for an additional 2-3 hours until at 600nm [ OD600 ]]The optical density of (2) is up to 0.6.

Invasion test: for bacterial invasion, Hela cells were plated at 200,000 cells/well in 6-well plates and cultured in 2ml of complete mediumOvernight. The bacterial cells were cultured to a medium exponential phase (optical density at 600nm [ OD600 ] in LB medium with ampicillin]0.6), and then centrifuged at 3,400rpm at 4 ℃ for 10 minutes. The bacterial pellet was resuspended in serum-or antibiotic-free MEM and the bacteria were added to the cells at an MOI of 1: 1000, 1: 500, 1: 250, 1: 125, or 1: 62.5 and allowed to stand at 37 ℃ with 5% CO₂Hela cells were challenged for 2 hours under conditions. Cells were washed 4 times with MEM containing 10% FBS and penicillin-streptomycin (100 IU penicillin and 100 μ g streptomycin per ml). Cells were incubated at 37 ℃ with 5% CO₂Further incubation for 48 hours in fresh complete medium, followed by isolation of total RNA by Qiagen RNeasy system using DNAse digestion on column or by TRIZOL extraction method.

siRNA transfection: one day prior to transfection, cells were plated in complete growth medium without antibiotics to achieve 30-50% confluence at the time of transfection. In 175. mu.l of Opti-MEM, various concentrations of siRNA were diluted from 20. mu.M stock. Mu.l of Oligofectamine was mixed into 15. mu.l of Opti-MEM, respectively. Mix gently and incubate at room temperature for 5-10 minutes. The diluted siRNA was mixed with diluted oligofectamine and incubated for 15-20 minutes at room temperature. In forming the complexes, the growth medium was removed from the cells and 800 μ l of serum-free medium was added to each well containing cells. To the cells were added 200. mu.l siRNA/oligofectamine complex and incubated at 37 ℃ for 4 hours. 1ml of growth medium containing 3 times the normal concentration of serum was added without removing the transfection mixture. Gene silencing was measured at 48 hours.

RT-PCR: quantitative real-time reverse transcription PCR (RT-PCR) was performed by TaqMan RT-PCR master Mix kit (Applied Biosystems) using the following primers and probe set for detection of HPV18E6E7 transcript.

A forward primer: 5'-CTGATCTGTGCACGGAACTGA-3' (148-168) (SEQ ID NO: 384)

Reverse primer: 5'-TGTCTAAGTTTTTCTGCTGGATTCA-3' (439-463) (SEQ ID NO: 385)

And (3) probe: 5'-TTGGAACTTACAGAGGTGCCTGCGC-3' (219-233 and 416-425) (SEQID NO: 386)

The probe was labeled at the 5 'end with the reporter fluorescent dye FAM and at the 3' end with the fluorescent dye quencher TAMRA. Human GAPDH transcripts were detected using GAPDH for normalization.

HPV shRNA sequence:

h1 (effective sequence)

5’-ggATCCTAGGTATTTGAATTTGCATTTCAAGAGAATGCAAATTCAAATACCTTTTgTCgAC(SEQ ID NO：387)

5’-GTCGACAAAAGGTATTTGAATTTGCATTCTCTTGAAATGCAAATTCAAATACCTAGGATCC(SEQ ID NO：388)

H2 (invalid sequence)

5’-ggATCCTCAGAAAAACTTAGACACCTTCAAGAGAGGTGTCTAAGTTTTTCTGTTTgTCgAC(SEQ ID NO：389)

5’-GTCGACAAACAGAAAAACTTAGACACCTCTCTTGAAGGTGTCTAAGTTTTTCTGAGGATCC(SEQ ID NO：390)

Western blotting: hela cells were lysed with 1X Cell lysis buffer (Cell Signaling Technology, Cat No. 9803). For electrophoresis, 2 XLoading buffer containing 50. mu.g total protein was added to each well of a 12% SDS-PAGE gel. After transfer, the blot was blocked and bound with primary antibody for 2 hours, then incubated with HRP-bound secondary antibody before detection by ECL. All primary antibodies were used at 1/1000 dilution, except HPV18E7 antibody at 1/250 dilution.

Anti-human pRb antibody: BD Pharmingen (Cat No.554136), secondary antibody: HRP-anti mouse

HPV18E 7: santa Cruz (Cat No. sc-1590), secondary antibody: donkey anti-goat IgG-HRP Cat No. sc 2020

p 53: santa Cruz (Cat No. sc-126), secondary antibody: HRP-anti mouse

p 21: santa Cruz (Cat No. sc-397), secondary antibody: HRP-anti-rabbit

c-Myc: cell Signaling Technology (Cat No.9402), secondary antibody: HRP-anti-rabbit

Colony formation assay: hela cells were harvested 2 hours after bacterial invasion. Control-treated cells or HPV shRNA-treated cells were washed 3 times with complete MEM medium and 1 time with PBS. Cells were then digested with pancreatin and counted. 500 cells of each treatment were added to a single well of a 6-well plate containing 2ml of complete growth medium. Cells were grown for 10 days and colonies were then fixed using giimsa staining.

MTT test: hela cells were harvested 2 hours after bacterial invasion. Control-treated cells or HPV shRNA-treated cells were washed 3 times with complete MEM medium and 1 time with PBS. Cells were then digested with pancreatin and counted. 5000 cells of each treatment were added in triplicate to a single well of a 96-well plate containing 100. mu.l of complete growth medium. The cells were incubated at 37 ℃ for 48-72 hours, then 10. mu.l of 0.5mg/ml MTT was added to each well. The plate was further incubated at 37 ℃ for 3 hours, and after aspirating the medium from the wells and incubating, 100. mu.l of MTT solubilization solution [ 10% Triton X-100 in acidic isopropanol (0.1N HCl) ] was added to each well to stop the reaction. The absorbance at 570nm was read on a plate reader.

In this example, inhibition of the HPV18E 6and E7 oncogenes by short hairpin RNAs was observed. Short hairpin RNAs were delivered by infecting human cervical cancer cells (Hela) with a short hairpin RNA-producing bacterial strain. The shRNA expression cassette comprises 19 nucleotides (nt) of the target sequence followed by the reverse complement of the circular sequence (TTCAAGAGAGA) (SEQ ID NO: 391) and 19 nt. For the 19nt, the efficacy of siRNA delivery and Gene silencing was determined using the two shRNA sequences disclosed in Cancer Gene Therapy (2006)13, 1023-. Briefly, Hela cells were plated at a cell density of about 40% confluence in antibiotic-free medium. The following day, siRNA was added to 6-well plates at various concentrations of 50, 100, 200 nM. Control siRNA was added at a single concentration of 100 nM.

As shown in PCT publication No. WO2008/156702 in fig. 7, oligofectamine transfection caused a reduction in E6 mRNA in Hela cells compared to control sirnas. siRNA (H1) showed a reduction of E6 mRNA by about 40%. The knockdown response was not dose-dependent.

Subsequently, the hairpin of siRNA (H1) was cloned into the TRIP vector. To determine whether gene silencing could be achieved by the transkingdom system, shRNA in human cervical cancer cells (Hela) was determined in an invasive assay. Briefly, Hela cells were plated at 2x10⁵Cells/well were plated in 6-well plates, allowed to grow overnight, and incubated the next day at different MOIs for 2 hours with bacteria engineered to produce hairpin RNA (Escherichia coli). The bacteria were washed 4 times with medium containing 10% FBS and Pen Strep and the mammalian cells were further incubated in complete medium for an additional 48 hours. Isolating RNA or protein from the bacteria.

PCT publication No. WO2008/156702 demonstrates in fig. 8 and 9 that sirnas down-regulate the expression of HPV E6 in Hela cells. Cells were plated in 6-well plates and grown to 40% confluence (approximately 40,000 cells). Oligofectamine/siRNA transfection complexes were prepared in serum-free Opti-MEM medium by mixing 4. mu.l Oligofectamine and siRNA (final concentration 50, 100, 200nM in 185. mu.l medium). Cells 48 hours post-transfection were collected and analyzed for target and GAPDH mRNA levels by real-time RT-PCR. Data were normalized to GAPDH signal. Two different negative control sirnas were used at a single concentration of 200 nM.

PCT publication WO2008/156702 shows the results of real-time PCR after the Hela cell invasion assay in sections A-C of FIG. 10. Hela cells were incubated for 2 hours at different multiplicity of infection (MOI) using BL21(DE3) expressing shRNA. 48 hours after transfection, cells were harvested and analyzed for mRNA levels of both target and GAPDH by real-time RT-PCR. Data were normalized to GAPDH signal. These data were then further normalized against untreated control cells.

PCT publication No. WO2008/156702 in fig. 11 shows the effect of downregulating HPV E6 and E7 genes on tumor suppressor pathways and other downregulated targets. Hela cells were incubated for 2 hours at different multiplicity of infection (MOI) using BL21(DE3) expressing shRNA. 48 hours after transfection, cells were harvested and analyzed by western blotting. As shown, 50 μ g of protein was loaded into each lane and separated by gel electrophoresis, transferred to a membrane and detected using antibodies specific for HPV18E7, p53, actin, p110Rb, p21, and c-myc.

PCT publication No. WO2008/156702 shows colony formation and MTT assays in fig. 12 and 13, respectively. Hela cells were incubated for 2 hours at different multiplicity of infection (MOI) using BL21(DE3) expressing shRNA. 2 hours after transfection, cells were washed, trypsinized and counted, and an equal amount of cells for each MOI was added to the wells of the 6-well plate (500 cells into each well of the 6-well plate for CFA, 5000 cells into each well of the 96-well plate for MTT). For colony formation, cells were cultured for 10 days and stained with giemsa (Geimsa), and MTT analysis was performed 72 hours after plating.

PCT publication No. WO2008/156702 shows the results of real-time PCR after Hela cell invasion assay in FIGS. 14 and 15. Hela cells were incubated for 2 hours at different multiplicity of infection (MOI) using BL21(DE3) expressing shRNA. 48 hours after transfection, cells were harvested and analyzed for mRNA levels of both target and GAPDH by real-time RT-PCR. Data were normalized to GAPDH signal. These data were then further normalized against untreated control cells.

PCT publication No. WO2008/156702 in fig. 16 shows the effect of downregulating HPV E6 and E7 genes on tumor suppressor pathways and other downregulated targets. Hela cells were incubated for 2 hours at different multiplicity of infection (MOI) using BL21(DE3) expressing shRNA. 48 hours after transfection, cells were harvested and analyzed by western blotting. As shown, 50 μ g of protein was loaded into each lane and separated by gel electrophoresis, transferred to a membrane and detected using antibodies specific to HPV18E7, p53, actin, p110 Rb.

PCT publication No. WO2008/156702 shows the results of real-time PCR after Hela cell invasion assay using BL21(DE3) frozen samples containing negative sHRNA control and HPV sHRNA in FIG. 17. Hela cells were incubated for 2 hours at different multiplicity of infection (MOI) using BL21(DE3) expressing shRNA. 48 hours after transfection, cells were harvested and analyzed for mRNA levels of both target and GAPDH by real-time RT-PCR. Data were normalized to GAPDH signal. These data were then further normalized against untreated control cells.

PCT publication No. WO2008/156702 shows the plating efficacy of BL21(DE3) frozen samples containing a negative srhrna control and HPVsHRNA in fig. 18. Thawing frozen bacteria and thawing at 3.38X10⁸Final concentration of cells/well resuspended. Invasion assay was performed using this concentration, at 2ml 3.38X10⁸The cells were treated as 1000 MOI. Several control bacteria or HPV bacterial stocks were serially diluted (1: 100) and plated on LB plates to assess the number and viability of the bacteria-treated cells at 48 hours. Gene silencing was analyzed by quantitative real-time PCR using delta Ct relative quantitation or by western blot analysis. HPVE6 mRNA levels were normalized to the internal control GAPDH. The final data were further normalized to RNA from untreated cells. For protein analysis, Cell lysates were prepared in Cell lysis buffer (Cell Signaling Technology) and protein concentrations were determined using the BCA kit from BioRad. For electrophoresis, protein expression was normalized against actin loading controls.

Example 8: evaluation of HPV E6 Gene knockdown by Western blotting Using HPV18E7 antibody

Hela cells were incubated for 2 hours with different multiplicity of infection (MOI) using BL21(DE3) (HPVH 1 construct below) expressing shRNA. 48 hours after transfection, cells were harvested and analyzed by western blotting. HPV E6-specific knockdown was compared to negative shRNA controls. Briefly, as shown, 50 μ g of protein was loaded into each lane and separated by gel electrophoresis, transferred to a membrane and detected using antibodies specific for HPV18E7 and actin.

HPVH1

5’-GATCC TAGGTATTTGAATTTGCAT TTCAAGAGA ATGCAAATTCAAATACCTTTT G-3’(SEQ ID NO：392)

3’-G ATCCATAAACTTAAACGTA AAGTTCTCT TACGTTTAAGTTTATGGAAAA CAGCT-5’(SEQ ID NO：393)

PCT publication No. WO2008/156702 shows HPV E6 gene knockdown evaluated by western blotting using HPV18E7 antibody in fig. 19. Hela cells were incubated for 2 hours at different multiplicity of infection (MOI) using BL21(DE3) expressing shRNA. 48 hours after transfection, cells were harvested and analyzed by western blotting. HPV E6-specific knockdown was compared to negative sHRNA controls. Briefly, as shown, 50 μ g of protein was loaded into each lane and separated by gel electrophoresis, transferred to a membrane and detected using antibodies specific for HPV18E7 and actin.

Example 9: CCL20 expression inhibition in CMT93 cells

A confluent T-175 flask of CMT93 cells was trypsinized in 10ml until the cells were shed. Pancreatin was inactivated by addition of 30mls DMEM (10% FCS, pen/strep) and the cells were thoroughly mixed by pipetting. From this solution 8ml were transferred to a 50ml sterile tube and 32ml of mem 10% was added. Cells were mixed well and 250uL was added to each well of a 48-well plate and incubated overnight at 37 ℃ to generate adherent cells at about 70% confluence the next morning. The next day, siRNA transfection complexes were generated by the following method.

Sequences (pre-annealed siRNA duplexes) were ordered from Qiagen. Each well was resuspended in 250ul siRNA buffer (Qiagen) to give a stock concentration of 20 uM. The plate was then placed in a water bath at 95 ℃ for 5 minutes and then allowed to cool slowly to resuspend the duplexes and separate the aggregates. The resuspended duplexes were then used in the transfection assay described in standard methods. The configuration was that each well of the 48-well plate contained 250uL of medium; each screen was repeated with three organisms, thus, the solution was prepared in 4 wells: 3 for transfection and 1 additional well.

0.3uL of the appropriate siRNA (from 20uM stock solution) was diluted in 47uL serum/antibiotic free medium and mixed. To this solution 3uL of HiPerfect transfection reagent (Qiagen) was added followed by simple vortexing and incubation at room temperature for 20 minutes. 50uL of the mixture containing the complexes was added to every 3 wells of a 48-well plate containing CMT93 cells. Transfection was performed at 37 ℃ for 24 h, at which time the medium was removed and replaced with 400uL of 400uLs DMEM/10% FCS containing 100ng/mL LPS. After stimulation, cells were washed and RNA was isolated for qRT-PCR (50 cycles) according to the Qiagen Quantitech method (see manufacturer's instructions).

PCT publication No. WO2008/156702 in fig. 20 shows the knock-down of CCL20 expression in CMT93 cells using various siRNA sequences. The siRNA sequences tested are listed in table 33.

Watch 33

Example 10: claudin-2 expression inhibition in CMT93 cells

A confluent T-175 flask of CMT93 cells was trypsinized in 10ml until the cells were shed. Pancreatin was inactivated by addition of 30mls DMEM (10% FCS, pen/strep) and the cells were thoroughly mixed by pipetting. From this solution 8ml were transferred to a 50ml sterile tube and 32ml of mem 10% was added. The cells were mixed well, 250uL was added to each well of the 48-well plate and incubated overnight at 37 ℃ to generate adherent cells with approximately 70% confluence the next morning. The next day, siRNA transfection complexes were generated by the following method.

0.3uL of the appropriate siRNA (from 20uM stock solution) was diluted in 47uL serum/antibiotic free medium and mixed. To this solution 3uL of HiPerfect transfection reagent (Qiagen) was added followed by simple vortexing and incubation at room temperature for 20 minutes. 50uL of the mixture containing the complexes was added to every 3 wells of a 48-well plate containing CMT93 cells. Transfection was carried out at 37 ℃ for 24 or 48 hours, at which time the cells were washed and RNA was isolated for qRT-PCR (50 cycles) according to the Qiagen Quantitech method (see manufacturer's instructions).

PCT publication No. WO2008/156702 shows, in FIG. 21, the knockdown of Claudin-2 expression in CMT93 cells by various siRNA sequences 24 hours after transfection. The siRNA sequences tested are listed in table 34.

Watch 34

Example 11: inhibition of IL6-Ra expression in CMT93 cells

0.3uL of the appropriate siRNA (from 20uM stock solution) was diluted in 47uL serum/antibiotic free medium and mixed. To this solution 3uL of HiPerfect transfection reagent (Qiagen) was added followed by simple vortexing and incubation at room temperature for 20 minutes. 50uL of the mixture containing the complexes was added to every 3 wells of a 48-well plate containing CMT93 cells. Transfection was performed at 37 ℃ for 24, 48 or 72 hours, at which time the cells were washed and RNA was isolated for qRT-PCR (40 cycles) according to the Qiagen Quantitech method (see manufacturer's instructions).

PCT publication No. WO2008/156702 at fig. 22 shows the knock down of IL6-RA expression in CMT93 cells by various siRNA sequences at 24 hours post transfection. The siRNA sequences tested are listed in table 35.

Watch 35

Example 12: inhibition of IL13-Ra1 expression in CMT93 cells

0.3uL of the appropriate siRNA (from 20uM stock solution) was diluted in 47uL serum/antibiotic free medium and mixed. To this solution 3uL of HiPerfect transfection reagent (Qiagen) was added followed by simple vortexing and incubation at room temperature for 20 minutes. 50uL of the mixture containing the complexes was added to every 3 wells of a 48-well plate containing CMT93 cells. Transfection was performed at 37 ℃ for 24 or 72 hours, at which time the cells were washed and RNA was isolated for qRT-PCR (40 cycles) according to the Qiagen Quantitech method (see manufacturer's instructions).

PCT publication No. WO2008/156702 in fig. 23 shows the knockdown of IL13-RA1 expression in CMT93 cells by various siRNA sequences at 24 hours post transfection. The siRNA sequences tested are listed in Table 36.

Watch 36

Example 13: inhibition of IL18 expression in CMT93 cells

0.3uL of the appropriate siRNA (from 20uM stock solution) was diluted in 47uL serum/antibiotic free medium and mixed. To this solution was added 3uL of Lipofectamine RNAiMAX transfection reagent (Qiagen), followed by simple vortexing and incubation at room temperature for 20 minutes. 50uL of the mixture containing the complexes was added to every 3 wells of a 48-well plate containing CMT93 cells. Transfection was performed at 37 ℃ for 24 or 72 hours, at which time the cells were washed and RNA was isolated for qRT-PCR (40 cycles) according to the Qiagen Quantitech method (see manufacturer's instructions).

PCT publication No. WO2008/156702 in fig. 24 shows the knockdown of IL18 expression in CMT93 cells by various siRNA sequences 24 hours after transfection. The siRNA sequences tested are listed in Table 37.

Watch 37

Example 14: IL-7 expression inhibition in CMT93 cells

Sequences (pre-annealed siRNA duplexes) were ordered from Qiagen. Each well was resuspended in 250ul siRNA buffer (Qiagen) to give a stock concentration of 20 uM. The plate was then placed in a water bath at 95 ℃ for 5 minutes and then allowed to cool slowly to resuspend the duplexes and separate the aggregates. The resuspended duplexes were then used in the transfection assay described in standard methods. The configuration was that each well of the 48-well plate contained 250uL of medium; each screen was repeated with three organisms, whereby the solution was prepared in 4 wells: 3 for transfection and 1 additional well.

0.3uL of the appropriate siRNA (from 20uM stock solution) was diluted in 47uL serum/antibiotic free medium and mixed. To this solution was added 3uL of Lipofectamine RNAiMAX transfection reagent (Qiagen), followed by simple vortexing and incubation at room temperature for 20 minutes. 50uL of the mixture containing the complexes was added to every 3 wells of a 48-well plate containing CMT93 cells. At 37 ℃ for 24 hours, at which time the cells were washed and RNA was isolated for qRT-PCR (40 cycles) according to the Qiagen Quantitech method (see manufacturer's instructions).

PCT publication No. WO2008/156702 at fig. 25 shows the knock-down of IL-7 expression in CMT93 cells using various siRNA sequences 24 hours after transfection. The siRNA sequences tested are listed in Table 38.

Watch 38

Example 15: inhibition of chitinase 3-like 1 enzyme (CH13L1) expression in CMT93 cells

In a 1.7ml microcentrifuge tube, 2.4. mu.l of 20. mu.M double-stranded RNA solution (Qiagen) was diluted in 394. mu.l of Opti-MEM serum-free medium (Invitrogen) containing 1. mu.l of Lipofectamine RNAImax (Invitrogen), mixed, and incubated at room temperature for 10 minutes to enable formation of the transfection complex. Mu.l of this mixture was added to each 3 wells of a 24-well tissue culture plate, onto which 500. mu.l of CMT-93 cells were plated to form a final volume of 600. mu.l per well and a final concentration of RNA of 20 nM. 24 hours after transfection, 0.1. mu.g/ml Lipopolysaccharide (LPS) (Sigma) was added to each well and the cells were incubated for an additional 24 hours to stimulate the production of CHI3L1, after which the cells were washed in PBS and harvested for RNA extraction. CMT-93 cells were prepared for transfection as follows. A confluent T-175 flask of CMT93 cells was trypsinized in 10ml until the cells were shed. Pancreatin was inactivated by addition of 30mlsDMEM (10% FCS) and the cells were thoroughly mixed by pipetting. From this solution, 10mL to 50mL of a sterile tube was transferred, and 40mL of DMEM 10% FBS was added. The cells were mixed well and 500. mu.L of each well of a 24-well plate was added. This cell concentration resulted in about 70% confluency after 24 hours of culture.

Figure 26 in WO2008/156702 shows the knock-down of CH13L1 expression in CMT93 cells using various siRNA sequences after 24 hours of transfection. The siRNA sequences tested are listed in Table 39.

Watch 39

Example 16: construction of CEQ200

CEQ200 has the following genotype: glnV44(AS), LAM^-，rfbC1，endA1，spoT1，thi-1，hsdR17，(r_k ^-m_k ⁺) creC 510. delta. dapA. MM294 had the following genotype: glnV44(AS), LAM^-，rfbC1，endA1，spoT1，thi-1，hsdR17，(r_k ^-m_k ⁺) creC 510. Plasmids were purchased from CGSC (see Datsenko et al, (2000) Proc. Natl. Acad. Sci. USA97, 6640-.

Derivatization of CEQ200

MM294 (from CGSC)

↓ transformation by plasmid pKD46

MM294(pKD46)

↓.

MM294ΔdapA::kan(pKD46)

↓ Retention of plasmid pKD46 by cell culture at 43 ℃

MM294ΔdapA::kan

↓ transformed with plasmid pCP20

MM294ΔdapA::kan(pCP20)

↓ plasmid pCP20 was retained and treated by inducing FLP recombinase at 43 deg.C

And deleting the kan gene

CEQ200

Example 17: construction of CEQ201

CEQ201 has the following genotype: CEQ200[ glnV44(AS), LAM^-，rfbC1，endA1，spoT1，thi-1，hsdR17，(r_k ^-m_k ⁺) creC510 Δ dapA Δ recA. MM294 had the following genotype: glnV44(AS), LAM^-，rfbC1，endA1，spoT1，thi-1，hsdR17，(r_k ^-m_k ⁺) creC 510. Plasmids were purchased from CGSC (see Datsenko et al, (2000) Proc. Natl. Acad. Sci. USA97, 6640-.

Derivatization of CEQ200

MM294 (from CGSC)

↓ transformation by plasmid pKD46

MM294(pKD46)

↓.

MM294ΔdapA::kan(pKD46)

↓.

MM294ΔdapA::kanΔrecA::cat(pKD46)

↓ Retention of plasmid pKD46 by cell culture at 43 ℃

MM294ΔdapA::kanΔrecA::cat

↓ transformed with plasmid CP20

MM294ΔdapA::kanΔrecA::cat(pCP20)

While deleting the kan and cat genes

CEQ201

Example 18: construction of BTPs (CEQ210) by deletion of the minC and/or minD Gene from MM294

MM294 (from CGSC)

↓ transformed with plasmid pKD46

MM294(pKD46)

↓.

MM294ΔminCD::kan(pKD46)

↓ retaining plasmid pKD46 by culturing cells at 43 ℃

MM294ΔminCD::kan

↓ transformed with plasmid pCP20

MM294ΔminCD::kan(pCP20)

Plasmid pCP20 was recovered and treated by inducing FLP recombinase at 43 ℃

↓ and deleting kan gene

CEQ210

Example 19: schematic representation of the pMBV40, pMBV43 and pMBV44 plasmids

The pMBV40, pMBV43 and pMBV44 plasmids were used as final or intermediate plasmids in the tkRNA system and can be constructed according to the following: pUC19 was digested with restriction enzyme PvuII. The resulting-2.4 kb fragment was ligated with a-200 bp DNA fragment generated by annealing 5 oligonucleotides to each other. The oligonucleotides have the following names and sequences:

OHTOP1：GACTTCATATACCCAAGCTTGGAAAATTTTTTTTAAAAAAGTCTTGACACTTTATGCTTCCGGCTCGTATAATGGATCCAGGAGTAACAATACAAATGGA(SEQ ID NO：556)

OHTOP2：TTCAAGAGATCCATTTGTATTGTTACTCCTTTTTTTTTTTGTCGACGATCCTTAGCGAAAGCTAAGGATTTTTTTTTTACTCGAGCGGATTACTACATAC(SEQ ID NO：557)

OHBOT1：GTATGTAGTAATCCGCTCGAGTAAAAAAAAAATCCTTAGCTTTCGCTAAGGATCGTCGACAAAAAAAAAA(SEQ ID NO：558)

OHBOT2：AGGAGTAACAATACAAATGGATCTCTTGAATCCATTTGTATTGTTACTCCTGGATCCATT(SEQ ID NO：559)

OHBOT3：ATACGAGCCGGAAGCATAAAGTGTCAAGACTTTTTTAAAAAAAATTTTCCAAGCTTGGGTATATGAAGTC(SEQ ID NO：560)

↓

transformation of the ligation mixture into Escherichia coli and selection of ampicillin-resistant transformants

And (4) digesting the seeds. The plasmid with the expected insert DNA sequence and restriction map from the transformant was used

The plasmid DNA was named pMBV 38.

↓

pMBV38 was digested with NdeI and digested with BamHI-SalI from plasmid pKSII-inv-hly

The generated 6kb fragment was blunt-ended

The predicted sequences are shown in Table 40.

Watch 40

↓

The ligation mixture was transformed into Escherichia coli and ampicillin-resistant transformants were selected. Will come from

Plasmid DNA of transformant having the insertion of inv and hly genes was designatedpMBV40。

↓

pMBV40 was digested with BspHI and the resulting 7.4kb DNA fragment was ligated with plasmid pKD4 (purchased from BspHI)

CGSC (see Datsenko et al, (2000) Proc. Natl. Acad. Sci. USA97, 6640-

Template generated PCR fragment ligation containing kan Gene fragment

↓

The ligation mixture was transformed into E.coli and kanamycin-resistant transformants were selected. Screening for limits thereof

And (3) preparing a sexual enzyme map. It has kan genes in two different orientations. Will have clockwise and counter-clockwise directions

Plasmids of the needle-oriented kan gene open reading frame were designated pMBV43 and pMBV44, respectively

As shown in figure 27 of PCT publication No. WO2008/156702, the pMBV40(amp selective, with H3 hairpin) or pMBV43 and pMBV44(kan selective, with H3 hairpin) plasmids had the following sequences, respectively.

Table 41

Table 42 contains 8427 base pairs of the predicted pMBV43 plasmid. The sequence shown contains the following regions: hly orf (682-2271 bp); inv orf (2994-; shRNA promoter (6303-6361 bp); sense strand (6362-6383 bp); loop (6384 and 6390 bp); the antisense strand (6391-6412 bp); terminator I (6413-; terminator II (6423-; the origin of replication (6720-7307bp) and kan orf (7498-8292 bp).

Watch 42

Table 43 contains the 8443 base pair sequence of the validated pMBV43 plasmid. The sequence shown contains the following regions: hly orf (682-2271 bp); inv orf (2992-; shRNA promoter (6317-6375 bp); sense strand (6376-6397 bp); loop (6398-; antisense strand (6405-6426 bp); terminator I (6427 and 6437 bp); terminator II (6438-6475 bp); the origin of replication (6735 and 7322 bp); and kan orf (7513-8307 bp).

Watch 43

Watch 44

Example 23: construction of pNJSZc plasmid

pNJSZ is a 10.4kb plasmid with the capacity required to induce tkRNAi. This plasmid contains two genes, inv and hly, that enable the bacteria to invade mammalian cells and escape into the vesicle. Expression of short hairpin RNAs differed between the starting Trip plasmid and pNJSZ. In pNJSZ, the expression of shRNA is under the control of a constitutive bacterial promoter, which allows for sustained expression. This is different from the starting Trip plasmid (which has an IPTG-inducible promoter controlling shRNA expression). In addition, pNJSZ and the starting Trip plasmid contain different antibiotic resistance genes. pNJSZ has a kanamycin resistance gene, while the starting Trip plasmid has an ampicillin resistance gene. pNJSZc was constructed by removing from pNJSZ any region of pNJSZ that is not required to maintain the plasmid or to induce tkRNAi.

Step 1 is shown in FIG. 28 of PCT publication No. WO 2008/156702. The other BamH1 at position 9778 was removed by digesting pNJSZ with both SpeI (9784) and XmaI (9772), filling both sites with T4 DNA polymerase, and then self-ligating the plasmids to yield pNJSZ Δ BamH 1.

Step 2 is shown in FIG. 29 of PCT publication No. WO 2008/156702. Both the other SalI site at position 972 and the f1 origin of replication were removed by digesting pNJSZ Δ BamH1 with BglI (208) and PmeI (982), filling both sites with T4 DNA polymerase, and then self-ligating the plasmid to produce pNJSZc.

The pNJSZc DNA sequence is shown in table 45.

TABLE 45

Example 24: deletion of rnc encoding RNAse III in CEQ200 (CEQ 221. DELTA. rnc)

Most bacteria contain a large amount of an RNA degrading enzyme RNase that degrades siRNA to cause a decrease in tkRNAi activity. In cases where the RNase of a particular bacterium may exhibit such siRNA degradation, targeted deletion of the gene encoding the RNase of interest (e.g., the rnc gene encoding RNase III) is performed to produce higher levels of siRNA per tkRNAi bacterium, resulting in more siRNA being delivered to the target cell and more efficient gene silencing of the target gene within the target cell.

Construction of Δ rnc

CEQ200

Using plasmid pKD46¹Transformation of

↓ performing Amp^rSelecting

CEQ200(pKD46)

The use of primers rncFP and rncRP and pKD3¹Prepared Delta rnc:. DELTA.rnc. cat PCR product transformation

X and x combined intoLine Cm^rSelecting

CEQ200Δrnc::cat(pKD46)

↓ retaining pKD46 by culturing cells at 43 ℃

CEQ200Δrnc::cat

↓ passing through plasmid pCP20¹Transformed cells

CEQ200Δrnc::cat(pCP20)

↓ retains pCP20 and deletes cat gene by treatment at 43 ℃ to induce FLP recombinase

CEQ200Δrnc

The strain is named CEQ221

¹From Datsenko and Wanner (2000) Proc. Natl. Acad. Sci. USA97, 6640-. These plasmids were purchased from CGSC.

The gel analysis shown demonstrates the structure of CEQ221 and confirms the Δ rnc genotype. Gel analysis demonstrated the expression of Proteus 23S rRNA in the Δ rnc strain and the lack of the ability to process 23S dsDNA. Total cellular RNA was extracted from both CEQ200 and CEQ221 carrying pPM2 and electrophoresed on a 1.5% agarose gel. The 23S rRNA transcribed from pPM2 has an insertion in helix 25 which is processed by RNase III during maturation to form a 23S rRNA fragment: 23S' and 23S ". RNase III deficient strain CEQ221 was unable to process this helix, thus showing that the protein 23S rRNA is intact. This is evidence that the Δ rnc strain CEQ221 has lost RNAse III activity.

The Δ rnc strain CEQ221 demonstrated increased shRNA production and increased delivery of higher amounts of shRNA into target cells. When transformed with the same plasmid, pNJSzc-H3, the Δ rnc bacterium (CEQ221) contained significantly more shRNA than the wt-rnc bacterium (CEQ200) transformed with the same plasmid. In vitro invasion assay experiments, Δ rnc bacteria deliver higher amounts of shRNA into cells.

Cells treated with CEQ221 (H3-. DELTA.rnc) contained higher levels of shRNA than cells treated with the same amount of CEQ200 (H3). SW480 cells were treated with either Escherichia coli- Δ rnc carrying the tkRNAi plasmid for the gene target (β -catenin H3) or with Escherichia coli carrying the same tkRNAi plasmid for β -catenin (H3). Cells were collected at the indicated time points and cell extracts were analyzed to calculate the amount of shRNA deposited intracellularly by the vector bacteria (carrier bacteria). The Δ rnc strain (CEQ 221-red column) is capable of depositing significantly higher amounts of shRNA into target cells than its wild-type rnc counterpart (blue column).

FIG. 1 shows enhanced gene silencing potency (maximal effect) and efficiency (IC)₅₀). Treatment with CEQ221(Δ rnc) achieved significantly higher levels of gene suppression compared to treatment with CEQ200(wt rnc). As shown in FIG. 1, Cos-7 cells were treated with higher amounts of bacteria carrying plasmid pNJSZc-H3 (or control plasmid pNJSZc-HPVb) and analyzed for expression of the beta-catenin target gene after 48 hours. The level of β -catenin gene expression (mRNA) is shown relative to cells that have been treated with the same bacterial load of control bacteria (containing plasmid pNJSZc-HPVb and producing shRNA against HPV). The results indicate that the observed beta-catenin gene expression levels had a dose-dependent decrease ("knockdown"). The efficacy of the- Δ rnc strain (CEQ221) was significantly higher than that of the wt-rnc strain (CEQ200), with the highest levels of gene silencing being 76% and 57%, respectively. These results indicate that CEQ221 is about 10-fold more efficacious than CEQ 200: IC with CEQ200₅₀Is 10⁷IC of CEQ221 vs cfu/ml₅₀Is 10⁶cfu/ml。

Example 25: designing RNAse III substrates as precursors to functional shRNAs for tkRNAi

In the previous examples, it was demonstrated that limiting RNase activity in the delivering bacteria is beneficial for protecting shRNA from adverse processing and degradation. In alternative embodiments, it would be beneficial to design hairpin RNAs that could be improved and more accurately and efficiently Dicer processed, which is critical for efficient RNA interference. Dicer is an RNase enzyme with activity specific for dsRNA, whereby the RNase III cleavage product contains a5 ' phosphate and a 3 ' hydroxyl terminus and a 2-nt overhang at the 3 ' terminus. Dicer products are further characterized by a discrete size of about 21 nt. Thus, this example provides hairpin RNA molecules that produce RNaseIII processing substrates in bacterial (tkRNAi) vectors, thereby forming Dicer processing substrates in host target cells.

RNaseIII enzymes can be divided into three types. The type I enzymes found in bacteria, phages and fungi contain a single RnaseIII domain and a dsRNA binding domain (dsRBD). The type II and type III enzymes were characterized by Drosha and Dicer, respectively. Dicer is the most complex Rnase III enzyme, typically comprising a DExD/H-box helicase domain, a small domain of unknown function (DUF283), a paz (piwi argonaute zwille) domain, two Rnase III domains in tandem (rnases IIIa and IIIb) and dsRBD. Some Dicer or Dicer-like proteins from lower eukaryotes have simpler domain structures; for example, Dicer protein from Giardia intestinalis (Giardia intestinalis) contains only PAZ and two RNase III domains. Previous mutations and enzymatic studies in E.coli RNase III and human Dicer have resulted in a "single processing center model" for RNase III cleavage. The model focuses on the formation of two Rnase III domains of the catalytic dimer: intermolecular homodimers for type I enzymes and intramolecular pseudodimers between rnases IIIa and IIIb for Dicer and Drosha. This dimerization creates a single processing center for dsRNA cleavage, where each RNase III domain cleaves one strand of the dsRNA. The distance between the two cleavage sites is controlled to produce a characteristic 2-nt 3' overhang. For Dicer, the distance between the end-binding PAZ domain and the Rnase III domain determines the length of the cleavage product (Du, Lee, Tjhen et al in PNAS 105(7) 2008).

The bacteria contain a type I Rnase III enzyme that cleaves dsRNA. There is evidence that this type I Rnase III recognizes specific motifs that determine where to cleave dsRNA. The enzyme then performs the cleavage in a manner that leaves a 2nt 3' overhang (see Pertzev and Nicholson Nucleic Acid Research vol.34(13)2006and reviewed by Nicholson in FEMS Micro Reviews 231999). Furthermore, sequences have been described that exclude the binding and cleavage of Rnase III: so-called anti-determinants (anti-determinants).

In the following examples, hairpin RNA in tkRNAi bacteria was processed using type I RNAseIII of hairpin RNA in tkRNAi bacteria prior to release to the cytoplasm of a mammal. The defined proximal and distal cassette sequences required for the defined bacterial RNAse III are placed "below" the pseudo-tetracyclic structure, which optionally as a variant of the design may be constructed with or without loops, and "spacers" above "the pseudo-tetracyclic ring, to extend the hairpin sequence by-21 nucleotides. The near/far box motif will comprise only the far box motif can comprise only-10 nt, whereby the remaining 11nt stretch adjacent to the silencing sequence should consist of all anti-determinant base-pairs. Bacterial RNAse III will recognize the distal and proximal cassette sequences and cleave dsRNA 2nt after at or below the proximal cassette (fig. 2), leaving a longer (i.e. more stable) hairpin structure. Furthermore, the presence of anti-determinant base pairing "before" the near/far box motif protects the hairpin from further processing/degradation and maintains the proper hairpin structure hairpin length, so that when Dicer processes the hairpin within the target bacterial cell, 21nt of silencing siRNA will be generated.

FIG. 2 shows a schematic representation of the structure of the RNase III substrate hairpin RNA with functional annotation.

FIG. 3 shows a schematic representation of the cleavage of hairpin precursors by bacterial type I RNase III. The cleavage is forward targeting occurring at about the 10nt terminus of the pseudo-tetracyclic structure, thereby forming the ideal Dicer-substrate precursor. This step occurs in bacteria prior to delivery to the target cell. Cleavage of type I Rnase III will form a hairpin of approximately 100 nucleotides comprising a2 nucleotide overhang at the 3' end, which leads to the next enzymatic processing step (see fig. 4).

Figure 4 shows the functional annotation of the second step of maturation (first Dicer cleavage step). This step occurs after release of the RNA hairpin molecule into the cytoplasm of the target cell. Processing of RNAse type I RNA III leaves a2 nucleotide overhang at the 3' end of the hairpin RNA structure that helps to direct and trigger Dicer to cleave the RNA structure upstream of 21 nucleotides (the cleavage site is indicated as the "first Dicer cleavage site" indicated by the arrow).

FIG. 5 shows a second Dicer cleavage step and maturation into active siRNA. This second Dicer cleavage occurs in the cytoplasm of the host cell and removes the hairpin loop structure, leaving behind a functional siRNA for loading into the RISC complex. The first Dicer cleavage also helps to guide Dicer leaving a 2-nt overhang at the 3' end of the RNA.

Time course experiments using bacterial type I RNase III cleavage of hairpin RNA resulted in a reduction from 150nt RNA to 100nt RNA. Single-stranded RNA containing hairpin sequences was synthesized from the plasmid template using the MEGAshortscript kit (Ambion). The RNA was then contacted with purified bacterial RNaseIII for the indicated time period, electrophoresed on a 10% TBE-Urea gel, and visualized by ethidium bromide staining. After 4 minutes of cleavage, approximately 100nt of RNA material appeared.

Example 26: construction of CEQ505

Drug candidate CEQ505 consisted of an MM 294-derived escherichia coli strain by deleting the dapA gene and the rnc gene. The inside of this escherichia coli strain was designated CEQ221 transformed with plasmid pNJSZc-H3, which is an expression plasmid encoding invasin expression by the inv gene, listeriolysin O expression by the hly gene, and short hairpin RNA targeting β -catenin mRNA by the shRNA expression cassette including hairpin sequence H3.

FACS analysis showed that surface expression of Yersinia invasin is required for CEQ200 Δ rnc pNJSzc H3 to enter mammalian cells. Yersinia and CEQ200 Δ rnc pNJSZc H3 both have surface expression of invasin.

CEQ200 Δ rnc pNJSZc H3 requires LLO activity to allow shRNA to escape mammalian cell endosomes. LLO activity was determined by a hemolysin test, which demonstrated that CEQ505 had hemolysin activity whereas CEQ221 without plasmid had no hemolysin activity.

shRNA H3 is required to silence β -catenin in mammalian cells. H3 hairpin relative expression was determined by PK, which demonstrated that CEQ505 expressed H3 shRNA, while untransformed strain CEQ221 did not express H3 shRNA.

FIG. 6 shows gene silencing using CEQ 505. Part A demonstrates that CEQ505 is able to silence mammalian β -catenin in Cos-7 cells by up to 90% in a dose-dependent manner. Part B demonstrates that CEQ221pNJSZc lamin (an equivalent strain targeting lamin genes) is able to silence mammalian lamin in SW480 cells in a dose-dependent manner up to 65%.

Example 26: modification of pMBV40, 43 and 44 to produce hairpins without a5 'or 3' tail

The original TRIP plasmid expressed the shRNA under the control of the T7RNA polymerase promoter, enhancer, and terminator. In this mode, transcription begins within the T7RNA polymerase promoter sequence. Thus, the T7 enhancer, BamHI site, SalI site and most of the terminator are transcribed. Whereas shRNA hairpins are about 55nt in length, transcripts predicted to be produced are about 115 bases in length. The enhancer and restriction sites for cloning form the 5 'tail, while the T7RNA polymerase terminator forms the 3' tail.

Thus, new promoter-terminator constructs (see example 19) were designed for use in pMBV40, 43, and 44 to form hairpins without a5 'or 3' tail. The BamHI site used to clone the hairpin was contained within the promoter element (immediately after the-10 consensus sequence) (Lisser and Margalit, 1993, nucleic acids Res.,21,1507-1516). The promoter is enhanced by the inclusion of an UP element (Estrem et al, 1998, Proc. Natl. Acad. Sci. USA)95，9761-9766；Meng et al.，2001，NucleicAcids Res.29,4166-4178). For efficient termination, a series of T segments were added after the hairpin and before the SalI site for cloning the hairpin (terminator I). Rho independentThe terminator of (a) comprises an a-rich sequence followed by a stem loop of 4-18bp, followed by a series of T segments (e.g. Lesnik et al, 2001, Nucleic Acids Res.29,3583-3594). Without the a-rich sequence, shRNA stem-loop lengths were 19-21bp, and since the gene was unusually small, it was difficult to predict the efficacy of this terminator. Thus, another rho-dependent terminator (terminator II) from the flagellin gene was also included. Due to the two terminators, two transcripts are predicted. Transcripts I and II are terminated by terminator I and terminator II, respectively.

Example 27: cloning of arabinose-inducible invasin Gene used in tkRNAi

In tkRNAi, intracellular delivery of a therapeutic shRNA is achieved by equipping the vector bacterium with an invasive protein that allows the bacterium to enter the host target cell by interacting with a host cell surface receptor. The invasin protein encoded by the inv gene of yersinia is an example of an invasive protein that interacts with host cell proteins called β -1-integrins to trigger uptake of the bacterium into the host cell. However, high levels of invasin protein expression can be toxic to bacterial vector strains. Therefore, in order to improve the efficacy and potential of tkRNAi-mediated gene silencing, bacterial strains capable of inducing the expression of invasin by adding arabinose to the bacterial culture medium were constructed.

The plasmid constructed had: an arabinose inducible invasin cassette comprising an AraC gene encoding an AraC protein (arabinose operon-repressor-activator); p regulated by metabolite inhibitory protein (CRP) and AraC protein_araBAD(arabinose promoter); and receive P_araBADThe promoter controls the cloned inv gene.

Invasin has different expression states: (1) in the presence of glucose and in the absence of arabinose, the promoter is inhibited by metabolite repression and AraC-mediated repression; (2) as there was no metabolite inhibition and no AraC mediated induction, an uninduced state occurred in the absence of any sugars (no glucose and no arabinose); and (3) the induction state occurs without glucose but with arabinose due to the AraC mediated induction but without metabolite inhibition.

The above state of invasin was determined according to the following method. The cells were cultured overnight in the presence of glucose, then diluted and cultured for 4 hours with glucose (inhibited) or without any sugar (both cultures). Induction with arabinose (10mM) A culture was cultured for 2 hours in the absence of arabinose. Cells were collected by centrifugation and the expression of invasin was determined by FACS. Escherichia coli without any plasmid was used as a negative control, and Yersinia cultured at 26 ℃ was used as a positive control.

In the presence of arabinose, a high level of invasin expression was found by FACS measurements, comparable to the level of invasin expression observed in the positive control (yersinia). Bacterial growth appeared to stop during the two hours of induction of invasin with arabinose, as determined by OD. The survival rate was also reduced by a factor of 100 in 2 hours, which is consistent with the inventors' initial assumptions. The inventors then optimized the experimental conditions to yield good invasin expression and good survival. The inventors found that, although there was a measurable reduction in growth rate, 0.3-1mM arabinose did not cause a detectable reduction in survival. According to FACS, it was also shown that the induction of invasin under these conditions was not different from that of 10mM arabinose. The results demonstrate that bacterial growth (as measured by OD) is a function of arabinose induction by invasin.

Example 28: alternative shRNA structures for tkRNAi

Transkingdom RNA interference (tkRNAi) uses vector bacteria to synthesize and deliver short hairpin RNA (shRNA) that resides in the cytoplasm of the target cell. To achieve this, bacteria are equipped with expression plasmids or chromosomal integrants to allow them to express at least three new properties: surface-expressed invasin markers (e.g., yersinia invasin protein encoded by the inv gene), endosomal release function (e.g., listeriolysin O protein-LLO encoded by the hly gene), and therapeutic payload, i.e., shRNA that triggers RNA interference upon delivery to the host cell cytoplasm. Experiments have shown that expression of large amounts of hairpin RNA burdens the bacteria and results in slower growth and/or modification of the plasmid or hairpin by the bacteria. The design of shrnas with higher structural energy makes it more difficult for the bacterial transcription machinery to separate the two strands, however this shRNA is more difficult to clone than shrnas with reduced structural energy. In this example, the inventors disclose a method of expressing alternative hairpin RNAs with lower energy coefficients to more easily maintain shRNA expression plasmids in bacteria while maintaining the ability to induce gene silencing by RNA interference.

The advantage of the design shown in figure 7 over existing shRNA structures is mainly the significantly lower structural energy to facilitate cloning of the tkRNAi plasmid, more stable maintenance of the plasmid in bacteria and sequencing of the plasmid. Introduction of a wobble at the 3 ' end of the sense strand (woble) [3 ' (S) wobble ] was tolerated and did not alter the silencing ability of the construct, while introduction of a5 ' (S) wobble might reduce the silencing ability. RNAi can be triggered by shRNA without the traditional double-stranded structure. Furthermore, the half-overlap structure can induce gene silencing AS long AS the Antisense Strand (AS) is full-length (19nt) and is covered at the 5' end by the sense strand.

Example 29: derepression of inv expression in TRIP and pNJSZc

The yersinia pseudotuberculosis surface-expressed invasin protein mediates entry into human cells by binding to members of the β -1 integrin family. For this reason, the present inventors cloned the invasin gene (inv) including its native promoter into pTRIP and pNJSZc plasmids to mediate internalization of escherichia coli into human intestinal epithelial cells. When H-NS (histone-like protein) binds to the inv promoter region together with YmoA, the expression of invasin in Yersinia pseudotuberculosis is suppressed. These two proteins form inhibitory complexes that reduce inv expression to basal levels. Upregulation of inv expression occurs when temperature regulated RovA (regulator of virulence A) binds within the same promoter region of inv (replacing H-NS/YmoA). Homologs of H-NS and YmoA exist in Escherichia coli. However, RovA is not present in Escherichia coli, which results in a constant basal level of invasin expression. It was reported that removal of the regulatory binding region of the inv promoter region results in constant up-regulation of inv. In this example, the inventors describe a method to remove the regulatory binding region from the inv promoter as cloned in pTRIP and pNJSZc to allow constant up-regulation of inv.

The primers shown in Table 46 were designed to delete 153 nucleotides which are located in the Inv promoter region of Yersinia pseudotuberculosis and are believed to be involved in the inhibition of inv in Escherichia coli. The primers were used in combination with pTRIP (template) and QuikChange Lightning Site-Directed Mutagenesis kit (Stratagen) to delete the regulatory binding regions shown in Table 46 cloned within the inv promoter region in TRIP containing H3 or the lamin hairpin.

TABLE 46

The resulting inv suppressor gene was sequence verified and cloned between the NruI and ScaI sites of the NJSZc.

Invasin expression was determined by FACS in which CEQ221/pTRIP and CEQ221/pNJSZc were cultured overnight at 37 ℃, washed with PBS and detected with anti-inv monoclonal antibody 3a 2. The positive control was Yersinia pseudotuberculosis cultured at 26 ℃ to obtain optimal expression of invasin.

FACS analysis showed that removal of the inv regulatory region resulted in increased expression of invasin in E.coli transformed with pTRIP (the derepression mutant was named pGB60) and pNJSZc (the derepression mutant was named pGB 69). The ability of the original plasmid and the de-repressed plasmid to induce internalization of Escherichia coli in Vero cells was determined by a standard gentamicin protection test. The data indicate that de-inhibition of inv leads to increased internalization of escherichia coli in Vero cells.

Example 30: opa 52-mediated invasion of Escherichia coli into T84 human intestinal epithelial cells

Opa52 was engineered for apical (apical), broad-range targeting of intestinal epithelial cells to deliver tkRNAi. Opa52 will bind to CEACAM1, 3, 5 and 6, with CEACAM1, -5 and-6 being expressed by epithelial cells. This allows delivery of tkRNAi into healthy and polarized epithelial cells. The following examples describe the construction and use of the Opa52 bacterial expression vector for the invasion of escherichia coli into highly polarized T84 human intestinal epithelial cells.

The opa52 gene sequence was obtained from GenBank (accession # Z18929) and modified to include a start codon, a signal sequence and a stop codon. The signal sequence is identical to the natural secretion signal of some other Opa proteins and is codon optimized for expression in escherichia coli. This gene was then placed under the control of a modified lacUV5 promoter containing a second lacO site to obtain enhanced transcriptional repression. The λ t0 terminator sequence is contained downstream of the opa52 stop codon. The complete DNA fragment WAs synthesized by Blue Heron Biotechnology (Bothell, WA), cloned into pUC19 and verified. To facilitate expression of Opa52 on a low copy ColE 1-compatible plasmid, the inventors subcloned the complete synthesis cassette from Blue Heron into the pACYC177 modification designated pJS 15. This is a small (2kb) and low copy (10-12 copies per cell) kanamycin resistant vector compatible with the ColE1 plasmid. The resulting opa52 construct designated pJS34 was 1040 base pairs in length and is shown in Table 47. The sequence comprises: restriction sites for KpnI (1-6), SpeI (101-106), NdeI (922-927), NotI (1023-1030) and PmeI (1033-1040); lacO sites (for LacI binding) (7-25 and 70-88); RBS (95-100); ptac-35(32-37) and-10 (56-62); a leader sequence (109-.

Watch 47

Table 48 shows the translated 270 amino acid sequence with the 23 amino acid leader peptide added. The leader peptide is amino acids 1-23.

Watch 48

Plating and culture of bacteria released from highly polarized T84 cells demonstrated that the ability of opa-expressing E.coli cells to invade was significantly higher than cells of the E.coli strain expressing invasin. Figure 8 shows data from replicate experiments.

To be able to target healthy (non-dysplastic) epithelial cells by tkRNAi, the inventors developed two alternative invasion strategies, both based on a single protein from an intestinal invasive pathogen targeting epithelial cell surface receptors.

The first is a member of the Opa family of proteins expressed by neisseria species and, depending on the Opa variant used, differentially targets a member of the carcinoembryonic antigen adhesion molecule (CEACAM) family expressed on the apical surface of epithelial cells (epithelial aspect). Primary binding is mediated by cilia, followed by a more intimate interaction by an opaque protein (Opa) present within the bacterial outer membrane. Opa proteins recognize specific receptors present in epithelial cells. Some Opa proteins bind to cell surface Heparan Sulfate Proteoglycan (HSPG) syndecans-1 and-4, while others bind to carcinoembryonic antigen (CEA) or members of the CD66 family (recently renamed the CEACAM family). CEACAM can be found on epithelial cells and neutrophils (two cell types targeted by neisserial strains during natural infection). The interaction between the Opa protein and the CEACAM family members is highly specific, i.e. each Opa variant shows a specific tropism only for certain members of the CEACAM receptor family.

The second of these targeting proteins is derived from Listeria monocytogenes, designated internalization protein A or InlA, and targets the adhesively linked E-cadherin protein component. InlA in combination with InlB enables listeria monocytogenes to invade a variety of non-phagocytic cells in a susceptible host. InlA promotes listeria monocytogenes entry into the intestinal epithelium by targeting the N-terminal domain of E-cadherin, the major molecule in the adhesion binding complex. Recent studies have shown that native InlA takes advantage of a temporary window in the maturation of intestinal epithelial cells and shedding from the apical end of the villus to enter the host. In other words, the intestinal epithelial cells are generated from stem-like cells that self-renew at the base of the villus pouch and progressively mature as they move from the pouch to the tip of the villus. After the intestinal epithelial cells reach the top of the villus, they are shed during normal turnover or by injury-induced apoptosis. In either case, transient exposure of the adhesion binding protein (i.e., E-cadherin) occurs, allowing InlA to bind and Listeria to enter the cell. Furthermore, under pathological conditions such as IBD, the integrity of the epithelial barrier around not only the site of the lesion, but also the uninvolved area, is affected. In this case, the affected barrier would expose the adhesive binding (e.g., E-cadherin) and thus enable the InlA-expressing delivery strain to enter cells within the inflammatory foci and surrounding epithelium. This is beneficial in that during active episodes of inflammation, the delivery strain preferentially invades and silences the target of the site of the inflamed/affected barrier, while not contacting the normal epithelial cell region. Thus, any tkRNAi delivery platform that relies on InlA can be used as a therapeutic agent for treatment during active inflammation.

The results have demonstrated that the E.coli cells carrying the Opa-expressing plasmid have a significantly higher invasive capacity than E.coli cells carrying the invasin-expressing plasmid.

Example 31: CEQ508 for the treatment of pathologies mediated by the upregulation of beta-catenin

The drug candidate CEQ508 consists of E.coli strain CEQ221 comprising the pMBV43-H3 plasmid. Strain CEQ221 was obtained from escherichia coli strain MM294 by the sequential deletion of dapA and rnc genes using the lambda phage Red recombination system with the aid of a 5-strain-gene disruption group (5-strain gene-disruption set, Datsenko and Wanner, 2000(proc.natl.acad.sci.usa 97, 6640)) purchased from CGSC. Plasmid pMBV43 in this example encodes the shRNA hairpin to target β -catenin mRNA by shRNA expression cassettes comprising the hairpin sequence "H3" (previously disclosed), yersinia pseudotuberculosis invasin (encoded by the inv gene), and listeria monocytogenes listeriolysin o (llo) (encoded by the hly gene). The pMBV43 plasmid was derived from pUC19 with the following changes:

1. the hairpin case has a modified P connected to an UP element_lacUV5A promoter and a set of two terminators.

2. The fragment containing the inv and hly genes was cloned from another existing plasmid, pKSII-inv-hly.

3. Kanamycin (kan) was used instead of amp as antibiotic resistance marker.

Modified promoter P used in the pMBV43 plasmid of this example_lacUV5Having at least three P's different from those used in the previously used pNJSZ plasmid_lacUV5Important differences in promoters.

1. 35 common elements (shaded from TTTACA to TTGACA);

2. adding an UP element upstream of the-35 element;

3. from P_lacUV5Elements of lacO (lac operator) were deleted and replaced with BamHI sites.

Watch 49

P_lacUV5The length of the promoter is 87Base pairs and as shown in table 49. P_lacUV5The-35 and-10 consensus sequences for the promoters were 7-12 and 31-37 base pairs, respectively. The lacO (lac operator operon) is shown as 43-64 base pairs.

Modified P_lacUV5The promoter was 65 base pairs in length and is shown in table 49. P_lacUV5The-35 and-10 consensus sequences for the promoters are 30-35 and 54-60 base pairs, respectively. The UP elements shown are 7-26 base pairs.

The pMBV43 plasmid is otherwise different in that it has two sets of terminators:

1. terminator 1: is a series of T segments that function as terminators when they follow the shRNA hairpin.

2. A terminator 2: identical to the Escherichia coli rrnC terminator and having the following sequence: GATCCTTAGCGAAAGCTAAGGATTTTTTTT (SEQ ID NO: 574).

In addition, the pNJSZ plasmid produces shRNA that comprises a total length of about 131-135 bases and consists of a5 'overhang of about 8 bases, a shRNA of 51 base pairs, and a 3' overhang consisting of about 72-76 bases. In contrast, the pMBV43 plasmid does not produce a5 'overhang, but rather a significantly smaller 3' overhang consisting of 2-5 bases and produces a 51 base pair shRNA, i.e., the total length of the shRNA is 53-70 bases. To function well, the 3' overhang requires at least 2 bases. Thus, the total length of the shRNA is 53-70 nucleotides in length, preferably 53-65 nucleotides in length, more preferably 53-58 nucleotides in length, and most preferably 53-55 nucleotides in length.

FACS analysis showed that entry of CEQ221 pMBV43-H3 into mammalian cells required surface expression of Yersinia invasin. Yersinia and CEQ221 pMBV43-H3(CEQ508) both have surface expression of invasin. CEQ221 without the pMBV43 plasmid showed no invasin expression. Negative control: yersinia without antibodies.

CEQ508 requires listeriolysin (LLO) activity to allow therapeutic cargo (shRNA) to escape mammalian cellular endosomes after invasion. The LLO activity was determined by the hemolysis assay described above. CEQ508 showed significant hemolytic activity while CEQ221 or PBS without plasmid showed no hemolytic activity.

Quantitative real-time PCR relative to H3 hairpin RNA expression demonstrated that CEQ508 contained about 20-fold more H3 shRNA than its precursor CEQ 505. As previously disclosed, CEQ505 was composed of escherichia coli from MM294, wherein the escherichia coli strain designated CEQ221 was generated by deletion of the dapA and rnc genes, followed by transformation with plasmid pNJSZc-H3, in which expression of invasin is encoded by the inv gene, expression of listeriolysin O by the hly gene and short hairpin RNA targeting β -catenin mRNA is encoded by the shRNA expression cassette comprising hairpin sequence H3. In contrast, CEQ508 consists of escherichia coli strain CEQ221 containing the pMBV43-H3 plasmid, encoding yersinia pseudotuberculosis invasin (encoded by the inv gene), listeriolysin o (llo) (encoded by the hly gene) of listeria monocytogenes, and shRNA hairpin sequence H3, as previously disclosed.

FIG. 9 shows the use of CEQ508 to silence genes in human cells (SW 480). Part a shows that CEQ508 is able to silence mammalian β -catenin mRNA up to 90% in SW480 cells in a dose-dependent manner. Controls were treated with CEQ221-pMBV43-lamin and CEQ221-pMBV 43-luciferase. Part B shows that CEQ508 is able to silence mammalian β -catenin in a dose-dependent manner up to 72% in SW480 cells. Controls were treated with CEQ221-pMBV 43-luciferase. Similar experiments performed by cells in DLD-1 indicate that CEQ508 is capable of silencing β -catenin mRNA by greater than 50%. Additional experiments demonstrated that β -catenin in HeLa cells was silenced using CEQ 508-H3. HeLa cells were cultured in standard DMEM (10% FBS, 1% Pen-Strep) at 37 ℃ and CEQ508-H3 was added to the culture when the cell confluence was 80%. Controls were treated with an E.coli strain of the same genetic background but expressing hairpin RNA targeting human lamin (hlam) rather than beta-catenin. Cells were treated with multiple multiplicity of infection (MOI) ranging from 1: 6.5 to 1: 51 for 2 hours and then washed four times. Fresh medium containing the antibiotics tetracycline and ofloxacin was then added and cells were harvested 48 hours after the challenge. RNA was extracted using TRIZOL (Invitrogen, Carlsbad, CA) and gene expression analysis was performed using quantitative real-time PCR. Dose-dependent silencing of β -catenin was observed in HeLa cells treated with CEQ508-H3, but not in cells treated with the control strain. At the highest MOI, β -catenin expression was reduced to 45% of basal.

Figure 10 shows that a decrease in gene expression was observed in SW480 cells at the protein level (by western blot) after a single treatment with CEQ508, but not in any control strain. SW480 cells were cultured in standard DMEM (10% FBS, 1% Pen-Strep) at 37 ℃. CEQ508 was added when the cell confluence was 70%. Using the same genetic background but expressing either: (a) a hairpin RNA targeting luciferase (luc), (b) bacteria with invasive properties invasin and listeriolysin but not expressing the hairpin RNA, or (c) escherichia coli CEQ221 not carrying the pMBV43 plasmid (not invasive) escherichia coli strain treated controls. Cells were treated with multiple multiplicity of infection (MOI) ranging from 1: 50 to 1: 150 for 2 hours and then washed four times. Fresh medium containing the antibiotics tetracycline and ofloxacin was added and cells were harvested 48 hours after challenge and whole protein was extracted using standard methods. Dose-dependent silencing of β -catenin was observed in SW480 cells treated with CEQ508, but not in cells treated with the control strain. At the highest MOI, β -catenin expression was reduced to a barely detectable level.

Furthermore, time course experiments were also performed and demonstrated that expression of β -catenin in SW480 cells after a single treatment with CEQ508 had long-term silencing of at least 50%, preferably at least 60%, more preferably at least 70% and most preferably at least 80% at least 1 day, preferably at least 2 days, more preferably at least 3 days and most preferably at least 4 days after administration. SW480 cells were cultured in standard DMEM (10% FBS, 1% Pen-Strep) at 37 ℃. CEQ508 was added when the cell confluence was 70%. Controls were treated with a bacterial strain with the same genetic background (escherichia coli strain CEQ221) but not carrying the pMBV43 plasmid (i.e. not carrying the genes for invasion and therefore not invasive). Cells were treated with MOI 1: 200 for 2 hours and then washed four times. Fresh medium containing the antibiotics tetracycline and ofloxacin was added, the cells were cultured and passaged when 90% confluence was reached. Cells from the parallel tubes were collected at designated time points after invasion. RNA was extracted using TRIZOL and gene expression analysis was performed using quantitative real-time PCR. The results show that there was substantial (> 80%) gene silencing following a single treatment with CEQ508, which was maintained for at least 4 days before the beta-catenin level slowly returned to normal. The apparent "overshoot" around 10 days is interpreted as an artifact caused by passage of the cell line.

With two different doses (low dose 10)⁷Per ml and high dose 10⁸/ml) treatment of CEQ508(CEQ221/pMBV43H3) resulted in no significant changes in gene expression of lamin in human cells (SW480) (as determined by quantitative real-time PCR), demonstrating no detrimental effects on other genes. By means of 10⁸Perml plasmid-free CEQ221 bacteria (plasmid-free, high), 10 each⁷A ratio of 10 to 10⁸CEQ221-pMBV43-luciferase (pMBV43luc, high and low) and 10 each/ml⁸A ratio of 10 to 10⁷Control treatments were performed on/ml CEQ221-pMBV43 (no hairpin pMBV43, high/low) that did not express short hairpin RNA, respectively. These data demonstrate that CEQ508 treatment does not cause problems for other genes.

Time-profiles were made to assess the presence of inflammatory cytokines 0-12 hours after a single oral feeding of CEQ508 in wild type mice and polyp-bearing APCmin mice. Animals (wild type and APCmin mice, 3-7 per group per time point) were treated with a single dose of CEQ508 and sera were collected at the indicated time points. Animals used as positive controls were injected intravenously with 400 μ g LPS. Inflammatory cytokines TNF α, IL6, IL12, IFN γ, MCP-1 and IL-10 were analyzed as indicators of inflammatory responses to oral administration of CEQ 508. In summary, none of the inflammatory cytokines detected after oral treatment with CEQ508 showed an increase, demonstrating that there was no systemic inflammatory cytokine response in wild-type or polyp-bearing APCmin mice after treatment with CEQ 508.

Table 50 demonstrates the pharmacokinetics of shRNA in gastrointestinal mucosa following oral feeding of CEQ508 or CEQ 501.

Watch 50

Mice were dosed with one or repeated CEQ508 or CEQ501 treatments and gastrointestinal mucosal tissues were analyzed to quantify the amount of shRNA deposited in GI mucosa at various time points after treatment. H3 RNA was detectable in the intestinal mucosa of mice dosed with CEQ501 or CEQ508, whereas PBS/glycerol treated mice lack H3 shRNA. Furthermore, the amount of H3 RNA detected in mucosa after treatment with CEQ508 was significantly higher compared to CEQ501, consistent with the Δ rnc mutation conferring higher yield and better stability of H3 shRNA. However, when tested 24 hours after the last dose (multiple daily dosing regimen), both CEQ501 and CEQ508 showed comparable levels of H3 shRNA in the intestinal mucosa, suggesting that both delivery platforms achieved equal steady-state levels of the delivered hairpins.

Table 51 shows the experimental groups used to evaluate the pharmacokinetics of live CEQ508 bacteria in mice after oral treatment

Watch 51

Mice were treated 1, 3 or 7 times with CEQ508 by oral gavage. Each dosageContaining 5x10⁹cfu CEQ 508. Tissues were analyzed 24 hours after the last dose. Tissues were removed under sterile conditions and assayed for the presence of viable therapeutic CEQ508 bacteria. Positive control animals were treated with i.v. injections of CEQ 508.

Table 52 shows the pharmacokinetics that demonstrate that no viable CEQ508 is recovered from any organ examined after these single or multiple (once a day, up to 7 days) oral treatments.

Table 52

These results demonstrate that CEQ508 is unable to escape the gastrointestinal tract in mice (except that two animals showed false positive bacteria after gavage injury). In wild-type mice (normal healthy mice with intact visceral barrier) and APCs with impaired epithelial barrier integrity due to dysplasia^minThis phenomenon was observed in all mice. However, CEQ508 was recovered in fecal samples collected 5 hours after dosing, demonstrating movement of viable bacteria throughout the length of the intestine. Consistent with expectations, the number of viable CEQ508 recovered from the stool-like sample by 24 hours post-dose decreased rapidly. Surviving CEQ508 was recovered in mice given a single intravenous injection via the tail vein. In these animals, CEQ508 was recovered in blood and organs tested 2 hours after injection. The amount of viable bacteria subsequently decreased but could still be recovered in the liver up to 96 hours after (iv) administration.

Received a single oral dose of CEQ508(5.0X 10)⁹cfu, gavage, total volume 0.2ml) animals collected fecal samples. Fecal samples were collected every hour for the first 6 hours, then every two hours up to 24 hours, then every 12 hours up to 108 hours. To enable the collection of fecal samples from a planned total of 24 animals, mice were divided into two groups, 12 per group and received a single oral dose of CEQ508 over a 12 hour cycle. The fecal samples were resuspended in 1ml of mediumIn bacterial PBS, diluted to 10mL with sterile PBS, 50 μ L of the suspension was plated on non-selective LB/DAP plates where all bacteria from the stool sample were expected to grow and only the therapeutic CEQ508 bacteria from the stool sample were expected to grow on the selective plates containing LB/Kan/DAP. Bacterial colonies were then counted. Mice were subdivided into the following groups according to the confluency of CEQ508 in fecal samples: 0CFUs, < 100CFUs, < 1000CFUs, > 1000 CFUs. Finally, as shown in table 53, the percentage of mice with a certain amount of bacteria was calculated.

Watch 53

Table 53 shows that mice began to shed viable bacteria in the first 2 hours after a single oral treatment with CEQ 508. The amount of CEQ508 in the fecal samples peaked at 5 hours (i.e., all mice shed > 1000CFU of viable CEQ508), remained elevated and then gradually decreased up to 8 hours after dosing. Most of the treated mice excreted only a small amount of CEQ508 (i.e., 33% < 1000CFU and 63% < 100CFUs) 24 hours after dosing. Of the total number of treated mice, only one third (33.3%) continued to shed viable CEQ508 (< 100CFUs) in the fecal samples 36 hours post-treatment, whereas no animals shed viable CEQ508 at 48, 60, 72, 84, 96 or 108 hours post-treatment. Taken together, these data demonstrate that CEQ508 not only passes through the gastrointestinal tract, but is also rapidly eliminated and is unable to reside and proliferate in the gut. CEQ508 peaked in the fecal samples 5 hours post-dose, remained elevated and then gradually decreased up to 8 hours post-dose, while no animals shed viable CEQ508 at 48, 60, 72, 84, 96, or 108 hours post-dose.

Example 32: CEQ509 for the treatment of pathologies mediated by the up-regulation of beta-catenin

The drug candidate CEQ509 consists of a Bacterial Therapeutic Particle (BTP), wherein the BTP is a minicell derived from escherichia coli strain CEQ210 comprising a pNJSZc plasmid. Strain CEQ210 was obtained from escherichia coli strain MM294 by deletion of the minC gene using the lambda phage Red recombination system with the aid of the 5-strain-gene disruption group purchased from CGSC (Datsenko and Wanner, 2000(proc.natl.acad.sci.usa 97, 6640)). The pNJSZc plasmid encodes the shRNA hairpin, yersinia pseudotuberculosis invasin encoded by the inv gene and listeria monocytogenes listeriolysin o (llo) encoded by the hly gene. BTP was purified by low speed centrifugation, yielding > 99.9% purity based on its ability to form colonies.

A number of experiments have been performed to evaluate tkRNAi activity of CEQ509, including experiments on LLO protein encoded by the hly gene of pNJSZc plasmid. It was demonstrated that CEQ509BTP has the same LLO activity as CEQ501 when equal amounts (equal biomass) of BTP are used.

CEQ509 requires listeriolysin o (llo) activity from listeria monocytogenes to escape the phagosome and release the hairpin into the cytoplasm. The LLO activity was determined by hemolysis at pH 5.5. Hemolysis was visually observed and quantified by measuring absorbance at 540 nm. For the absorbance determination, PBS treated samples were used as a blank in a spectrophotometer. CEQ509-H3 and CEQ509-HPV are BTPs containing H3 shRNA against β -catenin and HPV E6 protein (used as a control).

FACS analysis demonstrated the presence of invasin on the surface of CEQ509 BTP.

Entry of CEQ509BTP into mammalian cells requires the surface of CEQ509 to express yersinia pseudotuberculosis invasin.

Quantitative real-time pcr (qpcr) analysis demonstrated that CEQ509BTP did not contain any H3 shRNA and only produced a background signal.

Finally, BTP was determined in the tkRNAi assay as shown in figure 11. Figure 11 shows β -catenin silencing using CEQ 509. COS-7 cells were infected with CEQ509-H3 or CEQ509-HPV BTP at the multiplicity of infection (MOI) indicated. Cellular RNA was collected after 48 hours and qPCR-based quantification of β -catenin mRNA was performed. The Relative Quantitative (RQ) ratio of CEQ 509-H3-infected cells to CEQ 509-HPV-infected cells at the corresponding MOI was plotted. This and other data show that CEQ509 silences 30-50% of β -catenin. The assay demonstrated that the level of invasin expression was sufficient to induce tkRNAi-mediated gene silencing.

Claims

1. Invasive Escherichia coli comprising a prokaryotic vector comprising one or more DNA molecules encoding one or more siRNAs, a modified P_lacUV5A promoter, at least one Inv locus, and at least one HlyA gene, wherein the siRNA interferes with mRNA of β -catenin, and the invasive escherichia coli has reduced RNase III activity as compared to a wild-type escherichia coli.

2. Prokaryotic vector comprising one encoding one or more siRNAsOne or more DNA molecules, modified P_lacUV5A promoter, at least one Inv locus, and at least one HlyA gene, wherein the siRNA interferes with the mRNA of β -catenin.

3. A method of delivering one or more siRNA to a mammalian cell, the method comprising introducing into the mammalian cell at least one invasive escherichia coli bacterium of claim 1.

4. A method of modulating gene expression in a mammalian cell, the method comprising introducing into the mammalian cell at least one invasive escherichia coli bacterium of claim 1.

5. A method of treating or preventing a disease or disorder associated with β -catenin overexpression in a mammal in need thereof, the method comprising modulating β -catenin expression in the mammal, comprising introducing into cells of the mammal at least one invasive escherichia coli bacterium of claim 1.

6. The invasive Escherichia coli bacterium according to claim 1, wherein an rnc gene encoding RNase III is deleted in said bacterium.

7. The invasive Escherichia coli according to claim 1, wherein said RNase III activity is reduced at least 90% compared to wild-type Escherichia coli.

8. The invasive Escherichia coli according to claim 1, wherein said RNase III activity is reduced at least 95% compared to wild-type Escherichia coli.

9. The invasive Escherichia coli according to claim 1, wherein said RNase III activity is reduced at least 99% compared to wild-type Escherichia coli.

10. The invasive Escherichia coli bacterium of claim 1, wherein said one or more DNA molecules are transcribed into one or more shRNAs in said invasive bacterium.

11. The invasive escherichia coli bacterium of claim 10, wherein said one or more shRNA comprise a 3' overhang or a blunt end.

12. The invasive escherichia coli bacterium of claim 11, wherein said 3' overhang is 2-5 base pairs.

13. The invasive escherichia coli bacterium of claim 11, wherein said 3' overhang is no more than 2 base pairs.

14. The invasive Escherichia coli bacterium of claim 10, wherein said one or more shRNAs are processed into one or more siRNAs.

15. The invasive Escherichia coli bacterium of claim 1, wherein said prokaryotic vector further comprises at least one terminator sequence.

16. The invasive escherichia coli bacterium of claim 15, wherein said at least one terminator sequence comprises at least 5 contiguous thymidine base pairs.

17. The invasive escherichia coli bacterium of claim 15, wherein said bacterium further comprises a second terminator sequence.

18. The invasive escherichia coli bacterium of claim 17, wherein said second terminator sequence is an rrnC terminator sequence.

19. The invasive Escherichia coli bacterium of claim 1, wherein said prokaryotic promoter further comprises at least one UP element.

20. The invasive Escherichia coli bacterium according to claim 1, wherein said invasive bacterium is an attenuated, nonpathogenic or avirulent bacterium.

21. A composition comprising the invasive bacterium of claim 1 and a pharmaceutically acceptable carrier.

22. The method of claim 5, wherein about 10 is used³-10¹¹Invasive bacteria infect the mammalian cells.

23. The method of claim 22, wherein about 10 is used⁵-10⁹Invasive bacteria infect the mammalian cells.

24. The method of claim 5, wherein the concentration is from about 0.1 to about 10⁶The mammalian cells are infected with a range of multiplicity of infection.

25. The method of claim 25, wherein the amount is about 10²-10⁴The mammalian cells are infected with a range of multiplicity of infection.

26. The method of claim 5, wherein the expression of β -catenin is reduced compared to wild-type β -catenin expression or compared to β -catenin expression prior to introducing the invasive bacterium into the cell.

27. The method of claim 26, wherein said reduced expression of β -catenin is reduced expression of β -catenin mRNA.

28. The method of claim 26, wherein said reduced expression of β -catenin is reduced expression of β -catenin.

29. The method of claim 26, wherein the beta-catenin expression is reduced by at least 50% compared to wild-type beta-catenin expression or compared to beta-catenin expression prior to introducing the invasive bacterium into the cell.

30. The method of claim 26, wherein the beta-catenin expression is reduced by at least 75% as compared to wild-type beta-catenin expression or as compared to beta-catenin expression prior to introducing the invasive bacterium into the cell.

31. The method of claim 26, wherein the beta-catenin expression is reduced by at least 90% compared to wild-type beta-catenin expression or compared to beta-catenin expression prior to introducing the invasive bacterium into the cell.

32. The method of claim 5, wherein the disease or condition associated with β -catenin overexpression in the mammal is selected from the group consisting of: colon cancer, rectal cancer, colorectal cancer, Crohn's disease, ulcerative colitis, Familial Adenomatous Polyposis (FAP), Gardner syndrome, hepatocellular carcinoma (HCC), basal cell carcinoma, pilomas, medulloblastomas, and ovarian cancer.

33. The method of claim 5, wherein the mammalian cell is selected from the group consisting of: colon epithelial cells, rectal epithelial cells, small intestine epithelial cells, liver cells, skin epithelial cells, hair cells, nerve cells and ovarian cells.

34. The method of claim 5, wherein the mammal is selected from the group consisting of: human, bovine, ovine, porcine, feline, buffalo, canine, caprine, equine, donkey, deer, and primate.

35. The method of claim 34, wherein the mammal is a human.