HK1239733A1 - Enabling the use of long dsrna for gene targeting in mammalian and other selected animal cells - Google Patents

Description

Facilitating the use of long dsRNA for gene targeting in mammalian and other selected animal cells

This application is a divisional application of PCT application PCT/US2008/068866 filed on 30.6.2008, the date of entry into the chinese national phase was 2010, 3.1, with application number 200880105142.7, entitled "facilitating long dsRNA to be useful for gene targeting in mammals and other selected animal cells".

Cross Reference to Related Applications

This application claims priority and benefit of U.S. provisional patent application serial No. 60/947,311, filed on 29.6.2007, the contents of which are incorporated by reference in their entirety.

Background

RNA interference (RNAi) is a catalytic mechanism of gene-specific silencing in eukaryotes, which has profound significance to biology and medicine. However, inducers of the effective mechanisms of such gene targeting are distinct in mammalian and non-mammalian cells.

Long double-stranded rna (dsrna) triggers efficient sequence-specific gene silencing in nematodes (c. elegans) and drosophila melanogaster (drosophila melanogaster). In contrast, long dsrnas induce non-sequence-specific responses in mammalian cells due to activation of interferon-related pathways. The RNAi machinery is thought to be nonfunctional in mammalian cells until siRNA duplexes are found.

Messenger rna (mRNA) for degradation is selectively targeted using short interfering rna (sirna), resulting in silencing or knocking down the gene to be expressed by degrading the mRNA of interest. Typically, sirnas are double-stranded rnas (dsrnas) 20-25 nucleotides long with some unpaired overhang bases on each strand. Based on this model, molecular biology techniques using siRNA have become research tools and candidates for therapy (Dykxhoorn, Novina & Sharp. Nat. Rev. mol. cell biol.4: 457-.

While widely used for gene silencing in mammalian cells, siRNA has raised many problems that limit the potential of RNAi in biomedical research and RNA therapy development. Unlike long dsrnas in non-mammalian cells, designing sirnas has proven difficult. First, within a gene sequence, only certain sequence motifs can act as templates for siRNA. Despite the development of many algorithms over the past years, the identification of these motifs is almost a trial-and-error process. Second, unlike long dsRNA in non-mammalian cells, siRNA has a substantially low gene silencing efficiency in mammalian cells (Reynolds A et al Nature Biotech 22:326,2004; A. de Fougeroles et al Nat Rev drug Discov 6,443 (2007)). Finding highly efficient siRNA motifs along mRNA is difficult and impossible for some mRNAs, as there are an almost unlimited number of possible motifs of 20-25nt in the mRNA. Third, delivery of negatively charged siRNA to mammalian cells after siRNA design has proven to be a formidable challenge (Li CX, et al Cell Cycle 5:2103-2109 (2006)).

It is highly desirable if a technology can be used to make long dsrnas in mammalian cells. Due to strong cellular innate immune responses, such as non-specific Interference (IFN) responses, no successful case has been reported to date for the introduction of exogenous long dsRNA directly into target mammalian cells. (Yang, S. et al mol.cell.biol.21:7807-7816 (2001)).

Summary of The Invention

In one embodiment, the present invention provides a solution to the above problem by using a bacterial-mediated system. Bacterial innate immune responses are unresponsive to long dsrnas, and may act as a "shield" to prevent or mitigate the strong innate immune response of mammalian cells to long dsrnas. In accordance with the present invention, bacteria act as intermediaries to address the incompatibility between long dsrnas and mammalian cells by performing the work of preparing, processing, and presenting the processed long dsrnas and their products to a target mammalian cell (e.g., a mammal or other eukaryotic cell), thereby avoiding a strong innate immune response. To accomplish this, bacteria with cell-invasive properties are transformed with vectors that each encode a long dsRNA comprising a sequence that is substantially complementary to the messenger rna (mrna) sequence of a target eukaryotic or viral gene. In one embodiment, the dsRNA is non-coding or non-coding for a protein. When these bacteria, containing long dsRNA and its metabolites, invade the host cell, a mixture of such RNA duplexes is sequestered from recognition by immune receptors on the cell surface. When the bacteria eventually release the expressed RNA into the cytoplasm of the mammalian cell, it is likely that some or all of the expressed long dsRNA has been processed into a mixture of short RNA duplexes by the bacteria and the mammalian dicer and/or dicer-like enzyme. Because of its great diversity covering almost all motifs of mRNA, some bacterial contents that have been further processed by mammalian enzymes and organelles will begin to function as gene-silencing RNAs that produce a diverse degree of gene silencing effect. Thus, the expression of the target eukaryotic or viral gene is effectively reduced. In one embodiment, gene silencing is attributable to a mixture of short RNA duplexes resulting from bacterial transfection, but the invention should not be construed as being limited to this interpretation.

Because the method of the present invention does not require screening of effective siRNA motifs for specific genes within mRNA and enables simultaneous synthesis and delivery, the present invention allows significantly simplified and upgraded gene targeting through RNAi in gene function studies and gene targeting therapies of applicable bacteria. For example, using synthetic sirnas to trigger RNAi, each mRNA of the target gene must be screened for effective sirnas, which is a trial-and-error process. For some genes, identification of effective siRNA motifs was unsuccessful. In contrast, the present invention relies on long dsrnas, which in one embodiment produce a mixture of short RNA duplexes to exert the effect of sequence-specific gene silencing, thereby avoiding the pre-screening process.

Since metabolites from long dsrnas produced by bacteria cover almost all motifs of mRNA, the present invention also protects against frequently mutated disease genes, as in the case of some viral genes like HIV. After designing and developing an effective siRNA, mutations can occur in target motifs of the gene, which can cause complete failure of the siRNA. In contrast, because the processed long dsRNA targets many motifs of disease or viral genes, bacteria-mediated gene targeting using long dsRNA can circumvent this problem.

The present invention also provides a powerful tool for target Identification and Validation, and is referred to as the therapeutic pathway Identification and ValidationProvided is a technique. For example, the present invention enables genome-wide approaches to be providedLibrary (otherwise construction is not possible) targeted discovery. The present invention also provides techniques for in vivo gene targeting that enable discovery, validation and differentiation of therapeutic targets. The RNAi effect from the method of the invention proves to be more efficient and specific than currently available RNAi techniques. For example, the methods of the invention can cause gene silencing in cancer stem cells that are notoriously difficult to genetically manipulate.

Thus, in general, the present invention provides systems, materials and methods relating to: bacteria are genetically engineered to transcribe non-small hairpin RNAs (or non-short hairpin RNAs, both abbreviated as "non-shRNA"). The non-shRNA may be long double-stranded RNA (dsRNA), long hairpin RNA (lhRNA), or polycistronic shRNA, or a mixture of any of the above. The non-shRNA may be non-coding or non-protein coding. In one embodiment, the non-shRNA is processed in bacteria into a mixture of shorter RNA duplexes prior to presentation to a target eukaryotic cell. In one feature, bacteria having invasive properties are selected for the present invention. Metabolites from bacteria are capable of regulating gene expression in target cells.

In one aspect, the invention provides prokaryotic vectors encoding long double-stranded rna (dsrna) or lhRNA under the control of one or more prokaryotic promoters. The dsRNA comprises a sequence that is substantially complementary (including fully complementary) to a messenger rna (mrna) sequence of a target eukaryotic or viral gene. In one embodiment, the prokaryotic metabolite of the dsRNA is capable of modulating the expression of a eukaryotic gene or a viral gene. In one embodiment, the vector includes at least two prokaryotic promoters (e.g., T7) that may be identical. Each promoter controls the expression of one or the other of the two substantially complementary strands of the dsRNA. In one embodiment, the vector is a circular double-stranded plasmid and the two prokaryotic promoters are arranged on complementary strands of the plasmid. In another embodiment, the vector may be integrated into the bacterial chromosome. In another embodiment, the vector is present as a free gene in a bacterium. For example, the eukaryotic gene whose expression is targeted by the vector may be an oncogene or an HIV gene.

In another aspect, the invention provides a bacterium comprising (a) an RNA molecule having a double-stranded region, such as a long double-stranded RNA (dsrna) or lhRNA, or (b) a DNA molecule encoding said RNA. A single-stranded DNA molecule may consist of two complementary strands, each encoding a corresponding RNA strand. The RNA includes a sequence that is substantially complementary to the mRNA sequence of the target eukaryotic or viral gene. In one feature, the length of the double-stranded region of the RNA is at least 40bp, 70bp, 100bp, 200bp, 400bp or 1000bp, and in another feature, the length of the double-stranded region of the RNA is no more than 2000 bp. In one embodiment, the bacteria may be invasive, non-pathogenic, and/or therapeutic. In one embodiment, the bacterium is capable of processing RNA into a mixture of shorter RNA duplexes capable of modulating expression of a target gene. To this end, the bacteria also contain enzymes or ribozymes capable of processing RNA into a mixture of shorter RNA duplexes, for example one or more endonucleases, such as bacterial ribonuclease III or a dicer or both. In embodiments, the bacteria contain enzymes that aid in the transport of genetic material after its release from the bacteria into the cytoplasm of a target eukaryotic cell. The enzyme may be a Hly protein (e.g., listeriolysin O encoded by the Hly a gene). The same DNA molecule or a different DNA encoding RNA may encode the Hly gene. In another embodiment, the bacterium is capable of modulating expression of a eukaryotic gene or a viral gene in a eukaryotic cell following introduction into the eukaryotic cell, i.e., without the need for a pathway through a mixture of short RNA duplexes. One specific example of a DNA molecule encoding an RNA is the vector described immediately above.

In another aspect, the invention provides a bacterium comprising a mixture of short RNA duplexes capable of modulating expression of a eukaryotic gene or a viral gene. Mixtures of short RNA duplexes can be generated from non-small hairpin RNAs (non-shRNAs). In one embodiment, the mixture of short RNA duplexes is effective to reduce expression of eukaryotic or viral genes. The non-shRNA can be a long double-stranded RNA, a long hairpin RNA, or a polycistronic shRNA.

In another aspect, the invention providesLibrary of libraries, saidThe library comprises a plurality of vectors, each vector comprising a cDNA molecule or a cDNA fragment from a cDNA library, a first promoter and a second promoter. The first promoter controls expression of one strand of the cDNA molecule or cDNA fragment, and the second promoter controls expression of the other strand of the cDNA molecule or cDNA fragment. And multiple vectors can transform bacteria. The cDNA library may be derived from total mRNA of mammalian cells. In one embodiment, the cDNA fragments are generated by digestion of cDNA molecules with restriction enzymes or by PCR reactions. Aspects of the invention also relate to one or more vectors found in such libraries.

The invention also provides a method for producing a bacterial cellIn another embodiment of the library, the bacterial cells comprise a plurality of the vectors described above. In one embodiment, the transcript of the cDNA molecule or cDNA fragment may form a long dsRNA. In one embodiment, double stranded RNA transcribed from both strands of a cDNA molecule or cDNA fragment is processed into a mixture of shorter RNA duplexes. An inventive aspect of the invention also relates to one or more bacteria found in such libraries.

In another aspect, the invention also provides various methods of using the vectors, bacteria and libraries of the invention for therapeutic and research uses. For example, in one aspect, methods are provided for identifying therapeutic agents, the methods comprising infecting a population of cells with a library of the invention and selecting cells having a phenotypic change to identify a therapeutic target.

In one aspect, the invention provides a method of studying a pathway component in vitro, the method comprising: providing a eukaryotic cell capable of performing a biological pathway of interest; infecting cells with a bacterium of the invention whose target eukaryotic or viral gene is suspected to be a component of the pathway of interest; and subsequently analyzing the cells for any effect on the pathway of interest. In one embodiment, a long dsRNA expressed in the bacterium and its product in the bacterium interferes with the mRNA of the suspected component, thereby modulating the pathway. In one embodiment, the pathway affects cell survival, growth, differentiation, senescence, autophagy, division, or death. In another embodiment, the target affects the survival, proliferation, and pathogenicity of an infectious organism, such as a virus. The eukaryotic cell can be an animal cell, a stem cell, a cancer cell, and the like. In one embodiment, the cell is a cancer stem cell.

In one aspect, the invention provides a method of studying a therapeutic target in vivo, the method comprising: providing a living animal having cells exhibiting a disease; delivering the bacteria of the present invention to those animal cells for bacterial infection (bactofection); and subsequently collecting the infected cells to examine the effect of the treatment. The animal cells may comprise xenografts and/or tumor cells.

In one aspect, the invention provides methods of introducing a mixture of short RNA duplexes into animal cells, the methods comprising generating, processing, and presenting at least one long double-stranded RNA (dsrna) to the animal cells without eliciting a significant immune response from the animal. The dsRNA may be processed into a mixture of shorter RNA duplexes. In one embodiment, the method comprises producing and processing the dsRNA in bacteria, infecting animal cells with the bacteria, and lysing the bacteria to release its contents. According to the present method, the contents of the bacteria can be further processed in animal cells to become a mixture of shorter RNA duplexes.

The invention also provides methods of modulating gene expression in a target eukaryotic cell, which may be mammalian, avian, or other eukaryotic cell. The method comprises infecting a target cell with a bacterium of the invention, wherein the eukaryotic gene or viral gene targeted by the bacterium is the gene to be modulated.

The invention further provides a method of treating or preventing a cancer or cell proliferation disease in a subject, the method comprising infecting cells of the subject with a bacterium of the invention, wherein the bacterium-targeted eukaryotic gene or viral gene is a gene known to upregulate cell proliferation. In one embodiment, the subject is a mammal, bird, or other type of eukaryote.

The invention further provides methods of treating or preventing a disease caused by a viral infection in a subject. The method comprises the following steps: infecting cells of a subject with a bacterium of the invention, wherein the eukaryotic gene or viral gene targeted by the bacterium is involved in the pathogenicity of the virus.

The invention further provides a method of treating or preventing a disease caused by an altered gene in a subject, the method comprising infecting cells of the subject with a bacterium of the invention, wherein the bacterially targeted eukaryotic gene or viral gene is the altered gene. In one embodiment, the disease is caused at least in part by up-or down-regulation of the expression of the gene. In another embodiment, the disease is caused at least in part by one or more mutations in a gene.

Other features and advantages of the invention may be apparent from the description provided herein, including additional description in various embodiments. The examples provided illustrate the various components and methods used to practice the invention. The embodiments are not limiting of the claimed invention. Based on the disclosure, one can identify and use other components and methods for practicing the invention without departing from the principles of the invention. All embodiments may be used in combination with each other.

Brief Description of Drawings

FIG. 1 is a schematic representation of an embodiment of the present invention.

FIG. 2 is a block diagram showing how embodiments of the present invention may be constructed and usedSchematic representation of the library.

FIG. 3 is a schematic diagram of the construction of a duplex according to an embodiment of the present inventionPlasmidsSchematic representation of (a).

Fig. 4 includes FACS plots in the left and microscopic images in the right, demonstrating the isolation of Cancer Stem Cells (CSCs) according to an embodiment of the present invention.

Fig. 5 shows immunofluorescence images of CSCP1 protein expression analysis in CSCs treated with the bacterial targeting CSCP1 of the present invention (right panel) and a control (left panel).

Fig. 6 includes single-channel images (top panel) and intensity profiles (bottom panel) of CSCP3 protein expression analysis in CSCs treated with the bacterial targeted CSCP3 of the invention (right panel) and a control (left panel).

Fig. 7 includes microscopic images of annexin V-FITC staining assays in CSC populations treated with the bacterial targeting of the invention CSCP3 (right panel) and control (left panel).

Figure 8 shows another set of microscopy images of annexin V-FITC staining assays in CSC populations treated with the bacterial targeted CSCP3 of the present invention (right panel) and a control (left panel).

Fig. 9 includes microscope images of CSC spheres (spheres) treated with the bacteria-targeting CSCP3 of the present invention before (top left panel) and after (bottom left panel) addition of trypan blue. The right graph quantifies the effect of the bacteria of the invention on the CSC population drawn on the left.

Fig. 10 includes left CSCP3 protein expression western blot images, and right panels showing the viability statistics from differentiated cancer cells treated with the inventive bacterial targeted CSCP3 according to one experiment.

Detailed Description

As used herein, "modulate" refers to increasing or decreasing (e.g., silencing), in other words, up-regulating or down-regulating. As used herein, "introducing" or "delivering" a microorganism to a target cell refers to the process of infecting the target cell with the microorganism (e.g., a bacterium), and in some cases possibly releasing genetic material within the microorganism to a desired location (e.g., cytoplasm) of the target cell by lysing the microorganism.

The invention provides a platform technology for target discovery and gene silencing treatment. In one aspect, the present system produces and uses invasive bacteria to deliver DNA encoding long dsRNA or long dsRNA, or both, to a mammalian cell or other type of eukaryotic cell to exert an RNA interference (RNAi) effect in the eukaryotic cell. The RNA of the present invention is a non-small hairpin RNA (non-shRNA). In one embodiment, the RNA is non-coding. As used herein, "non-coding" or "non-protein-coding" means that the sequence is not translated into a protein. In one embodiment, the non-small hairpin RNA is long double-stranded RNA (dsRNA), long hairpin RNA (lhRNA), or polycistronic shRNA (Kim & Rossi, Nature Rev. Genet.8:173-184 (2007)). In a preferred embodiment, the non-shRNA is a non-coding long dsRNA which is digestible or processable into shorter fragments in bacterial cells. In one mechanism, the long dsRNA is processed into a mixture of shorter RNA duplexes. Genetic material processed by bacteria from long dsRNA can be introduced into an animal host without being detected by the host cell immune system and continues to regulate gene expression in the host cell through efficient post-transcriptional silencing and other mechanisms. The eukaryotic cell may be a mammalian, avian, or other eukaryotic cell. The gene of interest may be a mammalian, avian, bacterial, eukaryotic or viral gene.

By adopting a novel gene silencing technology, the invention provides a powerful tool for analyzing the gene function in vitro and in vivo; this tool is called Therapeutic Pathway Identification and ValidationProvided is a technique.

In one feature, the invention provides research tools for in vitro target discovery and validationThis aspect of the invention finds application in any cell population, including Cancer Stem Cells (CSCs) or other cell populations having unknown or yet not completely defined biological pathways, including non-cancer stem cells. Because the present invention is capable of causing gene silencing in CSCs, its confirmed potency is particularly advantageous for use in small cell populations, non-culturable cell populations, or those cell populations that are not amenable to routine genetic manipulation.

In another feature, the invention provides in vivo gene knockdown techniques that effectively identify, confirm, and/or differentiate therapeutic targets. The present invention is applicable to any in vivo disease model in which bacteria may be administered, however current knockdown techniques cannot be used for any established disease model.

In another feature, the invention also enables genome-wide approaches for target discovery. Use ofThe RNAi library constructed by the technique can be genome-wide or gene family library. Compared with the current synthetic siRNA library and RNAi vector library,libraries are less labor intensive and less expensive. In addition to this, the present invention is,libraries are able to find new genes and other libraries are not. Therefore, the invention provides a powerful tool for systematically detecting gene functions on a genome-wide scale and also provides a powerful method for screening potential therapeutic targets related to diseases.

In one advantageous aspect, the invention provides methods of producing and using bacteria, preferably non-pathogenic or therapeutic strains of bacteria, to present metabolites of non-shRNAs, in some cases mixtures of short RNA duplexes, into eukaryotic cells to exert RNA interference (RNAi) effects in eukaryotic cells. The non-shRNA may be a long double-stranded RNA (dsRNA), a long hairpin RNA (lhRNA), or a polycistronic shRNA. The non-shRNA has a double-stranded RNA region. The double stranded region is longer than the siRNA duplex, which is typically 20-25 bp. In one embodiment, the double stranded region is at least 40bp, 70bp, 100bp, 200bp, 400bp, 1000bp in length, and in another feature, no more than 2000bp in length.

As used herein, "mixture," "plurality," or "mixture" means at least two different sequences. For example, in one embodiment, the mixture of short RNA duplexes comprises more than 2,4, 8, 16, 50, 100, 200, 500, 1000, 2000, or 4000 short RNA duplexes that are not identical in sequence to each other. Conversely, "a plurality" means more than one, regardless of whether they are the same sequence.

In one embodiment, the short RNA duplex is generated by enzymatic or ribozyme digestion of the non-shRNA precursor. The enzyme may be an endonuclease. The endonuclease can be a member of the ribonuclease III family, such as bacterial ribonuclease III or a dicer, or a dicer-like enzyme.

In preferred embodiments, the non-shRNA is long double-stranded RNA (dsRNA), or long hairpin RNA (lhRNA). In one embodiment, the non-shRNA comprises a sequence that is substantially complementary to a sequence of the mRNA of the gene of interest in the target eukaryotic cell. Because they are substantially complementary, the two sequences need not be of the same or similar length, and 100% or complete complementarity is one example of substantial complementarity. In one embodiment, the non-shRNA precursor comprises an effective RNAi sequence against a gene of interest. In particular examples, the effective RNAi sequence is an siRNA sequence, but this is not always the case. Not every siRNA complementary to the target gene is effective in initiating RNAi degradation of the gene transcript. Indeed, time-consuming screening is often necessary to identify effective siRNA sequences. Genetic material comprising an "effective RNAi (or in one embodiment siRNA) sequence of a gene" or that can "effectively silence a gene" is capable of substantially reducing expression of the gene by at least 20%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90%, e.g., resulting in an observable phenotypic change.

The invention also provides bacteria, preferably non-pathogenic or therapeutic bacteria, for use in "shielding" or generating and processing non-shRNA into effective gene silencing products to circumvent the immune system of higher organisms. The bacterium of the present invention comprises (a) a non-shRNA, (b) a DNA encoding the non-shRNA, or (c) both of them. In one embodiment, the precursor comprises a non-encoded long dsRNA. The bacterium may further comprise an enzyme or ribozyme capable of processing the precursor into an siRNA. The enzyme may be an endonuclease such as bacterial ribonuclease III or a dicer. In one embodiment, the enzyme is endogenous to the bacterium. In another embodiment, the enzyme is exogenous to the bacterium and is introduced by a vector expressing the enzyme, e.g., a dicer-like enzyme.

Bacterial delivery is more attractive than viral delivery because it can be controlled by antibiotics and non-propagating attenuated bacterial strains. As such, bacteria are more amenable to genetic manipulation, which allows for the generation of vector strains specific for certain applications. In one embodiment of the invention, the method of the invention is used to produce bacteria that cause gene targeting in a tissue-specific manner.

The avirulent bacteria of the invention may enter mammalian host cells by a variety of mechanisms. Invasive bacterial strains have the ability to invade non-phagocytic host cells, as opposed to being taken up by specialized phagocytic cells, which often lead to destruction of the bacteria by specialized lysosomes. Natural examples of such bacteria are intracellular pathogens such as Listeria (Listeria), Shigella (Shigella) and Salmonella (Salmonella), but this property can also be transferred to other bacteria such as escherichia coli (e.coli) and bifidobacteria (bifidobacterium) including probiotics (p.courvalin, s.goussard, c.grillot-Courvalin, c.r.acad.sci.paris 318,1207(1995)) by transferring invasion-related genes. In other embodiments of the invention, the bacteria used to deliver interfering RNA to the host Cell include Shigella flexneri (Shigella flexneri) (D.R. Sizemore, A.A.Branstrom, J.C.Sadoff, Science 270,299(1995)), invasive E.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, Natechnol 16,862(1998)), Yersinia enterocolitis (Yersinia enterocolitica) (A.Al-Maririi A, A.Tibor, P.Lestresens, P.E.E.T.S.S.J.S.S.C.S.S.C.S.S.C.S.S.S.S.C.S.S.S.S.S.S.S.S.S.S.S.S.S.S.J.S.S.S.S.S.S.S.S.S.J.S.S.S.S.S.S.S.S.Ser. Ser. No. Ser. No. Ser. No. 6.No. Ser. No.6, No. Ser. No.6, No. Ser. No. Ser. No.8, No. Ser. 8, No. Ser. No.6, No. Ser. No. Ser. No.8, No.6, No. Ser. No.1, No.8, No.1, No. Ser. 8, No. Ser. Ser. Any invasive bacterium is used for DNA transfer into eukaryotic cells (s. weiss, t. chakraborty, CurrOpinion Biotechnol 12,467 (2001)).

Referring to FIG. 1 in embodiments of the invention, a mixture of multiple short RNA duplexes is produced in invasive bacteria, and preferably non-pathogenic bacteria, and then taken up by target eukaryotic cells. First, a prokaryotic vector, such as a plasmid, encoding non-shRNA containing a sequence substantially complementary to the mRNA sequence of the target gene is constructed and transformed into bacteria. Expression of non-shRNA is under the control of one or more prokaryotic promoters (e.g., T7). The plasmid shown in FIG. 1 has two prokaryotic promoters. In embodiments where the non-shRNA is a long dsRNA, each promoter controls the expression of one complementary strand of the dsRNA. Exemplary plasmids will be described in further detail in the examples section and with reference to fig. 3. Once the non-shRNA precursor is transcribed in the bacterium, it is digested into multiple fragments, e.g., short RNA duplexes, by endogenous bacterial ribonuclease III or exogenous dicer-like enzyme. Of course, at any given time, the bacterium may contain a mixture of non-shRNA, digestion fragments (e.g., short RNA duplexes), and DNA encoding RNA.

Still referring to fig. 1, in the process of bacterial invasion ("bacterial infection"), bacteria are taken up by the target eukaryote, for example, by endosomes. The bacterial content including the digestion product (e.g., short RNA fragment) of the non-shRNA is released in the cytoplasm upon bacterial lysis, resulting in targeted gene knockdown or silencing. Bacterial ribonuclease III can be replaced with dicer by expressing the dicer gene and deleting the ribonuclease III gene in the bacteria. Dicer has been reported to cleave long dsrnas into shorter fragments of 12-30 nucleotides using a so-called "size mechanism".

Alternatively, after the non-shRNA precursors and their bacterial digests are introduced into eukaryotic cells, they can be further processed and digested by eukaryotic enzymes, including dicers in eukaryotic cells. This may facilitate efficient RNAi by metabolites from non-shRNA.

Release of bacterial DNA and RNA from intracellular bacteria can occur by a variety of mechanisms depending on the bacterial strain. In one embodiment, the bacterial DNA and RNA may comprise a mixture of long dsRNA, short RNA duplexes, and/or dsRNA encoding plasmids. One mechanism involves the class III export system in salmonella typhimurium (s.typhimurium), which is a specialized polyprotein complex spanning the bacterial cell membrane whose function includes secretion of virulence factors extracellularly to allow signaling to the target cell, but which can also be used to deliver antigens into the target cell (russmann h.int J Med Microbiol,293:107-12(2003)) or by bacterial lysis and release of bacterial content into the cytoplasm. Lysis of intracellular bacteria is initiated by the addition of an intracellularly active antibiotic (tetracycline), or occurs naturally through attenuation of bacterial metabolism (auxotrophs) or through endosomes or lysosomes. After release of the eukaryotic transcript plasmid, dsRNA or siRNA is produced within the target cell, which in turn triggers a highly specific process of mRNA degradation, which leads to silencing of the target gene.

The present invention can be carried out using the naturally invasive pathogen Salmonella typhimurium. In one aspect of this embodiment, the strains of Salmonella typhimurium include SL 7207 and VNP20009(S.K. Hoiseth, B.A.D. Stacker, Nature 291,238 (1981); Pawelek JM, Low KB, Bermudes D.cancer Res.57(20):4537-44 (10/15 1997)).

In another embodiment of the invention, the invention is carried out using attenuated E.coli. In one example of this embodiment, the strain of E.coli is BM 2710(C.Grillot-Courvalin, S.Goussard, F.Huetz, D.M.Ojcius, P.Courvalin, Nat Biotechnol 16,862 (1998)). In a feature of this embodiment, the BM 2710 strain is engineered to have cell invasion properties by an invasion property particle, such as a plasmid encoding the Inv gene. According to another characteristic of the invention, the bacterium of the invention contains a vector having the gene Hly (listeriolysin O), since the Hly protein is considered important for the escape of genetic material from the input vesicle. Obviously, the vectors may be identical invasive plasmids. Thus, in one embodiment, the bacterium has a plasmid encoding the Inv and Hly genes. In one aspect of the invention, the plasmid is pGB2 inv-hly. In one example, the E.coli strain used in the present invention is BL21(DE3) pLysE.

The invention also includes the use of prokaryotic vectors or plasmids encoding non-shRNA precursors with invasive bacteria to cause RNA interference (RNAi) in eukaryotic cells. In one feature, the plasmid further comprises at least one prokaryotic promoter that controls expression of non-shRNA (e.g., non-coding long dsRNA).

The invention also provides methods of introducing a mixture of short RNA duplexes into eukaryotic cells using bacteria, preferably non-pathogenic or therapeutic strains of bacteria, to exert an RNA interference (RNAi) effect in the eukaryotic cells. The method can modulate or regulate gene expression in a target cell by effective post-transcriptional silencing. The eukaryotic cell may be a mammalian cell or an avian cell. The gene of interest may be a mammalian, avian, bacterial, eukaryotic or viral gene.

The invention has wide application as a research tool for identifying and confirming pathway components and therapeutic targets in vitro and in vivo. The present invention can be used for all types of cells, including cells that are difficult to study, such as Cancer Stem Cells (CSCs). The system of the invention can also be used to generate RNAi libraries as a tool for genome-wide or gene family-specific target discovery.

The invention is also useful for the development of therapeutic agents and drugs. Diseases and conditions treatable by the methods of the invention include cancer, cell proliferative disorders, viral interference, diseases caused by gene mutations, and the like.

RNA interference

RNA interference (abbreviated RNAi) is a double-stranded RNA (dsrna) -induced cellular process for targeted degradation of single-stranded RNA (ssrna). The ssRNA is a gene transcript, such as messenger RNA (mRNA). RNAi is a form of (most) post-transcriptional gene silencing in which dsRNA can specifically interfere with the expression of genes having sequences complementary to the dsRNA. The antisense RNA strand of dsRNA targets complementary gene transcripts, such as messenger RNA (mrna), for cleavage by ribonucleases in the RNA-induced silencing complex (RISC), which is a ribonuclease containing multiple protein complexes.

RNAi has been shown to be a common cellular process in many eukaryotes. RISC and dicer are conserved in the eukaryotic domain. RNAi is believed to play a role in the immune response to viruses and other foreign genetic material.

Double-stranded RNA (or dsRNA) is RNA having two complementary strands. dsRNA forms the genetic material of some viruses. In non-mammalian cells, where long dsrnas act as initiators for initiation of RNA interference processes, shorter RNA duplexes must be used to avoid non-specific innate immune responses. In one embodiment of the present invention, the non-shRNA has a double-stranded region of at least 40 bp. In another embodiment, the long dsRNA of the invention is equal to or longer than 30bp, 40bp, 45bp, 50bp, 70bp, 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 1000bp or 2000 bp. In one embodiment, the long dsRNA is no more than 600bp, 800bp, 1500bp or 2000 bp. In one embodiment, the dsRNA of the invention does not contain any mismatches or bulges. In another embodiment, the dsRNA of the invention comprises a mismatch and/or a bulge (bulbge).

Small interfering rnas (sirnas) are a class of short double-stranded rna (dsrna) molecules that play multiple roles in biology. More notably, it is involved in the RNA interference (RNAi) pathway, wherein the siRNA interferes with the expression of a specific gene. In addition, sirnas may also play a role in processes such as antiviral mechanisms or the formation of genomic chromatin structures. siRNA has a relatively short double-stranded rna (dsrna) region with 2-3 nucleotide overhangs with 5 '-phosphate and 3' -hydroxyl ends. As used herein, siRNA is about 20-25 nucleotides in length. Some short RNA duplexes generated from non-shRNA (e.g., long dsRNA) may be siRNA.

Hairpin RNA (hrna) is a single-stranded RNA molecule that contains two stems in the form of complementary sequences, and a loop sequence between the complementary fragments. Due to the complementarity of the sense and antisense fragments, such RNA molecules tend to be in the form of hairpin shapes having a single-stranded RNA (loop) region and a double-stranded RNA (dsrna) region. (see, e.g., Svoboda & Di Cara, cell. mol. Life Sci.63:901-918 (2006)).

As used herein, a short hairpin RNA (shrna) is a hairpin RNA that is 50nt or less in length. The shRNA can be processed into siRNA by dicer, which is then incorporated into the siRNA-induced silencing complex (RISC).

As used herein, long hairpin RNA (lhrna) is hairpin RNA that is greater than 60nt in length. The lhRNA can be processed into a variety of shorter RNA duplexes by dicer, which can comprise siRNA. In one embodiment, the lhRNA is equal to or longer than 70nt, 80nt, 100nt, 150nt, 200nt, 400nt, 700nt, 1000nt, 1500nt, 2000, 4000, or 8000nt in length. In alternative embodiments, the dsRNA region of the lhRNA has a length equal to or longer than 25bp, 30bp, 40bp, 50bp, 70bp, 100bp, 200bp, 300bp, 500bp, 600bp, 700bp, 1000bp, 2000bp, or 4000 bp. In one embodiment, the dsRNA region of the lhRNA of the invention does not contain any mismatch or bulge. In another embodiment, the dsRNA region of the lhRNA of the invention comprises a mismatch and/or a bulge. (as above)

Dicers are members of the ribonuclease III family of ribonucleases. Dicers cleave long double-stranded RNA (dsrna), pre-microrna (mirna), and other short hairpin RNA (shrna) into short double-stranded RNA fragments including siRNA. The dicer catalyzes the first step in the RNA interference pathway and initiates the formation of the RNA-induced silencing complex (RISC), the catalytic component argonaute, an endonuclease able to degrade messenger RNA (mrna), the sequence of which is complementary to that of the siRNA guide strand.

Ribonuclease III is also found in bacteria. Bacterial ribonuclease III cleaves long double-stranded RNA (dsRNA) into siRNA about 12-30 nucleotides long, with ends identical to those produced by dicer. siRNAs generated with bacterial ribonuclease III also initiate the formation of RNA-induced silencing complex (RISC) and initiate RNAi when delivered into animal cells. (Wang & Bechhofer, J Bacteriol.179: 7379-

The invention provides a mixture (or "diversity", "mixture") of digestion products, such as short RNA duplexes from a ribonuclease III digest of a dicer/long dsRNA comprising a sequence substantially complementary to a target messenger RNA (mrna) sequence. A mixture of short RNA duplexes is at least as effective in eliciting RNAi as using a single type of siRNA molecule with an effective siRNA sequence. The present invention thus advantageously eliminates the need for time-consuming screens, which are often necessary to identify effective siRNA sequences, and removes the delivery challenges for siRNA, on the one hand.

2. Bacteria producing, processing and delivering RNA or DNA encoding RNA to eukaryotic cells

In the present invention, bacteria are not simple delivery tools. Rather, the bacteria can synthesize, process, and deliver gene targeting RNAs. Any microorganism capable of synthesizing and delivering a molecule, such as an RNA molecule, into the cytoplasm of a target cell by crossing the cell membrane and entering the cytoplasm of the cell may be used to deliver RNA into such cells. In a preferred embodiment, the microorganism is a prokaryote. In an even more preferred embodiment, the prokaryote is a bacterium. Also within the scope of the invention are microorganisms other than bacteria that may be used to deliver RNA to cells. For example, the microorganism may be a fungus, such as Cryptococcis neofomans, protozoa, such as Trypanosoma cruzi (Trypanosomaticum ruzi), Toxoplasma gondii (Toxoplasma gondii), Leishmania donovani (Leishmania donovani) and Plasmodium falciparum (Plasmodia).

As used herein, the term "invasive" when referring to a microorganism, such as a bacterium, refers to a microorganism capable of delivering at least one molecule, such as RNA or a DNA molecule encoding RNA, to a target cell. An invasive microorganism may be one that is capable of passing through the cell membrane, thereby entering the cytoplasm of the cell and delivering at least some of its contents, such as RNA or DNA encoding RNA, to the target cell. The process of delivering at least one molecule to the target cell preferably does not significantly modify the invasive device. In a preferred embodiment, the microorganism is a bacterium. Preferred invasive bacteria are bacteria capable of delivering at least one molecule, e.g., RNA or a DNA molecule encoding RNA, to a target cell, e.g., by entering the cytoplasm of a eukaryotic cell. Preferred invasive bacteria are bacteria such as live invasive bacteria. Invasive microorganisms include microorganisms that are naturally capable of delivering at least one molecule to a target cell, such as by crossing a cell membrane, e.g., a eukaryotic cell membrane, and entering the cytoplasm, as well as microorganisms that are not naturally invasive but have been modified, e.g., genetically modified, to become invasive. In another preferred embodiment, a non-naturally invasive microorganism can be modified to become invasive by linking the bacterium to an "invasion factor", also known as an "entry factor" or "cytoplasmic targeting factor". As used herein, an "invasion factor" is a factor, such as a protein or a group of proteins, that when expressed by a non-invasive cell renders the bacterium invasive. As used herein, an "invasion factor" is encoded by a "cytoplasmic targeting gene". Naturally invasive microorganisms, such as bacteria, may have some tropism, i.e. towards preferred target cells. Alternatively, a microorganism, such as a bacterium, can be modified, e.g., genetically modified, to mimic the tropism of a second microorganism.

Delivery of at least one molecule to a target cell can be determined according to methods known in the art. For example, the presence of the molecule can be detected by hybridization or PCR methods, or by immunological methods including the use of antibodies, whereby the expression of the silenced RNA or protein is reduced. Determining whether the microorganism is sufficiently invasive for use in the present invention can include determining whether there is sufficient RNA delivered to the host cell relative to the number of microorganisms contacted with the host cell. If the amount of RNA is low relative to the number of microorganisms used, it may be desirable to further modify the microorganisms to increase their invasive potential.

Bacterial entry into cells can be determined by a variety of methods. Intracellular bacteria survive treatment with aminoglycoside antibiotics, while extracellular bacteria are rapidly killed. Quantitative assessment of bacterial uptake can be accomplished by inactivating extracellular bacteria by treating a monolayer of cells with the antibiotic gentamicin, and then removing the antibiotic before releasing viable intracellular organisms with mild detergents and determining viable numbers on standard bacteriological media. In addition, bacterial entry into cells is observed directly, for example, by thin-section-transmission electron microscopy of the cell layer or by immunofluorescence techniques (Falkow et al (1992) Annual Rev. cell biol.8: 333). Thus, a variety of techniques can be used to determine whether a particular bacterium is capable of invading a particular type of cell, or to verify bacterial invasion, e.g., after modification of a bacterium to modify its tropism to mimic the tropism of a second bacterium. The bacteria that deliver RNA according to the methods of the invention are preferably non-pathogenic. However, pathogenic bacteria may also be used as long as their pathogenicity has been attenuated, thereby rendering the bacteria harmless to the subject to which they are administered. As used herein, the term "attenuated bacteria" refers to bacteria that have been modified to significantly reduce or eliminate their damage to a subject. Pathogenic bacteria can be attenuated by a variety of methods as described below.

Without wishing to be bound by a particular mechanism of action, bacteria that deliver RNA into eukaryotic cells may enter multiple compartments of the cell, depending on the type of bacteria. For example, the bacteria may be in vesicles, for example phagocytic vesicles. Once inside the cell, the bacteria can be destroyed or lysed and their contents can be delivered into the eukaryotic cell. Bacteria can also be engineered to express phagosome degrading enzymes to allow RNA to leak from the phagosome. In some embodiments, the bacteria can remain viable in eukaryotic cells for different periods of time and can continue to produce RNA. The RNA or DNA encoding the RNA can then be released from the bacteria into the cell, for example by leakage. In certain embodiments of the invention, the bacteria may also replicate in eukaryotic cells. In a preferred embodiment, the bacterial replication is not significantly toxic to the host cell. The present invention is not limited to the delivery of RNA or RNA-encoding DNA by a particular mechanism and is intended to include methods and compositions that allow for the synthesis and/or delivery of RNA or RNA-encoding DNA by an independent bacterial delivery mechanism.

Described below are examples of bacteria that have been described in the literature as naturally invasive (part 2.1), and bacteria that have been described in the literature as naturally non-invasive (part 2.2), as well as naturally nonpathogenic or attenuated bacteria. Although some bacteria have been described as non-invasive (section 2.2), these are still sufficiently invasive to be used according to the invention. Whether or not conventionally described as naturally invasive or non-invasive, any bacterial strain may be modified to modulate, in particular increase, its invasive characteristics (e.g. as described in section 2.3).

2.1 Natural invasive bacteria

The particular naturally invasive bacteria used in the present invention is not critical. Examples of such naturally occurring invasive bacteria include, but are not limited to, Shigella species, Salmonella species, Listeria species, Rickettsia species (Rickettsia spp.), and enteroinvasive E.coli. The particular Shigella strain used is not critical to the present invention.

Examples of Shigella strains useful in the present invention include Shigella flexneri 2a (ATCC No.29903), Shigella sonnet (ATCC No.29930), and Shigella disteriae (ATCC No. 13313). Attenuated Shigella strains, such as Shigella flexneri 2a 2457T aroA virG mutant CVD 1203(Noriega et al, supra), Shigella flexneri M90T icsA mutant (Goldberg et al Infect Immun.,62:5664-5668(1994)), Shigella flexneri Y SFLl 14aroD mutant (Karnell et al Vacc,10:167-174(1992)) and Shigella flexneri aroD mutant (Verma et al Vacc,9:6-9(1991)) are preferred for use in the present invention. Alternatively, a new attenuated shigella species strain may be constructed by introducing an attenuating mutation alone or in combination with one or more additional attenuating mutations.

At least one advantage of the shigella RNA vaccine vector is its tropism for lymphoid tissues in the mucosal surface of the colon. Furthermore, the initial site of Shigella replication is thought to be located within dendritic cells and macrophages, which are typically found on the basolateral surface of M cells in mucosal lymphoid tissue (McGhee, J.R. et al Reproduction, vitality, & Development 6:369 (1994); reviewed in Pascual, D.W. et al immunology 5:56 (1994)). As such, shigella vectors can provide a means to express antigens in 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 al Science 270:299 (1995); Courvalin, P. et al, complexes Rendus de 1 academy des Sciences series II-Sciences deIa View-Life Sciences 318:1207 (1995); Powell, R.J. et al, Molecular adaptation of the control of infection diseases (1996), F.Brown, E.Norrby, D.Burton and J.Mekalanos, eds Cold Spring Harbor Laboratory Press, W.Neodyrk.183; Anderson, R.J. et al, for the extraction of the microorganism, 1997)). In practical terms, the severely restricted host specificity of shigella supports the prevention of the diffusion of shigella vectors into the food chain by intermediate hosts. In addition, highly attenuated strains have been developed in rodents, primates and self-growing (plants) (Anderson et al (1997) supra; Li, A. et al Vaccine 10:395 (1992); Li, A. et al Vaccine 11:180 (1993); Karnell, A. et al Vaccine 13:88 (1995); Sansonetti, P.J. and J.Aronde Vaccine 7:443 (1989); Fontaine, A. et al Research in Microbiology 141:907 (907); Sansonetti, P.J. et al (1991) Vaccine 9: 416; Norega, F.R. et al Infection & Immunity62:5168 (1994); Norega F.R. Imity & Immunity & 64 (3055); Fanfet al (1990); F.R. 4 & gt) (64: 1990); Imity & gt & 64: 1996) and J.R. et al Infection & gt 4: 1996; Koctionk & r.64: 1996). This recent recognition would allow the development of well-tolerated shigella vectors for use in humans.

Chemical non-specific mutagenesis may be used, such as with reagents such as N-methyl-N' -nitro-N-nitrosoguanidine; or non-specific mutagenesis using recombinant DNA techniques, classical genetic techniques (such as Tn10 mutagenesis, P22-mediated transduction, lambda phage-mediated exchange and conjugal transfer); or by introducing attenuating mutations into bacterial pathogens using recombinant DNA techniques of site-directed mutagenesis. Recombinant DNA technology is preferred because of its more well-defined construction of strains. Examples of such attenuating mutations include, but are not limited to: (i) auxotrophic mutations, such as aro (Hoiseth et al Nature,291:238-239(1981)), gua (McFarland et al Microbiol. Path.,3:129-141(1987)), nad (Park et al J. Bact,170:3725-3730(1988), thy (Nnalue et al infection. Immun.,55:955-962(1987)), and asd (Curtiss, supra);

(ii) mutations which inactivate global regulatory function, such as cya (Curtiss et al, infection. Immun.,55:3035-3043(1987)), crp (Curtiss et al (1987), supra), phoP/phoQ (Groisman 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 (Miller et al, J. Bact,172:2485-2490(1990)) or ompR (Dorman et al, infection. Immun.,57:2136-2140(1989)) mutations;

(iii) mutations that modify stress responses, such as recA (Buchmeier et al, MoI. 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,18:295-329(1984)) and groEL (Buchmeier et al, Sci.,248:730-732(1990)) mutations;

(iv) mutations in 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' Hautelle et al Mol. Micro.,6:833-841(1992)), plcA (Menglaud 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 the topology of DNA, such as top A (Galan et al, infection. Immun.,58: 1879-;

(vi) mutations that block or modify the Cell cycle, such as min (de Boer et al Cell,56:641-649 (1989)).

(vii) Introduction of genes encoding suicide systems, such as sacB (Recorbet et al, App. environ. micro.,59: 1361-. 1366 (1993); Quandt et al, Gene,127:15-21(1993)), nuc (Ahresholtz 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 the biological origin of lipopolysaccharide and/or lipid A, such as rFb (Raetz in Escherichia coli and Salmonella typhimurium, Neidhardt et al, ASM Press, Washington D.C. 1035-1063 (1996)), galE (Hone et al J. Infect. Dis.,156:164-167(1987)) and htrB (Raetz, supra), msbB (Reatz, supra)

(ix) Introduction of a phage lysis system, such as the lysosome encoded by P22 (Rennell et al Virol,143:280-289(1985)), lambda murein transglycosylase (Bienkowska-Szewczyk et al mol. Gen. Genet.,184:111-114(1981)) or the S gene (Reader et al Virol,43:623-628 (1971));

and

attenuating mutations may be constitutively expressed or under the control of inducible promoters, such as the temperature-sensitive heat shock family of promoters (Neidhardt et al, supra), or anaerobically inducible nirB promoters (Harbome et al Mol. Micro.,6: 2805. sup. 2813(1992)) or repressible promoters, such as uapA (Gorfikiel et al J.biol. chem.,268: 23376. sup. 23381(1993)) or gcv (Stauffer et al J.Bact,176: 6159. sup. 6164 (1994)).

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

Examples of Salmonella strains useful in the present invention include Salmonella typhi (ATCC No.7251) and Salmonella typhimurium (ATCC No. 13311). Attenuated salmonella strains are preferred for use in the present invention and include salmonella typhi (s.typhi) -aroC-aroD (phone et al vacc.9:810(1991) and salmonella typhimurium-axoA mutants (massoeni et al micro. pathol.13:477 (1992)).

The particular rickettsia strain used is not critical to the present invention. Examples of Rickettsia strains usable in the present invention include Rickettsia (Rickettsia Rickettsiae) (ATCC No. VR149 and VR891), Rickettsia prowaseckii (Rickettsia prowaseckii) (ATCC No. VR233), Rickettsia tsutsutsugamushi (ATCC No. VR312, VRl 50 and VR609), Rickettsia morganii (Rickettsia mooseri) (ATCC No. VR144), and Rickettsia sibirica (Rickettsia Rickettsiae)) (ATCCno. VR151) and Schizoctonia heliotropium (Rochalimaea quinata) (ATCC No. VR358). Attenuated rickettsia strains are preferred for use in the present invention and may be constructed by introducing one or more attenuating mutations in groups (i) to (vii) of the shigella species described above.

The particular enteroinvasive E.coli strain used is not critical to the present invention. Examples of enteroinvasive E.coli useful in the present invention include E.coli strains 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 E.coli strains are preferred for use in the present invention and may be constructed by introducing one or more attenuating mutations in groups (i) to (vii) of the Shigella species described above.

Furthermore, because certain microorganisms other than bacteria can also interact with integrin molecules (which are receptors for certain invasion factors) for cellular uptake, such microorganisms can also be used to introduce RNA into target cells. For example, viruses, such as the foot mouth disease virus, echovirus and adenovirus, and fungal pathogens, such as histoplasma capsulatum (histoplasma apsum) and Leishmania major (Leishmania major) interact with integrin molecules.

2.2 less invasive bacteria

Examples of bacteria that may be used in the present invention and that have been described in the literature as non-invasive or at least less invasive than the bacteria listed in the previous section (2.1) include, but are not limited to, Yersinia species (Yersinia spp.), Escherichia species (Escherichia spp.), Klebsiella species (Klebsiella spp.), Bordetella species (Bordetella spp.), Neisseria species (Neisseria spp.), aeromonas species (aeromonas spp.), francisella species (franciella spp.), corynebacterium species (corynebacterium spp.), citric acid bacterial species (Citrobacter spp.), Chlamydia species (rhodobacter spp.), rhodobacter sp., Mycobacterium species (brevibacterium sp.), and rhodobacter sp., rhodobacter sp, Pseudomonas sp, Helicobacter sp, Vibrio sp, Bacillus sp and Erysipelothrix sp. It is necessary to modify these bacteria to increase their invasive potential. The bacteria may also be in a semi-viable state for improved stability and/or efficiency. The particular yersinia strain employed is not critical to the present invention.

Examples of yersinia strains useful 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 YeO3-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 described above for shigella species in groups (i) to (vii).

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

The particular Klebsiella strain used is not critical to the present invention.

Examples of klebsiella strains that can be used in the present invention include klebsiella pneumoniae (k.pneumoniae) (atccno.13884). Attenuated strains of Klebsiella are preferred for use in the present invention and may be constructed by introducing one or more attenuating mutations described above for Shigella species in groups (i) to (vii).

The particular bordetella strain used is not critical to the present invention.

Examples of bordetella strains useful in the present invention include bordetella tracheitis (b. bronchinseptica) (ATCC No. 19395). Attenuated bordetella strains are preferred for use in the present invention and may be constructed by introducing one or more attenuating mutations described above for use in groups (i) to (vii) of 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 (n.meningidis) (ATCC No.13077) and neisseria gonorrhoeae (n.gonorrhoeae) (ATCC No. 19424). Attenuated strains of Neisseria, such as the Neisseria gonorrhoeae MS11 aro mutant (Chamberlain et al micro. Path.,15:51-63(1993)) are preferred for use in the present invention. Alternatively, a novel attenuated neisseria strain may be constructed by introducing one or more attenuating mutations described above for shigella species in groups (i) to (vii). The particular aeromonas strain employed is not critical to the present invention. Examples of strains of aeromonas useful in the present invention include a. eucrenophila (ATCC No. 23309). Alternatively, a new attenuated aeromonas strain may be constructed by introducing one or more attenuating mutations described above for use in groups (i) to (vii) of shigella species.

The particular Francisella strain used 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). An attenuated Francisella strain is preferred for use in the present invention and may be constructed by introducing one or more attenuating mutations described above for Shigella species in groups (i) to (vii).

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

The particular citric acid bacterial strain used is not critical to the present invention. Examples of the citric acid bacterium strain that can be used in the present invention include citric acid bacterium freundii (c.freundii) (ATCC No. 8090). Attenuated citric acid bacterial strains are preferred for use in the present invention and may be constructed by introducing one or more attenuating mutations described above for use in groups (i) to (vii) of shigella species.

The particular chlamydia strain used is not critical to the present invention. Examples of chlamydia strains useful in the present invention include c.pneumoniae (ATCC No. vrl 310). Attenuated chlamydia strains are preferred for use in the present invention and may be constructed by introducing one or more attenuating mutations described above for use in groups (i) to (vii) of shigella species.

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

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

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

The particular legionella strain used is not critical to the present invention. Examples of legionella strains that can be used in the present invention include l.pneumophila (ATCC No. 33156). An attenuated legionella strain, such as the L.pneumophila mutant (Ott, FEMS Micro.Rev.,14:161-176(1994)) is preferably used in the present invention. Alternatively, a new attenuated legionella strain may be constructed by introducing one or more attenuating mutations described above for shigella species in groups (i) to (vii).

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

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

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

The particular salmonella strain used is not critical to the present invention. Examples of Salmonella strains useful in the present invention include Salmonella typhimurium (ATCC No.7251) and Salmonella typhimurium (ATCC No. 13311). Attenuated Salmonella strains are preferred for use in the present invention and include Salmonella typhi aroC aroD (Hone et al Vacc,9: 810-491 (1991)) and Salmonella typhimurium aroA mutants (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 described above for shigella species in groups (i) to (vii). The particular vibrio strain used is not critical to the present invention.

Examples of Vibrio strains that can be used in the present invention include Vibrio cholerae (Vibrio cholerae) (ATCCNO.14035) and Vibrio cincinnati (Vibrio cincinnataensis) (ATCC No. 35912). Attenuated Vibrio strains are preferred for use in the present invention and include Vibrio cholerae RSI virulence mutants (Taylor et al J.Infect.Dis.,170: 1518-. Alternatively, a novel attenuated Vibrio strain may be constructed by introducing one or more attenuating mutations described above for Shigella species in groups (i) to (vii).

The particular bacillus strain used is not critical to the present invention. Examples of Bacillus strains useful in the present invention include Bacillus subtilis (ATCC No. 6051). Attenuated Bacillus strains are preferred for use in the present invention and include the 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 described above for shigella species in groups (i) to (vii).

The particular strain of erysipelothrix used is not critical to the present invention. Examples of the Erysipelothrix strain that can be used in the present invention include Erysipelothrix rhusiopathiae suis (ATCC No.19414) and Erysipelothrix tonsillus (ATCC No. 43339). Attenuated Salmonella strains are preferred for use in the present invention and include erysipelothrix rhusiopathiae Kg-Ia and Kg-2(Watarai et al J.Vet.Med.Sci.,55: 595-. Alternatively, a new attenuated erysipelothrix strain may be constructed by introducing one or more attenuating mutations described above for shigella species in groups (i) to (vii).

2.3 methods for increasing the invasive Properties of a Strain

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

Examples of such genes, referred to herein as "cytoplasmic targeting genes", include genes encoding proteins capable of invasion by Shigella, or a similar invasive gene of enteroinvasive E.coli, or Listeria lysin O of Listeria, as such techniques are known to produce a wide range of invasive bacteria capable of invading and entering the cytoplasm of animal cells (Fo. nal et al, Immun, 46:465 (1984); Bilececke et al Nature,345:175-176 (1990); Small et al, Microbiology-1986, pp 121-124, Levine et al, Amcan Society for Microbiology, Washington, D.C. (1986); Zychlinsky et al, Micro, 11:619-627 (1994); Gentsv et al (1995) Infeency & 63: 2; Isberg, R420262. and Nature R765: 1989; Cell R769: 769). Methods for transferring the above-described cytoplasmic targeting genes into bacterial strains are well known in the art. Another preferred gene encoding an invasin protein from Yersinia pseudotuberculosis (Yersinia pseudouberculosis) may be introduced into the bacterium to increase its invasion profile (Leong et al EMBO J.,9:1979 (1990)). Invasin may also be introduced in combination with listeriolysin, thereby further increasing the invasive characteristics of the bacteria relative to the introduction of either of these genes. For illustrative purposes, the above genes have been described; however, it is obvious to the person skilled in the art that any gene or combination of genes from one or more sources will suffice which is involved in the delivery of molecules, in particular RNA or DNA molecules encoding RNA, from a microorganism into the cytoplasm of a cell, for example an animal cell. Thus, such genes are not limited to bacterial genes, and include viral genes, such as influenza virus hemagglutinin HA-2, which promotes endosomal lysis (endomolysis) (Plank et al J.biol.chem.,269:12918-12924 (1994)). The above cytoplasm-targeting gene can also be obtained from DNA isolated from invasive bacteria carrying the desired cytoplasm-targeting gene by, for example, PCR amplification. Primers for PCR can be designed from nucleotide sequences available in the art, e.g., the references listed above and/or GenBank publicly available on the Internet (www.ncbi.nlm.nih.gov /). PCR primers can be designed to amplify a cytoplasmic targeted gene, a cytoplasmic targeted operon, a cytoplasmic targeted gene cluster, or a cytoplasmic targeted gene modulator. The PCR strategy used will depend on the genetic structure of the cytoplasmic targeted gene or genes in the target invasive bacterium. PCR primers are designed to contain sequences homologous to the DNA sequence at the beginning and end of the target DNA sequence. The Bacterial target may then be introduced into the cytoplasm, for example, by using Hfr transfer or plasmid mobilization (Miller, A Short Course in 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; and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.), and physical transformation techniques (Johnston et al supra; and Bothwell et al supra). The cytoplasmic targeting gene can be integrated into soluble phages (de Boer et al Cell,56:641-649(1989)), plasmid vectors s (Curtiss et al supra) or spliced into the chromosome of the target strain (Hone et al supra).

As mentioned above, in addition to genetically engineering bacteria to improve their invasive properties, bacteria can also be modified by attaching an invasion factor to the cell. Thus, in one embodiment, the bacteria are rendered more invasive by covalently or non-covalently coating the bacteria with an invasive factor, such as an invasin, invasin derivative or fragment thereof having sufficient invasiveness. In fact, it has been shown that non-invasive bacterial cells are coated with purified invasins from Yersinia pseudotuberculosis or that the carboxy-terminal 192 amino acids of the invasins are able to enter mammalian cells (Leong et al EMBO J.9:1979 (1990)). In addition, latex spheres coated with the carboxy-terminal region of an invasin are efficiently internalized by mammalian cells, such as Staphylococcus aureus (Staphylococcus aureus) strain (reviewed in Isberg and Trail van Nhieu Ann. Rev. Genet.27:395(1994)) which is immobilized with an antibody. Alternatively, the bacteria may also be coated with an antibody, variant thereof or fragment thereof that specifically binds to a surface molecule recognized by a bacterial entry factor. For example, it has been shown that if bacteria are coated with monoclonal antibodies directed against integrin molecules, such as α 5 β 1 (known as surface molecules that interact with bacterial invasin proteins), they are internalized (Isberg and Tran van Nhieu, supra). Such antibodies can be prepared according to methods known in the art. The efficacy of antibodies in modulating bacterial invasiveness is tested by, for example, coating the bacteria with an antibody, contacting the bacteria with eukaryotic cells having surface receptors recognized by the antibody, and monitoring the presence of intracellular bacteria according to the methods described above. Methods for attaching an invasion factor to a bacterial surface are known in the art, including cross-linking.

3. Target cell

The present invention provides methods for synthesizing, processing, and delivering RNA to any type of target cell. As used herein, the term "target cell" refers to a cell invaded by a bacterium, i.e., a cell having a surface receptor necessary for recognition by the bacterium.

Preferably, the target cell is a eukaryotic cell. Even more preferably, the target cell is an animal cell. An "animal cell" is defined as a nucleated, chloroplast-free cell derived from or present in a multicellular organism whose taxonomic (taxanomic) position is within the animal kingdom. The cells may be present in whole animals, primary cell cultures, explant cultures, or transformed cell lines. The particular tissue source of the cells is not critical to the invention. The recipient animal cells for use in the present invention are not critical thereto and include all organisms occurring in or derived from within animal kingdoms, such as those of mammals, fish, birds, reptiles.

Preferred animal cells are mammalian cells, such as human, bovine, ovine, porcine, feline, canine, caprine, equine and primate cells. Most preferably, the animal cell is a human cell.

In a preferred embodiment, the target cell is a mucosal surface. Certain enteropathogens, such as E.coli, Shigella, Listeria and Salmonella, are naturally suitable for this application because of the ability of these organisms to adhere to and invade the mucosal surfaces of the host (Kreig et al supra). Thus, in the present invention, such bacteria can deliver RNA molecules or DNA encoding RNA to cells in the mucosal compartment of the host.

Although certain types of bacteria have a certain consistency, i.e., tend towards preferred target cells, delivery of RNA or DNA encoding RNA to a certain type of cell can be accomplished by selecting bacteria that are tropic for the desired cell type or that are modified to be able to invade the desired cell type. Thus, as discussed above, for example, bacteria can be genetically engineered to mimic the tropism and invasive nature of mucosal tissue, thereby allowing the cells to invade mucosal tissue and deliver RNA or DNA encoding RNA to cells in these sites.

Bacteria can also target other types of cells. For example, bacteria can be targeted to human and primate red blood cells by modifying the bacteria to express Plasmodium vivax reticulocyte binding protein-1 or-2, or-1 and-2, on its surface, which specifically binds to red blood cells in humans and primates (Galinski et al Cell,69: 1213-. In another embodiment, the bacterium is modified to have on its surface an asialoglycoprotein that is a ligand for an 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 that has been shown to target plasmid uptake to cells with insulin receptors (Rosenkranz et al Expt. cell Res.,199:323-329 (1992)). Also within the scope of the invention are bacteria modified to have on their surface p60 of Listeria monocytogenes (Hess et al infection. Immun.,63: 2047-.

In another embodiment, the cell can be modified to become a target cell for an RNA-delivering bacterium. Thus, the cell may be modified to express a surface antigen recognized by the bacteria (i.e., a receptor for an invasion factor) for its entry into the cell. Cells can be modified by introducing into the cell a nucleic acid encoding a receptor for an invasion factor such that the surface antigen is expressed under desired conditions. Alternatively, cells may be coated with receptors for invasion factors. Receptors for invasion factors 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 NhieuAnn. Rev. Genet.27:395 (1994). Nucleotide sequences for integrin subunits can be found, for example, in GenBank, which is publicly available on the internet.

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

Examples of bacteria that naturally enter the cytoplasm of fish cells include, but are not limited to, Aeromonas (Aeromonas assalamicida) (ATCC No.33658) and Aeromonas salmonicida (Aeromonas schuberii) (ATCNo.43700). Attenuated bacteria are preferred for use in the present invention and include A.salmonidia vapA (Gustafson et al J.MoI.biol.,237:452-463(1994)) or A.salmonidia aromatic-dependent mutants (Vaughan et al infection. Immun.,61:2172-2181 (1993)).

Examples of bacteria that can 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 (S.galingarium) cya crp mutant (Curtiss et al (1987) supra) or the Salmonella enteritidis aroA aromatic-dependent mutant CVL30(Cooper et al infection. Immun.,62: 4739-.

Examples of bacteria that naturally enter the cytoplasm of a reptile cell include, but are not limited to, Salmonella typhimurium (ATCC No. 6994). Attenuated bacteria are preferred for use in the present invention and include attenuated strains such as the 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, so long as there are microorganisms that are native or have been modified to become invasive and then capable of invading such cells. Examples of microorganisms that can invade plant cells include Agrobacterium tumefaciens (Agrobacterium tumefaciens), which uses binding to a pilus-like structure of a plant cell by a specific receptor, and then delivers at least some of its contents into the plant cell by a process similar to bacterial binding.

Illustrated below are 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 No. CCL 62, CCL 159, HTB 151, 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 No. 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 No. CRL 6213, CCL 34, CRL 6202, CRL 6225, 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 equine derived cell lines include ATCC No. ccl 57 and CRL 6583.

Examples of deer cell lines include ATCC No. CRL 6193-6196.

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

3.1 cancer and non-cancer Stem cells

Cancer Stem Cells (CSCs) are a subpopulation of cancer cells that have characteristics commonly associated with stem cells, such as self-renewal and the ability to differentiate into multiple cell types. Recent studies have revealed that CSCs are highly tumorigenic, whereas a large number of cancer cells are non-tumorigenic. According to these studies, CSCs, although accounting for only a small fraction (typically less than 1%) of tumor cells, are the root cause of persistent malignant tumor growth and often are the initiator of metastasis. CSCs are susceptible to chemotherapy and other tumor-targeted therapies currently in common use in anti-hospitals.

CSCs are also difficult to isolate and also difficult to culture for laboratory purposes. Special isolation procedures are required and the cells tend to lose stem cell characteristics extremely rapidly in culture. Therefore, only narrow faces are typically used for in vitro CSC experiments. However, as shown in the examples described in the examples section below, the methods of the invention show surprising potency and specificity in causing gene silencing even in isolated stem cells.

Specifically, bacteria carrying the vector constructed according to the present invention significantly reduced the expression of the target genes (CSCP 1 and CSCP3, respectively). Phenotypic changes in the target cells, such as apoptosis (examples 3 and 4) and inhibition of sphere formation (example 5) also result from the treatment of the cells by the bacteria of the present invention, providing confirmation that the target genes play an important role in cell survival, division and death programs.

Because of the demonstrated ability to cause gene silencing in both minicell populations and non-culturable cell populations,the technology provides a platform for in vitro targeting/pathway discovery and for designing therapeutic regimens in any cell population that includes cancer stem cells and non-cancer stem cells that share some CSC properties.

The present invention also provides examples where substantial cell death in non-CSC cancer cells, i.e., "normal" or differentiated cancer cells, results from treatment with the bacteria of the present invention (example 6), further demonstrating the therapeutic potential of the present invention.

4. In vivo and in vitro research and drug development

The RNAi methods of the invention can also be used to generate transient "knockdown" genetic animal models, such as mouse models, as opposed to knockout models that are genetically engineered to discover and/or confirm gene function in vivo.

Currently, there is no good system for in vivo target validation. The current knockout animal model is time consuming, labor intensive, and requires gene targeting at the embryonic stage. Furthermore, current knockout animal models cannot be used to detect established tumors in vivo. Current RNAi delivery methods are also not suitable for in vivo target validation. In contrast, the bacteria-mediated RNAi methods of the present invention utilize systems suitable for rapid and efficient in vivo target validation, for example in the determination of tumors. In particular, bacteria are easier to control and target than viral vectors. In addition, the present methods mimic therapeutic intervention.

For example, transgenic mice with xenografted human tumors can be used as knock-down animal models to confirm the efficacy of candidate therapies by using the vectors and bacteria of the invention. Such non-pathogenic bacteria, once introduced into an animal model, will produce processed products of the non-shRNA, e.g., a mixture of short RNA duplexes of the mRNA for the target gene in tumor cells. This provides a tool for performing gene knockdown assays and detecting the anti-tumor activity of candidate treatments.

In vitro target validation can be readily accomplished using the present invention. In one embodiment, the assay comprises the following steps after the target of the phenotype (e.g., cancer growth) has been identified: constructing a vector encoding a non-shRNA (e.g., a long dsRNA) comprising a sequence substantially complementary to the mRNA of the target gene; transforming a live invasive bacterium with the vector; transfecting cancer cells in vitro with said live invasive bacterium; and observing whether cell proliferation is affected.

The methods of the invention are also useful as in vitro transfection tools for research and drug development.

These in vivo and in vitro methods use bacteria with desirable properties (invasiveness, attenuation, controllability). Such as Bifidobacteria (Bifidobacteria) and listeria, are used to perform the bacteria-mediated RNAi methods of the present invention. Plasmids are used to confer invasiveness and eukaryotic or prokaryotic transcripts of one or several dsrnas to bacteria.

5. Pharmaceutical composition

In a preferred embodiment of the invention, the invasive bacteria containing RNA molecules and/or DNA encoding such molecules are introduced into the animal by intravenous, intramuscular, intradermal, intraperitoneal, oral, intranasal, intraocular, intrarectal, intravaginal, intraosseous, buccal, immersion, and intraurethral infusion (infusion) routes.

The amount of live invasive bacteria of the invention to be administered to a subject varies depending on the class of the subject, as well as the disease or condition to be treated. Typically, the dose used will be about 103 to 1015 viable bacteria, preferably about 104 to 1012 viable bacteria per subject. The bacteria can be prepared in lyophilized or spore form for storage or pharmaceutical use.

The invasive bacteria of the present invention are typically administered together 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 buffered saline, buffers that buffer gastric acid in the stomach, such as citrate buffer with sucrose (pH 7.0), bicarbonate buffer 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 buffer with ascorbic acid, lactose and optionally aspartame (pH 7.0) (Levine et al Lancet,11:467-470 (1988)). Examples of carriers include proteins such as found in skim milk, sugars (e.g. sucrose) or polyvinylpyrrolidone. These carriers will generally be used in concentrations in the range of about 0.1-30% (w/v), but preferably 1-10% (w/v).

Shown below are other pharmaceutically acceptable carriers or diluents that can be used for the specific route of delivery. Any such carrier or diluent may be used to administer the bacteria of the present invention, so long as the bacteria are still able to invade the target cells. In vitro or in vivo testing for invasiveness can be performed to determine the appropriate diluents and carriers. The compositions of the present invention can be formulated for various types of administration, including systemic and local administration or localized administration. Lyophilized forms are also included, so long as the bacteria are invasive after contact with the target cells or administration to the subject. Techniques and formulations are commonly found in Remmington's Pharmaceutical Sciences, MeadePublishing co., Easton, Pa. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal and subcutaneous administration. For injection, the compositions of the invention, e.g., bacteria, can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution.

For oral administration, the pharmaceutical compositions may take the form, for example, by using pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or dibasic calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silicon dioxide); disintegrants (e.g., potato starch or sodium starch glycolate); or in the form of tablets or capsules prepared by conventional means with wetting agents such as 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. By using pharmaceutically acceptable additives such as suspending agents (e.g. sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous carriers (e.g. almond oil, oil esters, ethanol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl paraben or sorbic acid), such liquid formulations are prepared by conventional methods. The formulations also contain buffer salts, taste enhancers, colorants and, where appropriate, sweeteners.

Formulations for oral administration may be suitably formulated to give 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 for use according to the present invention are conventionally delivered in the form of a spray presentation 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, the dosage unit may be determined by providing a valve to deliver a measured amount. Capsules and cartridges of, for example, gelatin may be formulated for use in an inhaler or insufflator containing a powder mix of the composition, for example, bacteria, and a suitable powder base such as lactose or starch.

The pharmaceutical compositions may be formulated for parenteral administration by injection, for example by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., 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 water for injection, before use.

Pharmaceutical compositions may also be formulated in rectal, intravaginal or intraurethral compositions, for example suppositories or retention enemas containing conventional suppository bases such as cocoa butter or other glycerides. Systemic administration can also be by transmucosal or transdermal routes. 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 of bile salts and fusidic acid derivatives. In addition, detergents are also used to promote penetration. Transmucosal administration can be by nasal spray or by suppository. For topical administration, the bacteria of the present invention may be formulated into ointments, salves, gels, or creams as generally known in the art, so long as the bacteria remain invasive after contacting the target cells.

If desired, the compositions can be provided in a pack or dispenser device and/or kit containing one or more unit dosage forms containing the active ingredient. The package comprises, for example, a metal or plastic sheet, such as a blister pack. The packaging or dispensing device may have instructions for administration.

Invasive bacteria containing the RNA or DNA encoding the RNA to be introduced can be used to infect animal cells cultured in vitro, such as cells obtained from a subject. These in vitro infected cells can then be introduced into the animal, e.g., the subject from which they were originally derived, intravenously, intramuscularly, intradermally, or intraperitoneally, or by any infusion route that allows the cells to enter the host tissue. When delivering RNA into cells of an individual, the dose of living organism administered is about 0.1 to 106, preferably about 102 to 104 multiple infections of bacteria per cell.

In another embodiment of the invention, the bacteria may also deliver an RNA molecule encoding a protein to a cell, e.g., an animal cell from which the protein may then be collected or purified. For example, proteins can be produced in tissue culture cells.

6. Therapeutic and prophylactic uses

The RNAi methods of the invention are useful for the treatment and/or prevention of a variety of diseases, including those summarized in Dykxhoorn, Novina & Sharp. Nat. Rev. mol. cell biol.4:457-467 (2003); kim & Rossi, NatureRev.Genet.8:173-184 (2007); de Fougeroles, et al Nature Rev. drug Discov.6: 443-.

In one embodiment, the invention is useful as a cancer treatment or prevention. The method is effected by silencing or knocking down genes involved in cell proliferation or other cancer phenotypes. The bacterium of the present invention used for cancer treatment is preferably a bacterium modified to safely search out and kill tumors (Forbes, Nature Biot technology 24:1484-1485 (2006). the bacterium may be an obligate anaerobe such as Clostridium (Clostridium) novyi-NT, or a facultative anaerobe such as Salmonella typhimurium and Escherichia coli (supra).

Examples of these genes are k-Ras and β -catenin. In particular, k-Ras and β -catenin are targets for RNAi-based treatment of colon cancer. These oncogenes are viable and are relevant in most clinical situations. The bacteria-mediated RNAi method of the present invention is useful in the treatment and prevention of colon cancer that reaches the intestine. These methods are also useful for treating animals bearing xenograft tumors to treat and prevent cancer in the k-RasV12 model of intestinal tumorigenesis, and to prevent and treat tumors in the min mouse model of adenomatous polyposis coli (APC-min model). In this model, the mouse has a defective APC gene, resulting in the formation of many intestinal and colonic polyps, which is used as a familiar animal model for adenomatous polyposis coli (FAP) of intestinal tumorigenesis in humans.

The RNAi methods of the invention can also be used to treat or prevent viral diseases (e.g., hepatitis) and genetic disease conditions.

The RNAi methods of the invention may also be used to generate cancer-preventing "probiotics" for use, which have targets of the GI tract or liver, among others. The RNAi methods of the invention are useful as treatments for inflammatory diseases, such as hepatitis, Inflammatory Bowel Disease (IBD), or colitis. These methods are useful for silencing or knocking down non-oncogene targets (viral genes for the treatment and prevention of hepatitis B, hepatitis C; inflammatory genes for the treatment and prevention of inflammatory bowel disease) and other gene targets.

The RNAi methods of the invention are useful for delivering gene silencing into the digestive tract and colon (referred to as colon cancer treatment and prevention) and for oral application in the treatment of a variety of diseases. In another aspect of this embodiment, the delivery of gene silencing is parenteral.

Bacterially produced and/or delivered dsRNA can be used to treat viral infections, such as HIV, HBV, HCV, or other diseases, where genes causing a particular disease can be identified. Because the present invention does not require the identification of precise siRNA sequences effective against a gene causing a particular disease, whether endogenous or viral, the methods of the present invention are particularly advantageous for treating disease genes or strains that are frequently mutated or have many subgenotypes. A typical example is HIV carrying a frequently mutated gene.

7.Libraries

The invention also provides the term "Library "RNAi library, which may be genome-wide or gene family-specific.The library is a randomly constructed, double-stranded, self-mixing, size-controllable RNAi library. In vitro cell system RNAi library screening bacterial RNAi systems and microscopic imaging analysis based on the invention. In contrast to current siRNA/shRNA libraries, which require a priori knowledge of "good" siRNA sequences and have limited application to certain cell types (e.g., dividing cells and cells with viral receptors)The libraries can target known or unknown genes and can be applied to a wide range of cell types. Further, the inventionThe library is suitable for in vivo validation.

In an embodiment, referring to FIG. 2, of the inventionThe library comprises a plurality of vectors, each vector comprising one cDNA molecule or cDNA fragment from a cDNA library, a first promoter, and a second promoter; wherein the first promoter controls expression of one strand of the cDNA molecule or cDNA fragment and the second promoter controls expression of the other strand of the cDNA molecule or cDNA fragment. The cDNA fragments may be obtained from enzymatic digestion of cDNA molecules. In one embodiment, an approximately 500bp cDNA fragment is generated by enzymatic digestion. The constructed vector can be used for transforming bacterial cells. Once the vector transcribes the dsRNA from the cDNA sequence or cDNA fragment, the bacterial cell processes the dsRNA into a mixture of shorter RNA duplexes as previously described. After bacterial infection of the target cells, cells exhibiting the desired phenotypic change may be selected for further identification of the genetic target. The cDNA library or libraries may be derived from total mRNA of mammalian cells, or mRNA of a gene family or gene.

The invention further provides methods of screening RNAi libraries. The invention includesThe library infects mammalian cells and identifies mammalian cells having a change in at least one phenotype. The at least one phenotypic change is selected from the group consisting of nuclear number, nuclear morphology, cell death, cell proliferation, DNA fragmentation, cell surface markers, and mitotic index. The invention also includes sequencing the cDNA molecules of the vector in the identified mammalian cells.

RNAi libraries can be used to screen a wide range of disease-associated drug targets and identify potential therapeutic molecular drug targets. It can be used to assess the function of all human genes and perform functional genomics experiments with precursors unknown for the targeted gene. It can also be used to perform unbiased analysis in relevant cellular assays across different diseases and to infer multi-dimensional relationships of gene function in diseases.

RNAi libraries currently used in the art include mixed chemically synthesized siRNA libraries, viral vector-based shRNA libraries; and double-stranded RNA (dsRNA) in vitro digestion mixtures. These libraries need to be constructed or synthesized individually for known genes. They are typically limited in size and most of them are not even updateable. In the screening, these libraries are transfected into a cell system.

The present invention provides a facile method for generating and screening RNAi libraries. It simplifies the library construction process. In order to construct the RNAi library, it is not necessary to construct plasmids one by one. The present invention also eliminates time consuming and tedious purification steps of the product. In addition, the present invention can deliver RNAi directly through cell infection, thereby greatly reducing the cost of library screening.

The library may be operable across the entire genome or gene family. For each target, it has a self-mixing mixture of short RNA duplexes, rather than a single siRNA. Therefore, it is not necessary to synthesize siRNA one by one and to verify the effective order of siRNAAnd (4) columns. (Zhao HF, et al, Nature Methods 2: 967-973 (2005)). The present invention can avoid N-terminal function and thus lead to a thorough functional knock down.The screening of the library is a double blind selection based on selection and is unbiased, i.e. the genetic information is well balanced. In addition, useThe library is easily traceable to find candidate genes. In one embodiment, the use ofThe libraries were screened in transgenic and wild-type animals for different cancer-associated targets for therapeutic experiments.

The invention is further illustrated by the following examples which should not be construed as limiting in any way. The contents of all cited references, including documents cited in the entirety of this application, issued patents, published patent applications, are hereby expressly incorporated by reference, but are not to be considered prior art to the present invention. 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 explained in detail in the literature references. See, e.g., Molecular Cloning A Laboratory Manual, 2 nd edition (Cold spring harbor Laboratory Press:1989), by Sambrook, Fritsch and Maniatis; DNA Cloning, volumes I and II (d.n. glover, 1985); oligonucleotide Synthesis (m.j. gait, 1984); mullis et al U.S. Pat. Nos. 4,683,195; nucleic Acid Hybridization (B.D. Hames & S.J. Higgins, 1984); transcription AndTranslation (b.d. hames & s.j.higgins, 1984); culture Of Animal Cells (r.i. freshney, Alan r.loss, inc., 1987); immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the threading, Methods In Enzymology (Academic Press, Inc., N.Y.); gene Transfer Vectors for mammalian Cells (J.H.Miller and M.P.Calos, 1987, Cold Spring harbor laboratory); methods In Enzymology, volumes 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).

Examples

Examples are provided below to further illustrate various features of the present invention. The examples also illustrate useful methods for practicing the invention. These examples do not limit the invention.

Materials and methods

Plasmids

With reference to FIG. 3, buildThe plasmid may include an RNAi expression cassette with two T7 promoters. The desired DNA molecule was cloned into the plasmid via two XbaI sites.

For example, CSCP3/STAT3 and CSCP1/β catenin plasmids are purchased from origin technologies.A 782bp fragment (from nucleotide 1 to nucleotide 782) of the human STAT3 gene (GenBank accession No. NM-139276) or an approximately 300bp fragment (from nucleotide 11 to nucleotide 311) of CSCP3/STAT3 are inserted into an RNAi cassetteThe plasmid construct contained a 300bp fragment of the CSCP3/STAT3 gene, which was cloned into the ground using the following primersIn the plasmid:

CSCP3/STAT3-TPIV Forward 5'-GGATCTAGAATCAGCTACAGCAGC-3' (SEQ ID NO:1)

CSCP3/STAT3-TPIV reverse 5'-TCCTCTAGAGGGCAATCTCCATTG-3' (SEQ ID NO:2)

For CSCP1/βPlasmid, a 567 bp fragment (nucleotides 215 to 783) of the full-length human β catenin gene (GenBank accession NM-001904) was inserted using standard molecular cloning techniquesRNAi expression cassette in plasmid.

For vector construction, the T7 terminator was renatured and digested with BamHI/XbaI or XbaI/SalI according to standard molecular biology techniques. The terminator was then cloned intoBamHI/XbaI or XbaI/SalI sites of the vector:

BamHI positive direction:

5′ACGGATCCTCCTTTCAGCAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTTATTGCTCAGCGGTGGTCTAGAGGATCCAC 3’(SEQ ID NO：3)

BamHI reverse:

5’GTGGATCCTCTAGACCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGATCCGT 3’(SEQ ID NO：4)

SalI forward direction:

5’GCGTCGACTCTAGACCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGTCGACCG 3’(SEQ ID NO：5)

SalI is reversed:

5’CGGTCGACTCCTTTCAGCAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTTATTGCTCAGCGGTGGTCTAGAGTCGACGC 3’(SEQ ID NO：6)

CSC separation

Cancer Stem Cells (CSCs) are isolated by selecting for the presence of certain surface markers, such as CD44, and the absence of other specific surface markers, such as CD 24. Referring to FIG. 4, CD 44-cells and CD24-/CD44 high cells were isolated from FaDu human head and neck cancer cells by Fluorescence Activated Cell Sorting (FACS) (left panel). The cells were then cultured in the absence of adsorption and serum for a specified period of time to test their ability to form spheres (right panel).

The micrographic images in the right panel show that CD 44-cells do not have the ability to form spheres, whereas CD24-/CD 44-high cells have this ability, confirming that CD24-/CD 44-high cells are indeed cancer stem cells.

Example 1: RNAi against CSCP1 in Cancer Stem Cells (CSCs)

By carryingPlasmid E.coli cell BL21(DE3) pLysE treated SW480 human colon cancer Hoechst Subgroup (SP) cancer stem cells selected from the subgroup (side population). Using a carrying controlThe control cells (fig. 5, left panel) contained normal levels of CSCP1/β catenin, while CSCP 1-targeted bacteria triggered specific loss of CSCP1/β catenin by RNAi (right panel).

Example 2: RNAi against CSCP3 in Cancer Stem Cells (CSCs) selected by subpopulation

By means of a carrierPlasmid E.coli cells BL21(DE3) pLysE treated SW480 human colon cancer Hoechst Subgroup (SP) cancer stem cells prepared as in example 1. 24 hours after bacterial entry, cells were fixed and stained for CSCP3/STAT3 protein by standard immunofluorescence techniques. In fig. 6, the upper two panels show single channel immunofluorescence images. The next two figures show the intensity spectrum of CSCP3 with the pixel intensity scale provided in the far right figure. Cells treated with the targeted bacterium CSCP3/STAT3 (right panel) showed reduced CSCP3/STAT3 levels, demonstrating that RNAi was effectively performed by this bacterial-mediated system.

Example 3: RNAi against CSCP3 induced apoptosis in Cancer Stem Cells (CSCs)

The same cells as in example 2 and similarly treated cells were fixed and stained with annexin V-FITC 24 hours after bacterial entry to identify apoptotic (annexin V positive) cells. As shown in fig. 7, a significant numberThe treated cells entered apoptosis (right panel), while the control cells remained healthy (left panel). This illustrates the general therapeutic potential of using the present invention to treat cancer stem cells and cancers.

Example 4: RNAi against CSCP3 in Cancer Stem Cells (CSCs) selected by surface markers

Cancer stem cells were selected by surface labeling CD133 using FACS. Subsequently useOr controlThe obtained SW480 human colon cancer CD133+ cancer stem cells were processed. After 24 hours, cells were fixed and stained with annexin V-FITC to identify apoptotic cells. Similar results to example 3 were observed, much more relative to the control (left panel)Apoptosis occurred in the treated cells (fig. 8, right panel).

Example 5: RNAi against sphere formation inhibited by CSCP3 in Cancer Stem Cells (CSC)

CSCP3/STAT3RNA silencing using the methods of the invention also inhibited CSC sphere formation by up to 60% (fig. 9, right panel). By usingOr control TPIV treatment CD44 high FaDu cells isolated by FACS. Then, the cells were cultured for 5 days in the absence of attachment and serum to form spheres. Representative sphere images were captured before (top left) or after (bottom left) trypan blue addition to identify dead cells. Spheroid growth was scored by counting spheres with more than 50 cells.

Example 6: RNAi against CSCP3 in non-CSC cells

By usingOr controlPlasmid treatment of U2OS human osteosarcoma cells. Cells were harvested and the levels of CSCP3/STAT3 protein determined by western blot analysis 24 hours after cell infection (fig. 10, left panel). Cell viability was determined by routine MTT assay 72 hours after treatment (right panel) which showed 70% mortality in cancer cells with MOI of 300 and 6 with MOI00 showed a cell death rate of more than 80%. This provides evidence for the high efficiency of the bacteria-mediated RNAi approach of the invention against differentiated non-cancer stem cells.

Other embodiments are within the following claims. While several embodiments have been shown and described, various modifications may be made without departing from the spirit and scope of the invention.

Sequence listing

<110> Beijing Strong New Biotechnology Ltd

<120> enabling long dsRNA for gene targeting in mammalian and other selected animal cells

<150>US 60/947,311

<151>2007-06-29

<160>6

<170>PatentIn version 3.5

<210>1

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> DNA primer

<400>1

ggatctagaa tcagctacag cagc 24

<210>2

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> DNA primer

<400>2

tcctctagag ggcaatctcc attg 24

<210>3

<211>96

<212>DNA

<213> Artificial sequence

<220>

<223> DNA oligonucleotide

<400>3

acggatcctc ctttcagcaa aaaacccctc aagacccgtt tagaggcccc aaggggttat 60

gctagttatt gctcagcggt ggtctagagg atccac 96

<210>4

<211>96

<212>DNA

<213> Artificial sequence

<220>

<223> DNA oligonucleotide

<400>4

gtggatcctc tagaccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg 60

ggtcttgagg ggttttttgc tgaaaggagg atccgt 96

<210>5

<211>96

<212>DNA

<213> Artificial sequence

<220>

<223> DNA oligonucleotide

<400>5

gcgtcgactc tagaccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg 60

ggtcttgagg ggttttttgc tgaaaggagt cgaccg 96

<210>6

<211>96

<212>DNA

<213> Artificial sequence

<220>

<223> DNA oligonucleotide

<400>6

cggtcgactc ctttcagcaa aaaacccctc aagacccgtt tagaggcccc aaggggttat 60

gctagttatt gctcagcggt ggtctagagt cgacgc 96

Claims (29)

1. A bacterium comprising a long double-stranded RNA (dsRNA) consisting of two substantially complementary strands or a DNA molecule consisting of two complementary strands each encoding one corresponding RNA strand, and an enzyme capable of processing said dsRNA into a mixture of shorter RNA duplexes, wherein said dsRNA comprises a sequence substantially complementary to a messenger RNA (mrna) sequence encoded by a target mammalian gene or viral gene, wherein said dsRNA is a non-short hairpin RNA, wherein the double-stranded region of said dsRNA is longer than 70bp, wherein said mixture of short RNA duplexes is capable of modulating the expression of a mammalian gene or viral gene.

2. The bacterium of claim 1, wherein the enzyme is an endonuclease.

3. The bacterium of claim 1, wherein the enzyme is a bacterial ribonuclease III, dicer, or dicer-like enzyme.

4. The bacterium of claim 1, wherein the bacterium is capable of modulating expression of a mammalian gene or a viral gene in a mammalian cell upon introduction into the mammalian cell.

5. The bacterium of claim 1 wherein the dsRNA is at least 100bp in length.

6. The bacterium of claim 1 wherein the dsRNA is at least a length selected from the group consisting of 100bp, 200bp, 400bp, and 1000 bp.

7. The bacterium of claim 1 wherein said DNA molecule comprises one or more prokaryotic promoters controlling expression of said dsRNA.

8. The bacterium of claim 7, wherein at least one of the one or more prokaryotic promoters is a T7 promoter.

9. The bacterium of claim 7, wherein at least one of the one or more prokaryotic promoters is endogenous to the bacterium.

10. The bacterium of claim 1, wherein said DNA molecule comprises two prokaryotic promoters controlling expression of both strands of said DNA molecule.

11. The bacterium of claim 1, wherein said mammalian gene is associated with a mammalian disease caused at least in part by up-or down-regulation of expression of said gene.

12. The bacterium of claim 1, wherein the mammalian gene is associated with cancer, i.e., silencing or knocking down of the gene affects cell proliferation.

13. The bacterium of claim 1, wherein the mammalian gene is β -catenin.

14. The bacterium of claim 1, wherein the mammalian gene is k-Ras.

15. The bacterium of claim 1, wherein the viral gene is an HIV gene.

16. The bacterium of claim 1, wherein the bacterium is capable of invading a mammalian cell.

17. The bacterium of claim 1, wherein said bacterium is live.

18. The bacterium of claim 1, wherein said bacterium is a semi-inactive bacterium.

19. The bacterium of claim 1, wherein the bacterium is non-pathogenic.

20. The bacterium of claim 1, wherein the bacterium is a therapeutic bacterium.

21. The bacterium of claim 1, wherein said bacterium is an attenuated strain selected from the group consisting of Listeria, Shigella, Salmonella, Escherichia coli, and Bifidobacterium.

22. The bacterium of claim 1, wherein the DNA molecule comprises a circular double-stranded plasmid.

23. The bacterium of claim 1, wherein the DNA molecule is integrated into a bacterial chromosome.

24. The bacterium of claim 1 wherein the dsRNA comprises a sequence that is fully complementary to a mRNA sequence encoded by a mammalian gene or a viral gene.

25. A bacterium comprising a mixture of short RNA duplexes capable of modulating expression of a mammalian gene or a viral gene, wherein the mixture of short RNA duplexes is produced from a long double-stranded RNA (dsRNA) comprising a sequence substantially complementary to a messenger RNA (mrna) sequence encoded by a target mammalian gene or viral gene, wherein the dsRNA is a non-short hairpin RNA, wherein the double-stranded region of the dsRNA is longer than 70 bp.

26. The bacterium of claim 25, wherein said long double-stranded rna (dsrna) is at least 100bp in length.

27. The bacterium of claim 25, wherein said long double-stranded rna (dsrna) is at least a length selected from the group consisting of 100bp, 200bp, 400bp, and 1000 bp.

28. The bacterium of claim 1, further comprising an enzyme that aids in the transport of genetic material upon release from the bacterium into the cytoplasm of a target eukaryotic cell.

29. The bacterium of claim 28, wherein the enzyme is a Hly protein.