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WO2006130976A1 - Interfering rnas, methods for their production, and use - Google Patents

Interfering rnas, methods for their production, and use

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Publication number
WO2006130976A1
WO2006130976A1 PCT/CA2006/000932 CA2006000932W WO2006130976A1 WO 2006130976 A1 WO2006130976 A1 WO 2006130976A1 CA 2006000932 W CA2006000932 W CA 2006000932W WO 2006130976 A1 WO2006130976 A1 WO 2006130976A1
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rna
expression
interfering
bacterial
gene
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PCT/CA2006/000932
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French (fr)
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Shi-Hsiang Shen
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National Research Council Of Canada
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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111General methods applicable to biologically active non-coding nucleic acids
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    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00Preparation of compounds containing saccharide radicals
    • C12P19/26Preparation of nitrogen-containing carbohydrates
    • C12P19/28N-glycosides
    • C12P19/30Nucleotides
    • C12P19/34Polynucleotides, e.g. nucleic acids, oligoribonucleotides
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    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/10Type of nucleic acid
    • C12N2310/11Antisense
    • C12N2310/111Antisense spanning the whole gene, or a large part of it
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    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/10Type of nucleic acid
    • C12N2310/14Type of nucleic acid interfering N.A.
    • CCHEMISTRY; METALLURGY
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    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/50Physical structure
    • C12N2310/53Physical structure partially self-complementary or closed
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2330/00Production
    • C12N2330/30Production chemically synthesised

Abstract

Modulation of gene expression in eukaryotic cells may be achieved via RNA interference (RNAi), involving interfering RNAs such as small interfering RNA (siRNA) and short hairpin RNA (shRNA). Disclosed herein are methods for the production of interfering RNAs through the use of bacterial expression systems. Such methods are applicable, for example, for production of siRNA or shRNA on a large scale. Also disclosed are interfering RNAs generated by the methods of the invention, pharmaceutical compositions comprising such interfering RNAs, and their use for example in treating disease conditions.

Description

INTERFERING RNAs, METHODS FOR THEIR PRODUCTION, AND USE

FIELD OF THE INVENTION

The present invention relates to the field of the modulation of gene expression through RNA interference (RNAi) technology.

BACKGROUND TO THE INVENTION

RNA interference or "RNAi" was a term initially coined by Fire and co- workers to describe the observation that double-stranded RNA (dsRNA) can block gene expression when it is introduced into nematodes (Fire et al. (1998) Nature 391 , 806-811, incorporated herein by reference). RNAi is also known as posttranscriptional gene silencing or PTGS. It is now understood that dsRNA can direct gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided an important new tool for studying gene function. RNAi involves mRN A degradation, but many of the biochemical mechanisms underlying this interference are unknown. The use of RNAi has been further described in Carthew et al. (2001) Current Opinions in Cell Biology 13, 244-248, Elbashir et al. (2001) Nature 411, 494-498, and Paddison & Hannon, Cancer Cell (2002) 2: 17-2, all of which are incorporated herein by reference. RNAi appears to be mediated by RNA-induced silencing complex (RISC), a sequence-specific, multicomponent nuclease that destroys messenger RNAs homologous to the introduced dsRNA, which effectively acts as the gene silencing trigger. RISC is known to contain short RNAs (approximately 22 nucleotides) derived from the double-stranded RNA trigger. RNAi has become the method of choice for loss-of-function investigations in numerous systems, including C. elegans, Drosophila, fungi, plants, and even mammalian cell lines. In such assays, RNAi agents corresponding to the gene of interest, e.g., synthetic double stranded siRNA molecules having a sequence homologous to a sequence found in a target mRNA transcribed from the gene of interest, are introduced into a cell that contains the gene of interest and the phenotype of the cell is then determined. Any deviation in observed phenotype to the control wild type phenotype is then used to assess a function of the gene of interest, since the observed phenotype may be derived from the siRNA-mediated inactivation of the gene of interest.

In addition to the above applications, RNAi has potential in therapeutic applications. Many disease conditions are caused, at least in part, by aberrant or inappropriate gene expression. For example, in cancer aberrant overexpression of oncogenes can result in unregulated cell growth. In some cases, RNAi may present a viable therapeutic option to treat various forms of cancer (see for example Milner J, Expert Opin Biol Ther. 2003 Jun;3(3):459-67).

The application of RNAi for laboratory or clinical use presents unique challenges. Although dsRNA is more stable than ssRNA, dsRNA is nonetheless prone to degradation for example upon exposure to RNases. To date, methods applicable for large scale production of dsRNAs and RNAs suitable for use in RNAi have not been forthcoming. There remains a continuing need for methods of conducting RNAi, and methods of producing interfering RNAs having an activity and quality suitable for use in a laboratory or clinical setting.

SUMMARY OF THE INVENTION

It is an object of the present invention, at least in preferred embodiments, to provide interfering RNA suitable for use in vitro or in vivo to cause at least partial gene silencing.

It is another object of the present invention, at least in preferred embodiments, to provide a method for producing interfering RNAs.

In one aspect of the invention there is provided a method for producing an interfering RNA that is able to modulate expression of a gene in a target cell, the method comprising the steps of: transforming a bacterial cell with an expression vector encoding said interfering RNA in operable association with a bacterial promoter; and culturing said bacterial cell in a culture under conditions suitable for expression of said interfering RNA in said bacterial cell. Preferably, the method further comprises the step of: purifying said interfering RNA from said bacterial cell and / or other components of the bacterial culture.

Preferably, said target cell is a eukaryotic cell. Preferably, said bacterial cell is a strain of E. coli. Preferably, said bacterial promoter is a T3, T7, or SP6 promoter.

Preferably,"said bacterial promoter including a binding site for an RNA polymerase, wherein the expression of the RNA polymerase or components thereof is under the control of an inducible promoter.

In preferred aspects, where the method includes a step of purifying, the step of purifying comprises gel electrophoresis, high performance liquid chromatography, or adherence and elution from a solid substrate.

Preferably, in any method of the invention, said interfering RNA is siRNA or shRNA. More preferably, when said interfering RNA is shRNA, the method further comprises the step of: processing said shRNA with an enzyme suitable to cleave the hairpin loop of said shRNA, or a portion of said shRNA comprising the hairpin loop, thereby to generate siRNA. Preferably, said processing comprises exposing said shRNA to an RNase. More preferably, said RNase is RNase Tl or Dicer. More preferably, the RNase is co-expressed in said bacterial cell with said interfering RNA. More preferably, said RNase is encoded on said expression vector. Alternatively, said interfering RNA is exposed to and processed by said RNase in vitro following purification from said bacterial cell and / or said bacterial culture.

In another aspect the present invention provides for a method for producing an interfering RNA for use in contacting a cell, and modulating expression of a target gene in said cell, the method comprising the steps of: generating an expression vector encoding said interfering RNA in operable association with a bacterial promoter; and combining said expression vector with a bacterial RNA polymerase, ribonucleotides, ATP, and other factors required to achieve in vitro transcription thereby to generate said interfering RNA. In another aspect the invention provides for a use of a bacterium to generate interfering RNAs.

In another aspect the invention provides for a use of a bacterial promoter in operable association with a DNA segment encoding an interfering RNA, together with a bacterial RNA polymerase corresponding to said bacterial promoter, to generate interfering RNAs. Preferably, said bacterial promoter is selected from the group consisting of: a T3 promoter, a T7 promoter, and an SP6 promoter. Preferably, said RNAs are generated within a bacterial cell.

In another aspect the invention provides an interfering RNA generated by the any method of the present invention.

In another aspect the invention provides for a use of the interfering RNA of the present invention, for modulating expression of a target gene in a eukaryotic cell. Preferably, said eukaryotic cell is in a cell or tissue culture.

In another aspect the invention provides for a use of the interfering RNA of the invention, for the manufacture of a medicament to modulate expression of a target gene in a patient. Preferably, the expression of said target gene in said patient at least in part causes, or may be expected to cause, a disease condition. In another aspect the invention provides for a pharmaceutical composition comprising the interfering RNA of the invention, together with at least one pharmaceutically acceptable diluent, carrier or excipient. Preferably, said interfering RNA modulates expression of a target gene in a patient that if unchecked will cause a disease in said patient.

In another aspect of the invention there is provided a vector comprising a bacterial promoter and at least one cloning site for receiving in operative association with the bacterial promoter an insert encoding an interfering RNA, wherein transformation of the vector into a bacterial cell results in expression of said interfering RNA.

In another aspect of the invention there is provided a construct comprising the vector of the invention, with an insert encoding an interfering RNA ligated into the cloning site. Preferably, the construct further comprising a terminator downstream from the insert, and an RNase cleave site between the insert and the terminator. More preferably, the RNase cleavage site is an RNase Tl cleavage site comprising four consecutive guanines.

In another aspect of the invention there is provided a method for generating inferring RNA, the method comprising the steps of: generating the construct of the present invention; expressing from the construct said insert thereby to generate said interfering RNA or a precursor thereof; and if necessary contacting said precursor with an RNase thereby to cleave terminator sequence and / or to cleave a hairpin loop from said precursor thereby to generate said interfering RNA.

In another aspect of the invention there is provided a kit comprising the vector of the invention, optionally together with instructions for use in the production of interfering RNAs.

In another aspect of the invention there is provided a kit comprising the construct of the present invention, optionally together with instructions for use in the production of interfering RNAs.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Expression of siRNAs from E. coli. (a) The construct containing both an expression cassette for the sense strand of siMVP17 and an expression cassette for the antisense strand of siMVP17 was transformed into E. coli BL21-Gold (DE3). Following 4 h induction with IPTG, abundant doubled stranded RNA (siMVP17) was assembled in E. coli. Lane - IPTG, 1 μg of total RNA extract from uninduced bacteria; lane + IPTG, 2.5 μg of total RNA extracts from IPTG-induced bacteria; lane siRNA, 300 ng of chemically synthesized siRNA (23bp, Dharmacon). (b) siMVP (4OnM) was co-transfected with a plasmid pEGFP-MVP containing the expression cassette for the EGFP-MVP fusion transcript into HeLa cells. Photomicrographs of the cells showing EGFP expression were taken 48 h after the transfection. pEGFP-MVP, plasmid alone, no siRNA; siMVP17-E.coli, siRNA produced from E. coli; siMVP 17-Dharma, siRNA chemically synthesized by Dharmacon; HBl, siHBl, an unrelated siRNA against the gene encoding the hepatitis B surface antigen, synthesized in vitro using the Silencer siRNA Construction kit (Ambion). The numbers in brackets are the percentage values of fluorescence measured by the Fluorescence Plate Reader, based on HBl being 100%.

Figure 2. Expression of shRNAs from E. coli. (a) An expression cassette for an EGFP-specific shRNA (shEGFP) was transformed into E. coli BL21-Gold (DE3) and shRNA was highly expressed following IPTG induction, with a molecular weight higher than that of the control siRNA (siEGFP, 21-nt), which was synthesized in vitro using the Silencer siRNA Construction kit (Ambion) and mixed with the extracted total RNAs before loading on an agarose gel. (b) Identity of the expressed shEGFP was confirmed by Northern blot with the designed 32P-labelled sense strand probe, (c) The shEGFP was isolated from agarose gel and transfected with plasmid pEGFP-Cl into HeLa cells. Control siEGFP was synthesized in vitro using the Silencer Construction kit. Photomicrographs of the cells showing EGFP expression were taken 48 h after the transfection.

Figure 3. Co-expression of shEGFP with RNAse Tl fusion protein, (a) An E. coli- expressed RNase Tl fusion protein extracted from bacteria was subjected to Western blot analysis with an antibody specifically against the S-Tag epitope located at the N-terminus of the Tl fusion protein, (b) shEGFP with or without co-expression of the RNase Tl fusion protein was subjected to electrophoresis in agarose gel. Lanes: Marker, 25-bp molecular weight marker; +Tl, shEGFP was co-expressed with RNase Tl; - Tl, shEGFP was expressed without co-expression of RNase Tl . (c) shEGFPs with or without co-expression of RNase Tl were gel -purified and co- tansfected with plasmid pEGFP-Cl (60ng/well in 96-well plate) into HeLa cells for knocking down the expression of the EGFP gene. Photomicrographs of the cells showing EGFP expression were taken 48 h after the transfection.

Figure 4. Processing of shEGFP in vitro with RNase Dicer, (a) An E. co/z-expressed shEGFP was gel-purified and digested with recombinant RNase Dicer under the conditions described in Methods. The Dicer-processed siEGFP migrated to a position around 25 bp. Lanes: Marker, 25-bp molecular weight marker; -Dicer, undigested shEGFP; + Dicer, Dicer-digested shEGFP (siEGFP). (b) shEGFP and Dicer-digested shEGFP (siEGFP) were co-tansfected with plasmid pEGFP-Cl (60ng/well in 96-well plate) into HeLa cells for knocking down the expression of the EGFP gene. Photomicrographs of the cells showing EGFP expression were taken 48 h after the transfection. The numbers in bracket are the values of fluorescence- activated cell sorting (FACS) analysis. pEGFP-Cl, plasmid alone; HBl, siHBl, an unrelated siRNA against the gene encoding the hepatitis B surface antigen; EGFP, shEGFP; EGFP + Dicer; shEGFP was digested with RNase Dicer.

Figure 5. Processing of shMVP2 and shMVP3 with RNase Tl. (a) The E. coli- expressed shMVPs were purified from gel as before and digested with RNase Tl under the conditions described in the Methods and materials section. The RNase Tl processed shMVP2 and shMVP3 migrated at the positions around 25 bp as indicated, (b) HeLa cells were transfected either with gel-purified shMVPs or with RNase Tl digested shMVPs. Cell lysates were made and subjected to Western blot analysis with anti-MVP antibody (MVP). Control, an un-related shRNA (siHBl); shMVP-Tl, no RNase Tl digestion; shMVP+Tl, shMVP was digested with RNase Tl .

Figure 6. Sequences involved in the construction of siRNA producing vector.

Figure 7a. Flow chart to illustrate a preferred method of the invention for producing interfering RNAs.

Figure 7b. Flow chart to illustrate a preferred method of the invention for producing interfering RNAs. DEFINITIONS:

"DNA segment" refers to a linear fragment of single- or double-stranded deoxyribonucleic acid (DNA), which can be derived from any source, "expressed" means the generation of a RNA molecule from a DNA molecule (i.e., a complementary RNA molecule generated from the DNA molecule by the process of transcription) and / or the generation of a polypeptide or protein molecule from a DNA molecule via a RNA intermediate (i.e., by the processes of transcription and translation), "expression" of a gene or nucleic acid encompasses not only cellular gene expression, but also the transcription and translation of nucleic acid(s) in cloning systems and in any other context such as in vitro expression.

"construct", as used herein refers to an engineered DNA molecule including one or more nucleotide sequences from different sources. A preferred construct includes at least an interfering RNA-encoding region operably linked to a promoter sequence, "kit" is any manufacture (e.g. a package or container) comprising at least one reagent, e.g. a construct, for activating RNAi in a cell or organism, the manufacture being promoted, distributed, or sold as a unit for performing the methods of the present invention.

"gene" includes genomic DNA, cDNA, RNA, or other polynucleotides that encode gene products, and in the case of a polynucleotide may or may not include promoter or enhancer sequences.

"target gene", as used herein, refers to a gene intended for modulation via RNA interference ("RNAi").

"modulation" or "modulate" refers to an influence upon gene expression (including transcription and / or translation) that may include maintenance and / or upregulation and / or down regulation of the gene expression.

"target protein" refers to a protein the expression of which is intended for modulation via RNAi.

"target RNA" refers to an RNA molecule intended for degradation by RNAi. An exemplary "target RNA" is a coding RNA molecule (e.g., a mRNA molecule). "promoter" refers to a DNA sequence to which RNA polymerase can bind and initiate transcription. An "inducible promoter" is a DNA sequence which, when operably linked with a DNA sequence encoding a specific gene product, causes the gene product to be substantially produced in a cell only when an inducer which corresponds to the promoter is present in the cell. The term "Pol III promoter" refers to an RNA polymerase III promoter. Exemplary Pol III promoters include, but are not limited to, the U6 promoter, the Hl promoter, and the tRNA promoters. The term "Pol II promoter" refers to an RNA polymerase II promoter. Exemplary Pol II promoters include, but are not limited to, the CMV promoter and the Ubiquitin C promoter. The term "promoter" may also encompass any "bacterial promoter". The expression "bacterial promoter" includes any promoter that exhibits some degree of functional activity in a bacterium of any kind, and therefore encompasses any wild- type or artificially modified promoter of bacterial or viral derivation. Such a promoter may be selected from the following non-limiting group: a T3 RNA polymerase promoter, a T7 RNA polymerase promoter, and an SP6 RNA polymerase promoter. "RNA interference" or "RNAi", as used herein, refers generally to a sequence- specific or selective process by which a target molecule (e.g., a target gene, protein or RNA) is modulated. In specific embodiments, the process of "RNA interference" or "RNAi" features degradation of RNA molecules, e.g., RNA molecules within a cell, said degradation being triggered by an RNA agent. Degradation is catalyzed by an enzymatic, RNA-induced silencing complex (RISC). RNAi occurs in cells naturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAi is know to proceed via fragments cleaved from free dsRNA which direct the degradative mechanism to other similar RNA sequences. Alternatively, RNAi can be initiated artificially, for example, to modulate or silence the expression of target genes. "RNA agent", as used herein, refers to an RNA (or analog thereof), comprising a sequence having sufficient complimentary sequence to a target RNA (i.e., the RNA being degraded) to direct RNAi. A sequence having a "sufficiently complementary sequence to a target RNA sequence to direct RNAi" means that the RNA agent has a sequence sufficient to trigger the destruction of the target RNA by the RNAi machinery (e.g., the RISC complex) or process. "RNA" or "RNA molecule" or "ribonucleic acid molecule" refers to a polymer of ribonucleotides. "DNA" or "DNA molecule" or "deoxyribonucleic acid molecule" refers to a polymer of deoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., by DNA replication or transcription of DNA, respectively). RNA can be post-transcriptionally modified. DNA and RNA can also be chemically synthesized. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g., double-stranded, i.e., dsRNA and dsDNA, respectively). "mRNA" or "messenger RNA" refers to a single-stranded RNA that specifies the amino acid sequence of one or more polypeptide chains. This information is translated during protein synthesis when ribosomes bind to the mRNA. "gene product" refers primarily to proteins and polypeptides, or fragments thereof, encoded by other nucleic acids (e.g., non-coding and regulatory RNAs such as tRNA, sRNPs). "regulation of expression" refers to events or molecules that increase or decrease the synthesis, degradation, availability or activity of a given gene product, "transcript" refers to a RNA molecule transcribed from a DNA or RNA template by a RNA polymerase template. The term "transcript" includes RNAs that encode polypeptides (i.e., mRNAs) as well as noncoding RNAs ("ncRNAs"). "small interfering RNA" ("siRNA") (also referred to in the art as "short interfering RNAs") refers to an RNA agent, preferably a double-stranded agent, of about 10-50 nucleotides in length (the term "nucleotides" including nucleotide analogs), preferably between about 15-25 nucleotides in length, more preferably about 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, the strands optionally having overhanging ends comprising, for example, 1 , 2 or 3 overhanging nucleotides (or nucleotide analogs), which is capable of directing or mediating RNA interference. Naturally-occurring siRNAs are sometimes generated from longer dsRNA molecules (e.g., >25 nucleotides in length) by RNase Dicer . "shRNA", as used herein, refers to an RNA agent having a stem-loop structure, comprising a first and second region of complementary sequence, the degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop optionally resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region, "treatment", as used herein, is defined as the application or administration of a therapeutic agent to a subject, or application or administration of a therapeutic agent to an isolated tissue or cell line from a subject, who has a disease or disorder, a symptom of a disease or disorder, or a predisposition toward a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, the symptoms of the disease or disorder, or the predisposition toward a disease or disorder. A therapeutic agent includes, but is not limited to a composition comprising at least one of interfering RNA, small molecules, peptides, antibodies, ribozymes, antisense oligonucleotides, and chemotherapeutic agents, "effective amount", as used here in, is defined as that amount necessary or sufficient to treat or prevent a disorder. The effective amount can vary depending on such factors as the size and weight of the subject, the type of illness, or the particular agent being administered. One of ordinary skill in the art would be able to study the aforementioned factors and make the determination regarding the effective amount of the agent without undue experimentation. "nucleoside" refers to a molecule having a purine or pyrimidine base covalently linked to a ribose or deoxyribose sugar. Exemplary nucleosides include adenosine, guanosine, cytidine, uridine and thymidine. The term "nucleotide" refers to a nucleoside having one or more phosphate groups joined in ester linkages to the sugar moiety. Exemplary nucleotides include nucleoside monophosphates, diphosphates and triphosphates. The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein and refer to a polymer of nucleotides joined together by a phosphodi ester linkage between 5' and 3' carbon atoms.

"mutation" refers to a substitution, addition, or deletion of a nucleotide within a gene sequence resulting in aberrant production (e.g., misregulated production) of the protein encoded by the gene sequence. A "gain-of- function" mutation is a mutation that results in production of a protein having aberrant function as compared to the wild-type or normal protein encoded by a gene sequence.

"pharmaceutical composition" as used herein, refers to an agent formulated with one or more compatible solid or liquid filler diluents or encapsulating substances which are suitable for administration to a human or other animal.

"involved" in a disorder includes a gene, the normal or aberrant expression or function of which effects or causes a disease or disorder or at least one symptom of said disease or disorder, "examining / analyzing the function of a gene in a cell or organism" refers to examining or studying the expression, activity, function or phenotype arising therefrom. Various methodologies of the instant invention include a step that involves comparing a value, level, feature, characteristic, property, etc. to a "suitable control", referred to interchangeably herein as an "appropriate control". A "suitable control" or "appropriate control" is any control or standard familiar to one of ordinary skill in the art useful for comparison purposes. In one embodiment, a "suitable control" or "appropriate control" is a value, level, feature, characteristic, property, etc. determined prior to performing an RNAi methodology, as described herein. For example, a transcription rate, mRNA level, translation rate, protein level, biological activity, cellular characteristic or property, genotype, phenotype, etc. can be determined prior to introducing an RNAi agent of the invention into a cell or organism. In another embodiment, a "suitable control" or "appropriate control" is a value, level, feature, characteristic, property, etc. determined in a cell or organism, e.g., a control or normal cell or organism, exhibiting, for example, normal traits. In yet another embodiment, a "suitable control" or "appropriate control" is a predefined value, level, feature, characteristic, property, etc.

"transforming" or "transformation" refers to a process by which polynucleotides such as DNA or RNA are caused to enter a prokaryotic organism. Preferably, following entry the polynucleotides reside in a stable or transient manner in the prokaryote, and RNA is expressed from the polynucleotide where and / or when appropriate. Preferably, the process of transforming involves a technique that is well known in the art including but not limited to electroporation, heat shock techniques, and other techniques involving ballistic acceleration of microparticles.

"Transfecting" or "transfection" refers to a process by which polynucleotides such as

DNA are caused to enter a eukaryotic cell or organism. Preferably, following entry the polynucleotides reside in a stable or transient manner in the eukaryotic cell or organism, and RNA is expressed from the polynucleotide where and / or when appropriate. Preferably, the process of transfection involves a technique that is well known in the art including but not limited to the use of viral or liposome vector (e.g.

Lipofectamine), and other techniques involving ballistic acceleration of microparticles.

"upstream" refers to nucleotide sequences that precede, e.g., are on the 5' side, of a reference sequence.

"downstream" refers to nucleotide sequences that follow, e.g., are on the 3' side, of a reference sequence. "preferred" or "preferably" refers to preferred features of the broadest embodiments of the invention, unless otherwise stated.

The terms used herein are not limiting to the invention as described and claimed.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

RNA interference (RNAi) is an evolutionarily conserved mechanism for silencing gene expression via sequence- specific degradation of target RNA. In recent years, RNAi has revolutionized experimental biology and gained recognition as a powerful tool for the study of functional genomics. The sequence-specific selectivity and robust capacity of RNAi to inactivate genes in vivo has also led to the development of RNAi-based new therapeutics.

It has been demonstrated that both small interfering RNA (siRNA) and short hairpin RNA (shRNA) produced by in vitro methods can effectively trigger RNAi in mammalian cells via transfection. At present, for research purposes siRNAs and shRNAs are mainly produced by two in vitro methods, namely chemical synthesis and biochemical synthesis with T7 RNA polymerase. However, since siRNA-based therapeutics is under way, the demand of siRNAs for drug discovery and therapeutics is expected to rapidly increase. This could make the current in vitro methods for production of siRNAs unsuitable for such therapeutic applications, since siRNAs produced by these methods are very expensive, and their scale of synthesis may be below the amounts required for therapy. Hence, the development of alternative and inexpensive methods for large-scale production of siRNA is highly desired. It has been reported that long double-stranded RNAs (dsRNAs) could be synthesized in E. coli and delivered into C. elegans via ingestion for RNAi induction (Timmons et al, 2001). A similar method has been used in the past to produce long dsRNAs in vitro, and then digest the long dsRNAs with RNases, such as Dicer or RNase III to obtain shorter dsRNAs (siRNAs) for RNAi. However, this method is more expensive than the methods disclosed herein. In addition, RNAs produced by such an approach are mixtures of various dsRNAs and may be not suitable for therapeutic purposes. Long dsRNA synthesized in E. coli may not be directly suited for RNAi in mammalian cells because long dsRNAs evoke strong interferon responses. There is described herein a cost-effective method to produce both siRNAs and shRNAs from bacterial cells, and corresponding cultures. Concrete evidence is presented to show that both siRNA and shRNA produced directly from bacterial cells such as E. coli may effectively trigger RNAi in transfected cells. Furthermore, shRNA expressed from bacterial cells can be processed into the correspondent functional siRNA by in vitro digestion with RNases. The inventor has endeavoured to develop systems and methods for the manufacture of interfering RNAs, which are preferably suitable for use'in modulation of gene expression or gene silencing, for example either in laboratory experiments or in the treatment of patients. The invention represents the first time that interfering RNAs have been successfully produced in prokaryotic cells and systems. In particularly preferred aspects of the invention, the interfering RNAs are of a type and quality suitable for application to eukaryotic cells or organisms, to achieve modulation of gene expression or gene silencing in those eukaryotic cells or organisms. Other aspects of the invention will become apparent upon reading and understanding the present specification in its entirety. The invention presents important opportunities for larger scale, or even industrial scale, production of interfering RNA suitable for use in the laboratory or clinic, via prokaryotic systems.

In selected embodiments the invention provides a method for expression, preferably high-level expression, of either small interfering RNA (siRNA) and / or short hairpin RNAs (shRNAs) in prokaryotes. The methods are applicable to any prokaryotic system, but in preferred embodiments the prokaryote may be of a type that is commonly utilized for industrial or biotechnology purposes such as for example strains of E. coli. In some instances, the interfering RNA may be expressed using an RNA polymerase promoter such as the T7, T3, or SP6 RNA polymerase promoters, to name a few examples. For siRNA expression, the sense and anti-sense strand may be separately expressed and assembled into RNA duplexes in vivo

(within the bacterial cells) or in vitro (exterior to the bacterial cells). For shRNA, the expressed sense and antisense strand may be joined together with a loop sequence during expression.

Importantly, the present application discloses concrete evidence to show that both siRNAs and shRNAs, isolated from bacterial cells in accordance with the methods of the invention, are functional in triggering RNAi via transfection into eukaryotic cells. In selected embodiments, the expressed shRNAs, where present, may be further processed in vitro into the correspondent siRNAs, for example using an RNase such as for example Dicer or RNase Tl . The processed siRNAs, as with the shRNAs, are shown to be capable of specifically knocking down both the transgenic and endogenous genes efficiently.

The methods of the present invention include methods for producing an interfering RNA that is able to modulate expression of a gene in a target cell, the method comprising the steps of: transforming a bacterial cell with an expression vector encoding said interfering RNA in operable association with a bacterial promoter; and culturing said bacterial cell in a culture under conditions suitable for expression of said interfering RNA in said bacterial cell. Such methods of transforming and culturing of bacterial cells are well known in the art, and examples of such methods are described for example in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Press, Cold Spring Harbour,

N.Y.(1989) to name just one example of a standard text of the subject. The methods may also include purification of the interfering RNA from said bacterial cell and / or other components of the bacterial culture. Again, such purification techniques are widely known, and kits are commercially available for RNA extraction from bacterial cultures. Further examples of purification may include gel electrophoresis, high performance liquid chromatography, or adherence and elution from a solid substrate.

In preferred embodiments, the interfering RNAs generated by the methods of the invention are of a type and quality suitable for application to eukaryotic cells to cause gene modulation by RNAi in those cells.

Any bacterial prokaryotic cell may be utilized to generate the interfering RNAs in accordance with the present invention. Although commonly available strains such as those of the species E. coli are preferred, the invention is not limited in this regard. Th skilled artisan will appreciate that a wide range of bacterial cells may be utilized without deviating from the invention. All such prokaryotic systems are expected to allow for the production of interfering RNAs. Preferably, the methods of the invention employ a bacterial promoter for interfering RNA expression that is commonly known in the art. As described in the definitions section, a bacterial promoter may be selected from any promoter that exhibits some degree of activity in a bacterial cell, and may include any suitable promoter of bacterial or viral origin that includes wild-type or artificially modified sequence. Examples of such promoters include those that bind T3, T7, or SP6 RNA polymerases.

In preferred embodiments the expression of the RNA polymerase (or components thereof) responsible for effecting interfering RNA expression is itself regulated by an inducible promoter.

The methods of the invention are applicable to the production of either siRNA or shRNA. In selected embodiments, the shRNA may be further processed using an RNase to generate siRNA. Although RNase Tl and Dicer are discussed herein an represent preferred RNases, any RNase may be utilized to cleave the hairpin loop structure in the shRNA. The RNase may be applied to the shRNA following purification from the bacterial culture. Alternatively, the RNase may be co-expressed with the interfering RNA such that processing of shRNA to siRNA occurs in vivo prior to purification. For example, the expression vector encoding the interfering RNA may be bicistronic and include further sequence for co-expression of the RNase.

In other embodiments the invention provides a method for producing an interfering RNA for use in contacting a cell, and modulating expression of a target gene in said cell, the method comprising the steps of: generating an expression vector encoding said interfering RNA in operable association with a bacterial promoter; and combining said expression vector with a bacterial RNA polymerase, ribonucleotides, ATP, and other factors required to achieve in vitro transcription thereby to generate said interfering RNA. Therefore, the invention includes the option of utilizing bacterial promoters and their corresponding RNA polymerases in in vitro systems for manufacture of interfering RNAs via in vitro transcription. Also within the scope of the present invention is the use of any bacterium or prokaryote for the production of interfering-RNAs of any kind. Also within the scope of the invention is the use of any bacterial promoter (including any such promoter of bacterial or viral origin) in operable association with a DNA segment encoding an interfering RNA, together with a bacterial RNA polymerase corresponding to said bacterial promoter, to generate interfering RNAs. Importantly, the invention further encompasses any interfering RNA produced in accordance with the methods of the invention, and the use thereof. For example, such an interfering RNA may be used for modulating expression of any target gene in a eukaryotic cell, including a eukaryotic cell in cell or tissue culture, or a cell of a patient. In this way, the interfering RNA may be used for laboratory experiments, for example to test gene function during culture experiments. Furthermore, the interfering RNA may be used in the manufacture of pharmaceuticals intended for use in prophylactic and / or therapeutic treatments of patients, including both human and animal patients. In this way, pharmaceutical compositions of the invention that include interfering RNAs maybe used to target disease conditions arising from aberrant expression (such as over expression) of one or more specific genes.

The invention further encompasses any vector comprising any bacterial promoter and at least one cloning site for receiving downstream from the bacterial promoter an insert or DNA segment encoding an interfering RNA, wherein transformation of the vector into a bacterial cell results in expression of said interfering RNA. Further encompassed is a kit comprising the vector, optionally with instructions for use in generating interfering RNA using a prokaryotic cell.

The following examples illustrate preferred embodiments of the invention, and are in no way intended to limit the scope of the present invention as disclosed herein, and covered by the appended claims:

EXAMPLE 1 - High level expression of functional siRNA

In bacterial cells such as E. coli there are various nucleases that degrade RNAs, especially single stranded RNAs, rapidly. To make host E. coli more suitable for production of siRNA high-level expression of single stranded RNA single stranded RNAs are preferably assembled into double stranded RNA (siRNA), which show a great resistance to nucleases. For cost-effective production of siRNA in E. coli, constructs were designed and generated, in which the expression cassette for the sense strand RNA and the expression cassette for the antisense RNA were in tandem under control of a T7 RNA polymerase promoter. It will be appreciated that any suitable promoter may be employed and", in light of the disclosure herein it is within the competence of one skilled in the art to select a suitable promoter. A suitable promoter will have the capability to direct the synthesis of small RNA efficiently in E. coli. Other suitable promoters, for example, include the bacteriophage T3 and Sp6 RNA polymerase promoters. The constructs were designed such that expression from the RNA polymerase promoter generates siRNA against an endogenous gene encoding the major vault protein (MVP) (Mossink et ah, 2003). The constructs for expression of a siRNA (siMVP17, see Table 1) were transformed into E. coli BL21- GoId (DE3) strain. Following 4-hour induction with IPTG for expression of the single strand RNAs, abundant double stranded RNAs, siMVP17, were produced (Fig. Ia). The assembled siRNAs were gel-purified from a total RNA preparation and co-transfected with a plasmid expressing an EGFP-MVP fusion transcript into HeLa cells. Figure Ib shows that the potency of E. coli-produced siMVP17 was, at least, as strong as the chemically synthesized siRNA in knocking down the expression of the EGFP gene.

EXAMPLE 2 - Expression of functional shRN As

It was examined whether shRNAs synthesized in bacterial cells could be functional in eukaryotic cells. For this purpose, an shRNA sequence, namely shEGFP, specifically targeting the enhanced green fluorescence protein (EGFP) gene with a 9-nt loop sequence TTCAAGAGA (Brummelkamp et al., 2002; SEQ ID NO: 1) was expressed in E. coli BL21-Gold (DE3) strain under control of the T7 RNA polymerase promoter. Analysis of total RNAs extracted from E. coli revealed that an RNA band with a size larger than the control of 21-nt siRNA was highly expressed following IPTG induction for 3 h (Fig. 2a). Northern blotting with a probe specific to the antisense sequence of the shRNA confirmed that this dominant RNA band corresponded to the designed shRNA sequence (Fig. 2b). To test its RNAi potency, the expressed shRNA specific to EGFP (shEGFP) was purified from agarose gel and co-transfected with plasmid pEGFP-Cl containing the EGFP gene expression cassette into HeLa cells. As shown in Fig.2c, the in vivo synthesized shEGFP, like the in vitro transcribed siEGFPl (see Table 1), was able to efficiently knock down expression of the transfected EGFP gene.

Further, two shRNAs, shMVP2 and shMVP3 were expressed. These two shRNAs had a newly designed loop sequence GTCAAGACG (SEQ ID NO: 2) against an endogenous gene, the major vault protein (MVP) gene (see Table 1). The gel-purified shMVP2 and shMVP3 were able to knock down the MVP expression by more than 80% as evaluated with Western blot analysis (see Fig. 4 below).

EXAMPLE 3 - Processing of shRNAs in vivo into siRNAs by co-expression of RNase Tl Ribonuclease ("RNase Tl") Tl from Aspergillus oryzae is a small single- domain protein consisting of 104 amino acids that cleaves single stranded RNA specifically at the 3'-side of guanosine (G). To make a small recombinant RNase Tl protein more stable in E. coli, a fused RNAse Tl protein was expressed. For this, a fragment was constructed in such a way that 54 amino acids located at the N- terminus of HIV gp41 protein was preceded on the RNase Tl protein. The fragment encoding for the fusion Tl protein was inserted into pET29a vector for protein expression under the control of the T7 RNA polymerase promoter. The expression level of the fused RNase Tl protein could be further justified by mutation of the T7 promoter when required. shEGFP was co-expressed by insertion of its T7 promoter- directed expression cassette into the same vector pET vector at the Hindlll site. Expression of both RNase Tl fusion protein and shEGFP was induced in E. coli BL21-Gold (DE3) strain by addition of IPTG for 3 h. The expression of RNase Tl fusion protein was confirmed by Western blot analysis with an antibody specifically against the S-Tag located at the N-terminus of the RNase Tl fusion protein (Fig. 3a). Total RNAs were extracted from the induced bacteria with or without the co- expression of RNAse Tl . As shown in Fig. 3b, when shEGFP was expressed alone in E. coli, the expressed product migrated to a position of higher than 25 bp as observed in Figure Ia. However, when shEGFP was co-expressed with RNase Tl in E. coli, it migrated to a position of approximately 25 bp, an expected size lower than that of unprocessed shEGFP. The products expressed in E. coli with or without RNase Tl expression were gel-purified and co-transfected with plasmid pEGFP-Cl into HeLa cells for functional assay as before. Figure 3 c shows that the both products greatly knocked down the expression of the transfected EGFP gene when compared with control that was transfected with unrelated siRNA (siHBl), suggesting that shEGFP, when processed in vivo by co-expressed RNAse Tl, is functional.

EXAMPLE 4 - Processing ofshRNAs in vitro into siRNAs by RNase Dicer Long double stranded RNAs (dsRNA), upon entering the cells, are first processed by an RNase Ill-like enzyme named Dicer producing so-called small interfering RNA duplexes (siRNA) of 21-25 nucleotides in length with two- nucleotide 3' end overhangs. The produced siRNA can be further integrated into the RNA-induced silencing complex termed RISC to trigger RNAi in vivo. It was desired to determine whether shRNAs, when transfected into mammalian cells, are processed by Dicer or other appropriate RNases into siRNAs, as this would imply that E. coli -produced shRNAs, such as shEGFP and shMVP, are also effective for gene silencing following their processing into siRNAs in vitro by appropriate RNases. In this regard, RNase Dicer was used to process the E. co/j-synthesized shEGFP and tested the function of the processed product for gene silencing in vivo. As shown in Fig. 4a, after digestion of shEGFP with Dicer, the processed product migrated to a position of an expected size, approximately 25 bp. The processed product siEGFP was gel -purified and co-transfected with plasmid pEGFP-Cl into HeLa cells for functional assay as before. Figure 4b shows that the potency of the Dicer-processed siEGFP was comparable to that of the unprocessed shEGFP for knockdown of the transfected EGFP gene. EXAMPLE 5 - Processing ofshRNAs in vitro into siRNAs with RNase Tl

To test whether RNase Tl is able to process shRNAs, two shRNAs against the MVP gene, shMVP2 and shMVP3 (see Table 1) with a newly designed loop sequence GTCAAGACG, were expressed in E. coli. Total E. co/z'-expressed RNA or gel- purified shRNA were processed in vitro with RNase Tl . Digestion of shMVP2 and shMVP3 with RNase Tl resulted in production of RNA bands migrating to a position around 25 bp, the expected processed products (Figure 5a).

The processed siMVP2 and siMVP3 were gel-purified for evaluation of their RNAi potencies. For this, both the shRNAs and the siRNAs were transfected into HeLa cells. After 48 hrs following the transfection, the transfected cells were lysed and cell lysates were subjected to Western blot analysis with anti-MVP antibody. As shown in Fig. 5b, both the unprocessed shRNAs and processed siRNAs were able to knock down the expression of the MVP gene by over 80% when compared with control that was transfected with unrelated siRNA (siHBl).

Thus, there are disclosed herein methods for the production of siRNA and shRNA in E. coli strain with very high yields.

An expression host will preferably have an inducible gene encoding a functional RNA polymerase capable of directing efficient production of small RNA in the host cell. When E. coli is the expression host, an inducible T7 RNA polymerase gene is desired. For example, E. coli BL21 (DE3) strains contain the T7 RNA polymerase gene expression of which is induced by adding IPTG. The E. coli- expressed siRNA and shRNA were functional in triggering RNAi in mammalian cells as demonstrated in efficiently knocking down both the transgenic gene (EGFP) and the endogenous MVP gene via transfection. Further, the E. co/z-produced shRNA could be processed into the correspondent siRNA either by the in vivo co- expressed RNAse Tl , or by the in vitro digestion of RNase Dicer/RNase Tl . As with the shRNAs, the RNase-processed product siRNAs were also functional in triggering RNAi in cells. EXAMPLE 6 - Typical methods of the present invention

Figures 7 a and 7b include flow charts to describe the basic steps of selected methods of the invention. In Figure 7a there is illustrated a method for producing an interfering RNA that is able to modulate expression of a gene in a target cell, the method comprising the steps of: in step 101 transforming a bacterial cell with an expression vector encoding said interfering RNA in operable association with a bacterial promoter; and in step 102 culturing said bacterial cell in a culture under conditions suitable for expression of said interfering RNA in said bacterial cell. In Figure 7b there is illustrated a method for producing an interfering RNA for use in contacting a cell, and modulating expression of a target gene in said cell, the method comprising the steps of: in step 201 generating an expression vector encoding said interfering RNA in operable association with a bacterial promoter; and in step 202 combining said expression vector with a bacterial RNA polymerase, ribonucleotides, ATP, and other factors required to achieve in vitro transcription thereby to generate said interfering RNA.

METHODS AND MATERIALS Reagents and materials'. E. coli BL21-Gold (DE3) strain was from Novagen.

Recombinant RNase Dicer was from Gene Therapy System (San Diego). RNase Tl was purchased from Fermentas Canada Inc. (Burlington, On Canada). Anti-MVP antibody was from BD Biosciences Transduction (San Jose). Construction of siRNA-producing vector. The BgIII and HindIII sites of pEGFP-Cl were destroyed. In order to facilitate RNase Tl -mediated in vitro or in vivo processing of E. co //-expressed shRNAs and siRNAs, 4 guanines (GGGG) were added in front of the T7 terminator or the class II termination signal CATCTGTTTT (J. Biol Chem 273, 18802-18811, 1998) . Oligonucleotides containing 4 G and the T7 terminator/the class II termination signal with BgIII and Bbsl sites at 5' end and BamHI site at 3' end were inserted at Asel site (see Figure 6). Engineering four guanines before the RNA polymerase terminators (both T7 and the second II termination signal) was to facilitate the in vitro and in vivo processing of shRNA/siRNA by RNAse Tl. This is because Tl cleaves single stranded RNA specifically at the 3'-side of guanosine (G) (see EXAMPLE 3), so that the additional sequence from the terminator (attaching to the tail of shRNA/siRNA) will be cleaved by Tl .

Oligos containing the T7 promoter followed the siRNA sense sequence and the T7 promoter followed the antisense sequence were separately inserted into BgIII and Bbsl sites. To make the sense expression cassette and the antisense expression cassette in tandem, the antisense cassette was isolated from the latter vector by BgIII and BamHI digestion, and inserted into the BamHI site of the vector containing the sense expression cassette, resulting in a construct containing the sense expression cassette followed by the antisense expression cassette in a head-to-tail orientation.

A construct for the co-expression of a fused RNase Tl with shRNAs and siRNAs in E. coli.

To express a fused RNase Tl, a fragment encoding 54 amino acids at the N-terminus of HIV gp41 protein was cloned upstream to the RNase Tl gene. The entire fragment containing 54 amino acids of gp41 and the RNase Tl was cloned into the EcoKl and Xhol sites of pET29a. The expression of the Tl fusion protein is under the control of either the wild type T7 promoter or a mutated T7 promoter for reducing the expression level of the fused RNase Tl protein. For co-expression of shRNA/siRNA with the fusion Tl protein, the T7 promoter-directed expression cassette for shRNAs and siRNAs was inserted into the Hindϊll site at pET vector.

A construct for expression of an EGFP-MVP fused transcript. To express an EGFP-MVP fusion transcript, the MVP gene was cloned 3' adjacent to the stop codon of the EGFP gene. The expression of the entire fusion transcript was under the control of the human cytomegalovirus promoter (CMV). Plate reading of fluorescence: Fluorescence was measured by reading the 96-well plates in a Perkin Elmer Envision 2100 Multilabel Reader, 40 hours after transfection.

In vitro synthesis ofsiRNA duplex, and expression and purification of shRNA from bacteria:

SiRNA duplexes were chemically synthesized by Dharmacon Inc., or designed and synthesized in vitro by T7 RNA polymerase using the Silencer siRNA Construction kit from Ambion according to the manufacturer's protocol. To express shRNAs in E. coli, DNA oligonucleotides were designed and synthesized by a protocol similar to that reported previously (Brummelkamp et al., 2002), except that the synthesized oligonucleotides were inserted between the T7 promoter and the T7 terminator that were cloned at the unique Ase I site of pEGFP-Cl vector (Clontech). The constructs were transformed into E. coli BL21-Gold (DE3) strain and shRNAs were expressed by induction with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37°C for 3 h. Total RNAs were extracted with TRIZOL (Invitrogen), and applied to agarose gel for electrophoresis, and siRNAs/shRNAs were isolated from agarose gel. Both the in vitro synthesized siRNAs and the bacteria-expressed shRNAs were transfected with Lipofectamine 2000 (Invitrogen) into mammalian cells at a concentration of 40 nM shRNA (or siRNA) per well in 96-well plates for RNAi assay. Knockdown of EGFP was monitored by fluorescence microscopy observation or by fluorescence plate reading or by fluorescence-activated cell sorting (FACS) analysis.

The potencies of the shMVP and the processed siMVP with RN ase Tl were evaluated by directly knocking down the expression of the endogenous MVP gene as visualized through Western-blot analysis with an anti-MVP antibody. For this, shMVP and siMVP (1.4 μg/well) were transfected with Lipofectamine 2000 (Invitrogen) into HeLa cells in 6-well plates. Processing of shRNAs in vitro into siRNAs

Total RNA preparation was run on 3% NuSieve / 1% agarose gel. ShRNA was cut, extracted from agarose gel via centrifugation on glass wool plugs in microcentrifuge tubes. For the in vitro processing of shRNAs with RNase Tl or Dicer, total RNA or RNA extracted from agarose gel were further purified using phenol-CHCl3 and ethanol precipitation. Ten μg of shRNA was digested with 1,000 U of RNase Tl for one hour at 37°C, or 15 μg shRNA was digested with 20 U of Dicer enzyme O/N at 37°C. After digestion, the products were purified using filter cartridges for siRNA purification from Silencer siRNA Construction kit (Ambion) according to the instruction manual.

References:

1) Brummelkamp, T.R., Bernards, R. and Agami, R. Science 296, 550-553 (2002). 2) Mossink, M.H. et al. Oncogene 22, 7458-7467 (2003).

3) Timmons, L., Court, D.L. and Fire, A. Gene 263, 103-112 (2001).

Table 1, shRNA and siRNA Sequences: Designed siRNA and shRNA sequences used in the experiments

SiEGFP3: AAGCTGACCCTGAAGTTCATC (SEQ ID NO: 7) ShEGFPb: AAGCTGACCCTGAAGTTCATC TTCAAGAGA GATGAACTTCAGGGTCAGC (SEQ ID NO: 8) SiMVP 1 T: AATGGAAC AAGGC ATCCAGGATG (SEQ ID NO: 9) ShMVP2b: ACATCCGGCAGGACAATGA GTCAAGACG TCATTGTCCTGCCGGATGT (SEQ ID NO: 10) ShMVP3b: GCTTGATTTTGAGGATAAA GTCAAGACG TTTATCCTCAAAATCAAGC (SEQ ID NO: 11)

SEQUENCE LISTING FREE TEXT:

SEQ ID NO: 1 Loop sequence 1 SEQ ID NO: 2 Loop sequence 2

SEQ ID NO: 3 Oligo with 4G and T7 terminator (forward) SEQ ID NO: 4 Oligo with 4G and T7 terminator (reverse) SEQ ID NO: 5 Oligo with 4G and Class II terminator (forward) SEQ ID NO: 6 Oligo with 4G and Class II terminator (reverse) SEQ ID NO: 7 siRNAs with UU overhangs at 3' end were synthesized in vitro by T7 RNA polymerase using the Silencer Construction kit from Ambion according to the manufacturer's protocol, or chemically synthesized by Dharmacon. Only the sense strand of DNA sequence is shown.

SEQ ID NO: 8 the designed shRNA sequences were cloned into the T7 promoter- directed expression cassette for expression in E. coli. SEQ ID NO: 9 siRNAs with UU overhangs at 3' end were synthesized in vitro by T7 RNA polymerase using the Silencer Construction kit from Ambion according to the manufacturer's protocol, or chemically synthesized by Dharmacon. Only the sense strand of DNA sequence is shown.

SEQ ID NO: 10 the designed shRNA sequences were cloned into the T7 promoter- directed expression cassette for expression in E. coli. SEQ ID NO: 11 the designed shRNA sequences were cloned into the T7 promoter- directed expression cassette for expression in E. coli.

Claims

CLAIMS:
1. A method for producing an interfering RNA that is able to modulate expression of a gene in a target cell, the method comprising the steps of: transforming a bacterial cell with an expression vector encoding said interfering RNA in operable association with a bacterial promoter; and culturing said bacterial cell in a culture under conditions suitable for expression of said interfering RNA in said bacterial cell.
2. The method of claim 1, further comprising the step of: purifying said interfering RNA from said bacterial cell and / or other components of the bacterial culture.
3. The method of claim 1, wherein said target cell is a eukaryotic cell.
4. The method of claim 1 , wherein said bacterial cell is a strain of E. coli.
5. The method of claim 1, wherein said bacterial promoter is a T3, T7, or SP6 promoter.
6. The method of claim 1, said bacterial promoter including a binding site for an RNA polymerase, wherein the expression of the RNA polymerase or components thereof is under the control of an inducible promoter.
7. The method of claim 2, wherein the step of purifying comprises gel electrophoresis, high performance liquid chromatography, or adherence and elution from a solid substrate.
8. The method of claim 1, wherein said interfering RNA is siRNA or shRNA.
9. The method of claim 8, wherein said interfering RNA is shRNA, the method further comprising the step of: processing said shRNA with an enzyme suitable to cleave the hairpin loop of said shRNA, or a portion of said shRNA comprising the hairpin loop, thereby to generate siRNA.
10. The method of claim 9, wherein said processing comprises exposing said shRNA to an RNase.
11. The method of claim 10, wherein said RNase is RNase Tl or Dicer.
12. The method of claim 10, wherein the RNase is co-expressed in said bacterial cell with said interfering RNA.
13. The method of claim 12, wherein said RNase is encoded on said expression vector.
14. The method of claim 13, wherein said interfering RNA is exposed to and processed by said RNase in vitro following purification from said bacterial cell and / or said bacterial culture.
15. Use of a bacterium to generate interfering RNAs.
16. Use of a bacterial promoter in operable association with a DNA segment encoding an interfering RNA, together with a bacterial RNA polymerase corresponding to said bacterial promoter, to generate interfering RNAs
17. Use of claim 16, wherein said bacterial promoter is selected from the group consisting of: a T3 promoter, a T7 promoter, and an SP6 promoter.
18. Use of claim 16, wherein said RNAs are generated within a bacterial cell.
19. An interfering RNA generated by the method of any one of claims 1 to 14.
20. Use of the interfering RNA of claim 19, for modulating expression of a target gene in a eukaryotic cell.
21. Use of claim 20, wherein said eukaryotic cell is in a cell or tissue culture.
22. Use of the interfering RNA of claim 19, for the manufacture of a medicament to modulate expression of a target gene in a patient.
23. Use of claim 22, wherein expression of said target gene in said patient at least in part causes, or may be expected to cause, a disease condition.
24. A pharmaceutical composition comprising the interfering RNA of claim 19, together with at least one pharmaceutically acceptable diluent, carrier or excipient.
25. The pharmaceutical composition of claim 23 , wherein said interfering RNA modulates expression of a target gene in a patient, that if unchecked will cause a disease in said patient.
26. A vector comprising a bacterial promoter and at least one cloning site for receiving in operative association with the bacterial promoter an insert encoding an interfering RNA, wherein transformation of the vector into a bacterial cell results in expression of said interfering RNA.
27. A construct comprising the vector of claim 26, with an insert encoding an interfering RNA ligated into the cloning site.
28. The construct of claim 27, further comprising a terminator downstream from the insert, and an RNase cleave site between the insert and the terminator.
29. The construct of claim 28, wherein the RNase cleavage site is an RNase Tl cleavage site comprising four consecutive guanines.
30. A method for generating inferring RNA, the method comprising the steps of: generating the construct of claim 28; expressing from the construct said insert thereby to generate said interfering RNA or a precursor thereof; and if necessary contacting said precursor with an RNase thereby to cleave terminator sequence and / or to cleave a hairpin loop from said precursor thereby to generate said interfering RNA.
31. A kit comprising the vector of claim 26, optionally together with instructions for use in the production of interfering RNAs.
32. A kit comprising the construct of claim 27, optionally together with instructions for use in the production of interfering RNAs.
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