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WO2006091112A1 - Compositions pour l’administration de molécules d’interférence arn et leur procédés d’utilisation - Google Patents

Compositions pour l’administration de molécules d’interférence arn et leur procédés d’utilisation Download PDF

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WO2006091112A1
WO2006091112A1 PCT/NZ2006/000026 NZ2006000026W WO2006091112A1 WO 2006091112 A1 WO2006091112 A1 WO 2006091112A1 NZ 2006000026 W NZ2006000026 W NZ 2006000026W WO 2006091112 A1 WO2006091112 A1 WO 2006091112A1
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composition
target
sina
binding agent
cell
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Grant Munro
Nevin Abernethy
Ilkka J. Havukkala
Glen Reid
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Genesis Research and Development Corp Ltd
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Genesis Research and Development Corp Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/12—Mucolytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]

Definitions

  • the present invention relates to the treatment of disorders by means of RNA interference (RNAi). More specifically, the present invention relates to the targeted delivery of small nucleic acid molecules that are capable of mediating RNAi against genes that are active in key pathways involved in disorders such as those of the airways and cancer.
  • RNAi RNA interference
  • Cystic fibrosis is the most common fatal genetic disorder in the United States, affecting approximately 30,000 children and young adults. The disorder causes the body to produce abnormally thick mucus in the lungs, sinuses and pancreas, which leads to airway obstruction, and subsequent colonization and infection by inhaled pathogenic microorganisms. Cystic fibrosis airway epithelia have been shown to exhibit defective cAMP-mediated chloride secretion (Johnson et al, J. Clin. Invest. 95:1377-1382, 1995). Mucus overproduction and small airway plugging is also one of the hallmarks of asthma and is a cause of death from asthma. In addition, excess mucus production is associated with chronic bronchitis, which is a common form of chronic obstructive pulmonary disease (COPD). Another disorder characterized by excess mucus production is chronic sinusitis.
  • COPD chronic obstructive pulmonary disease
  • HCLCAl human calcium-dependent chloride channel- 1
  • a Phase II clinical trial of a small molecule inhibitor of HCLCAl demonstrated maintenance of existing lung function in cystic fibrosis patients receiving the small molecule, compared to a decline in those receiving placebo (reported at the North American Cystic Fibrosis Conference, Anaheim, CA, October 2003; see, for example, the website of Genaera Corp.).
  • Kamada et a ⁇ Genes Immun. 5:540-547, 2004 have described studies indicating that variations in the HCLCAl gene affect patient's susceptibility for asthma.
  • Chemokines are considered to be principal mediators in the initiation and maintenance of inflammation.
  • the chemokine cell surface receptor CXCR4 is used by eosinophils, mast cells, T cells, pre-B cells and metastatic tumor cells as a signal for blood vessel cell wall attachment and escape, and for migration into tissues and organs.
  • the ligand for CXCR4 is stromal cell-derived factor (SDF-I; also known as CXCL12 and as pre-B cell growth stimulating factor (PBSF)).
  • SDF-I exists as two forms, alpha and beta, which are identical with the exception of four additional C- terminal residues in SDF-l ⁇ .
  • the CXCR4/SDF-1 receptor/ligand pairing is absolutely unique and non-promiscuous.
  • SDF-I is expressed by several tumors, including pancreatic cancer and glioblastoma, and both SDF-I and its receptor CXCR4 are believed to be important in tumor neovascularization and metastatic spread of tumors.
  • a small molecule inhibitor of SDF-I known as NSC 651016, has been shown to have antiangiogeneic properties (Schneider et ah, Clin. Cancer Res. 8:3955-3960, 2002), while blockage of CXCR4 with the soluble inhibitor AMD3100 has been shown to reduce a number of pathological parameters related to asthmatic- type inflammation in a mouse model (Lukacs et ah, Am. J. Pathol. 160:1353-1360, 2002) and to inhibit autoimmune joint inflammation in IFN- ⁇ receptor-deficient mice (Matthys et ah, J. Immunol. 167:4686-4692, 2001).
  • RNA interference is a post-transcriptional RNA silencing phenomenon used by most eukaryotic organisms as a defense mechanism against viral attack and transposable factors. This RNA silencing process was first identified in plants, where it is referred to as post-transcriptional gene silencing (PTGS), and was subsequently observed in the nematode C. elegans by Fire and Mello ⁇ Nature 391:806-811, 1998).
  • RNAi involves the use of small interfering nucleic acid or RNA molecules (siRNAs) that selectively bind with complementary mRNA sequences, targeting them for degradation and thus inhibiting corresponding protein production. More recently it has been shown that siRNAs can induce de novo methylation and silencing of promoter sequences, known as transcriptional gene silencing (TGS).
  • TGS transcriptional gene silencing
  • dsRNA double-stranded RNA
  • Dicer a member of the RNase III family of dsRNA-specific ribonucleases
  • siRNAs small interfering RNAs
  • Each siRNA consists of two separate, annealed, single strands of nucleotides, with each strand having a 2-3 nucleotide 3' overhang, hi the effector step, siRNA duplexes bind to a nuclease complex to form an RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • the RISC then targets the endogenous mRNA complementary to the siRNA within the complex, and cleaves the endogenous mRNA approximately twelve nucleotides from the 3' terminus of the siRNA. Degradation of the endogenous mRNA is then completed by exonucleases. An amplification step may also exist within the RNAi pathway in some organisms, for example by copying of the input dsRNAs or by replication of the siRNAs themselves. Transfection of long dsRNA molecules of greater than 30 nucleotides into most mammalian cells causes nonspecific suppression of gene expression, as opposed to the gene-specific suppression seen in other organisms.
  • siRNAs targeting oncogenes are effective in reducing tumors in mice (Cancer Cell 2:243-247, 2002). More recent studies have indicated that synthetic dsRNA molecules 25-30 nucleotides in length may be more potent than dsRNA molecules 21 nucleotides in length (Kim et al. Nature Biotech. 23:222-226, 2005) and that use of dsRNA molecules having a combination of an asymmetric 3'- overhang two nucleotides in length and 3'-DNA residues on the blunt end may be advantageous (Rose et al. Nucl. Acids Res. 33:4140-4156, 2005).
  • RNAi has several advantages over other gene silencing techniques, such as the use of antisense oligonucleotides (ODN). RNAi techniques result in more specific inhibition of gene expression than ODN and are able to induce the same level of silencing as ODN at much lower concentrations of reagent. Also, siRNAs are more resistant to nuclease degradation than ODN. Bertrand et al. (Biochem. Biophys. Res. Commun. 296:1000, 2002) have demonstrated that, in mice, siRNA silencing is more effective than antisense suppression.
  • ODN antisense oligonucleotides
  • siRNAs in order to be effective in suppressing expression of a gene of interest to a high degree, siRNAs must be designed so that they are specific to the target gene.
  • a delivery system that specifically delivers the siRNA to the desired target is preferably employed. Delivery of siRNA to cells by means of exogenous delivery of preformed siRNAs or via promoter-based expression of siRNAs or shRNAs has been described. Genetic constructs for the delivery of siRNA molecules are described, for example, in US Patent 6,573,099. The delivery of short RNA fragments to cells in vivo in mammals can be problematic due to the rapid degradation of the RNA. The use of chemically modified siRNA molecules, as discussed below, can aid in overcoming this problem.
  • Short hairpin RNA are nucleic acid molecules that mimic the structure of the RNAi duplex and that can be produced in cells following delivery of expression vectors encoding the shRNA.
  • shRNA expression plasmids to reduce gene expression in vivo in rats has been described by Zhang et al. (J. Gene Med. 5:1039-1045, 2003).
  • the present invention provides compositions for the treatment and/or prevention of a disorder in a mammal by means of RNA interference, together with methods for the use of such compositions.
  • the inventive compositions comprise a small interfering nucleic acid molecule (siNA) that suppresses expression of a target gene within the target cell, wherein the target gene is selected from the group consisting of: CXCR4, SDF-I and HCLCAl.
  • Such compositions may further comprise a binding agent that specifically binds to a target internalizable antigen that is expressed on the surface of a target cell of interest, whereby, after binding to the target antigen, the binding agent and siNA are internalized into the cell, and the siNA released.
  • the cDNA sequences for CXCR4 isoforms a and b are provided in SEQ ID NO: 1 and 2, respectively, with the cDNA sequences for SDF-l ⁇ and SDF-l ⁇ being provided in SEQ ID NO: 3 and 4, respectively.
  • the cDNA sequence for HCLCAl is provided in SEQ ID NO: 5.
  • Examples of siNAs that are capable of suppressing expression of HCLCAl include the siRNA sequences corresponding to the target sequences provided in SEQ ID NO: 6-15.
  • Examples of siNAs that are capable of suppressing expression of SDF-I include the siRNA sequences corresponding to the target sequences provided in SEQ ID NO: 22-26 and 29-96.
  • the present invention provides compositions comprising a genetic construct that is capable of expressing a siNA that suppresses expression of a target gene within the target cell, wherein the target gene is CXCR4, SDF-I and/or HCLCAl.
  • Such compositions may additionally comprise a binding agent that specifically binds to a target internalizable antigen that is expressed on the surface of a target cell of interest whereby, after binding to the target antigen, the binding agent and genetic construct are internalized into the cell, and the siNA is expressed by the genetic construct.
  • the siNA is under the control of an RNA polymerase Ill-dependent or a tissue-specific RNA polymerase II-dependent promoter.
  • the inventive compositions comprise a genetic construct that is capable of expressing a siNA that suppresses expression of a CXCR4, SDF-I and/or HCLCAl gene within the target cell, wherein the genetic construct is packaged within a viral vector which, upon infection of the target cell, releases its genetic material enabling expression of the genetic construct.
  • the viral vector is an adeno virus-associated vector (AAV).
  • AAV adeno virus-associated vector
  • viral capsid proteins may act as a binding agent thereby targeting the siNA to specific cells and/or tissues.
  • HCLCAl is a promiscuous "chemokine sink" receptor present on a number of cell types that is, importantly, non-signalling. It mops up and internalizes a wide variety of chemokines (Weber et ah, MoI. Biol. Cell. 15:2492- 2508, 2004).
  • internalizable antigens located on the surface of cells that express HCLCAl include members of the integrin superfamily (for example ⁇ v ⁇ l), FGF receptors and the transferrin receptor.
  • the present invention provides compositions that comprise a siNA capable of suppressing expression of HCLCAl (or a genetic construct capable of expressing such a siNA) in combination with a binding agent that specifically binds to ⁇ v ⁇ l integrin. Delivery of compositions that are capable of suppressing expression of
  • CXCR4 and/or SDF-I is preferably targeted to eosinophils, mast cells, T cells, pre-B cells, epithelial cells, endothelial cells and/or metastatic tumor cells.
  • an internalizable antigen present on metastatic tumor cells is the antigen HER2neu.
  • target antigens that are expressed on the surface of a mast cell and that facilitate internalization of a complex bound to the target antigen include Fc ⁇ Rl and CXCR4 itself, as CXCR4-bound complexes are known to be internalized and degraded by the cell.
  • Examples of internalizable antigens located on the surface of T cells and pre-B cells include integrins, adhesion molecules and CD 19.
  • the present invention provides compositions that comprise a siNA capable of suppressing expression of CXCR4 and/or SDF-I (or a genetic construct capable of expressing such a siNA) in combination with a binding agent that specifically binds to ⁇ 4 ⁇ 7 integrin, HER2neu, Fc ⁇ Rl and CXCR4.
  • the binding agent employed in the inventive compositions is an antibody, or an antigen-binding fragment thereof.
  • Other binding agents that may be effectively employed in the inventive compositions include cell- specific ligands, and peptides or small molecules that specifically bind to cell-specific receptors. Viral (capsid) proteins may also be employed as binding agents.
  • binding agents that target the HER2neu antigen may be employed, such as those described in US patent 5,677,171, the disclosure of which is hereby incorporated by reference.
  • the binding agent employed in such compositions is the humanized monoclonal antibody trastuzumab (also known as HerceptinTM; Genentech, South San Francisco, CA; US Patent 6,800,738, the disclosure of which is hereby incorporated by reference).
  • the binding agent is linked to the siNA, genetic construct or viral vector by means of a streptavidin-biotin linker as described below.
  • the siNA, genetic construct or viral vector is complexed to a lipid carrier, such as a cationic lipid carrier, which in turn may be linked to a binding agent.
  • the siNA, genetic construct or viral vector is encapsulated within a liposome. A binding agent, or the antigen-binding portion thereof, may be present on the surface of the liposome.
  • the siNA within the genetic construct of the present invention is operably linked to a promoter that is specific to the target cell, whereby suppression of gene expression in non-target cells is reduced.
  • the present invention provides methods for reducing the expression of a target gene in a cell comprising contacting the cell with a siNA directed against the target gene, wherein the target gene is selected from the group consisting of: CXCR4, SDF- 1 and HCLCAl, together with methods for treating disorders characterized by undesirable levels of CXCR4, SDF-I and/orHCLCAl.
  • the present invention provides methods for the reduction of airway inflammation such as eosinophilia in a patient, such methods comprising administering at least one of the compositions disclosed herein.
  • the reduction in airway inflammation and eosinophilia will vary between about 20% and about 80%, preferably between 80% and 100%, and most preferably between 90% and 100%.
  • the percentage of reduction in lung inflammation and eosinophilia can be determined by measuring the number of leucocytes, including eosinophils in bronchoalveolar lavage fluid before and after treatment.
  • the present invention provides methods for the treatment or prevention of disorders by administering compositions disclosed herein that are directed against HCLCAl.
  • disorders include, but are not limited to: disorders characterized by excess production of mucus; chronic obstructive pulmonary diseases (COPD), such as asthma, chronic bronchitis and emphysema; inflammatory lung diseases; cystic fibrosis; and acute or chronic respiratory infectious diseases.
  • COPD chronic obstructive pulmonary diseases
  • the present invention provides methods for the treatment of disorders including, but not limited to, cancers, in particular metastatic cancers; autoimmune disorders; and allergic disorders, such as asthma, by administering an inventive composition directed against CXCR4 and/or SDF-I.
  • Fig. 1 shows an exemplary RNAi vector of the present invention.
  • Fig. 2 shows the inhibition of murine Sdfl gene expression by RNA interference in vitro in murine HC-11 mammary epithelial cells.
  • Fig. 3 shows the inhibition of murine Sdfl gene expression by RNA interference in vitro hi murine 6AVS bone marrow stromal cells.
  • the present invention is generally directed to compositions and methods for the treatment or prevention of disorders by means of RNA interference.
  • Disorders that may be effectively treated and/or prevented using the inventive compositions include, but are not limited to: diseases that benefit from the reduction of inflammation, including eosinophilia in the tissues of the respiratory system; disorders characterized by excess production of mucus; chronic obstructive pulmonary diseases (COPD), such as asthma, chronic bronchitis and emphysema; inflammatory lung diseases; cystic fibrosis; acute or chronic respiratory infectious diseases; cancers, in particular metastatic cancers; and autoimmune disorders.
  • COPD chronic obstructive pulmonary diseases
  • the inventive compositions comprise a "naked” or modified small interfering nucleic acid molecule (siNA) directed against a target gene, or a genetic construct that expresses the siNA under the control of a tissue-specific promoter, wherein the target gene is selected from the group consisting of: CXCR.4; SDF-I; and HCLCAl.
  • the inventive compositions may also comprise a binding agent, such as an antibody, that specifically binds to a target antigen which is present on the surface of a target cell of interest.
  • the target antigen recognizes and internalizes certain specific biological molecules, such that, on binding of a siNA-binding agent or genetic construct-binding agent complex to the target antigen, the complex is internalized into the target cell by endocytosis, the siNA or genetic construct is released from the complex, and the siNA reduces expression of the target gene by means of RNA interference.
  • target gene refers to a polynucleotide that comprises a region that encodes a polypeptide of interest, and/or a polynucleotide region that regulates replication, transcription, translation or other processes important to expression of the polypeptide.
  • small interfering nucleic acid molecule refers to any nucleic acid molecule that is capable of modulating the expression of a gene by RNA interference (RNAi), and thus encompasses short interfering RNA (siRNA), short interfering DNA (siDNA), double-stranded RNA (dsRNA), double- stranded DNA (dsDNA), complementary RNA/DNA hybrids, nucleic acid molecules containing modified (for example, semi-synthetic) base/nucleoside or nucleotide analogues, which may or may not be further modified by conjugation to non-nucleic acid molecules, custom modified primary or precursor micro-RNA (miRNA), short hairpin RNA (shRNA) molecules, and longer (up to one kb or more) dsRNA or hairpin RNA molecules, as long as these do not activate non-specific interference, for example via interferon.
  • RNAi short interfering RNA
  • siDNA short interfering DNA
  • dsRNA double-stranded
  • the hairpin loop region may be short (e.g. 6 nucleotides), long (undefined length), or may include an intron that is efficiently spliced in the targeted cells or tissues. Additionally, multiple tandem repeats in one orientation (for example, three or more short sense repeats) are included under the definition of siRNA, as these can elicit a potent RNAi like response in some systems.
  • siNAs examples include those corresponding to the target DNA sequences provided in SEQ ID NO: 6-15, 22-26 and 29-96.
  • an RNA sequence when comparing an RNA sequence to a DNA sequence, an RNA sequence will contain ribonucleotides where the DNA sequence contains deoxyribonucleotides, and further that the RNA sequence will typically contain a uracil at positions where the DNA sequence contains thymidine.
  • siRNA will be used throughout this disclosure as a prototypical small interfering nucleic acid molecule, with or without a hair pin structure.
  • the siNA is generated from an introduced DNA molecule that contains promoter and terminator sequences responsible for transcribing the nucleic acid sequences that comprise the siNA.
  • the introduced DNA may be in the form of a covalently-closed linear or circular plasmid or a PCR product, and these will preferably contain little or no DNA of prokaryotic origin.
  • Promoters may be of the type activated by RNA polymerase III or RNA polymerase II. Those of the former type include U6, tRNAval, Hl and versions of these promoters modified to achieve higher levels of transcription.
  • Promoters activated by RNA polymerase II may be constitutive (such as the widely used CMV and EF l ⁇ promoters), or may be transcribed in a preferred manner in a single cell, cell type, tissue type, or biochemical event. These latter promoters may be chosen for high level or low-level expression. When a hairpin or custom miRNA is used, a single specific promoter may be employed. When two custom microRNAs with complementary target regions are employed, or when dsRNAs are to be formed from two separate strands, specific, or combinations of constitutive and specific, promoters may be employed. In circumstances where reduced, but not eliminated, expression levels are desired, this may be achieved using completely or incompletely homologous siNAs, or using promoters of varying transcriptional activity. Alternatively, siNA may be targeted to regions of mRNA that are either highly affected, or less completely affected, by an siNA, or more than one siNA sequence directed to the target gene, genes or a pathway may be used to achieve stronger interference.
  • the siNA may be targeted to the 5' untranslated region, the coding region, or the 3' untranslated region of the target gene or message. Additionally, regions of the promoter of a target gene, or regions usually upstream of a gene may be targeted for
  • RNAi assisted heterochromatin formation RNAi assisted heterochromatin formation, epigenetic silencing/activation or other events affecting the function of the gene.
  • the siNA is between 19 to 30 nucleotides in length (for example, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides), more preferably 19-27 nucleotides in length, such as 21, 25 or 27 nucleotides in length, and comprises an oligonucleotide strand, also referred to as the antisense strand, that includes an antisense region about 19 to about 23 nucleotides in length that is complementary to at least a portion of a nucleotide sequence, such as a mRNA sequence, corresponding to a target DNA sequence.
  • the siNA may also contain a second oligonucleotide strand, referred to as the sense strand, that is sufficiently complementary to the first strand to anneal to the first, or antisense, strand, for example under biological conditions, such as those found in the cytoplasm of a cell.
  • the sense and antisense strands may be separate, distinct sequences, as in a dsRNA molecule, or may be linked, as in, for example, a shRNA molecule.
  • the siNA of the present invention is a dsRNA molecule.
  • the first and second strands of the dsRNA may be separate or may be linked, for example by a chemical linker or by one or more nucleotides, and may be of the same or different lengths.
  • the dsRNA may have an overhang of 1-5 (for example, 1, 2, 3, 4, or 5) nucleotides on at least one of the 3 '-terminal and 5'- terminal ends of the antisense and/or sense strands.
  • both strands of the dsRNA may be blunt-ended.
  • the 3' end of the sense strand of the dsRNA may be modified to allow for Dicer binding and processing as described, for example in US published patent application no. US 2005-0244858, the disclosure of which is hereby incorporated by reference.
  • An siNA can be unmodified or may be chemically-modified in order to increase resistance to nuclease degradation and/or to reduce the risk of unwanted immunostimulatory effects, caused, for example by activation of the interferon response.
  • some or all of the nucleotides of a siNA may comprise modified nucleic acid residues, or analogs of nucleic acid residues. Modifications can be incorporated in the 3 '-terminal region and/or the 5 '-terminal region, or throughout the siNA sequence, and can be made to the bases, sugar moieties and/or phosphate backbone. Examples of modifications that may be effectively employed in the inventive siNAs include those described in US published patent applications no.
  • Modifications that may be made to the phosphate backbone include, for example, phosphonate, such as methylphosphonate, phosphorothioate, phosphotriester and alkylphosphotriester modifications.
  • Modifications that may be made to the sugar moieties include, for example, 2'-alkyl pyrimidine, such as 2'-O-methyl, 2'-fluoro, amino and deoxy modifications.
  • Modifications that may be made to the base groups include, for example, abasic sugars, 2-O-alkyl modified pyrimidines, 4-thiouracil, 5-bromouracil, 5-iodouracil, and 5-(3-aminoallyl)-uracil modifications.
  • the hybridization characteristics of the modified siNA may be similar to or improved compared to the corresponding unmodified siNA. Such modifications can also improve the efficacy and safety of in vivo therapy by changing the stability, lifetime and circulation of the siNAs in the human body.
  • siNAs directed against target sequences disclosed herein can yield siNAs that hybridize strongly and specifically to the target nucleic acid.
  • siNAs directed against target sequences that are shifted by one to four nucleotides 5' or 3' of the sequences disclosed herein may be effective. It is useful to administer more than one such variant to a target area, or a combination of several different siNAs targeting different regions in and around the desired gene (e.g., exons, introns, promoter, or intergenic regions).
  • selected target sequences are sensitive to down regulation by low concentrations of siRNA.
  • Guidelines for the design of siNA include those provided in Ambion's Technical Bulletin #506 (available from Ambion Inc., Austin, TX), and are described below.
  • siRNA for example, nanomolar or sub-nanomolar concentrations
  • Assessing whether a gene has been downregulated, and the extent of downregulation, can be performed using, for example, real-time PCR, PCR, western blotting, flow cytometry or ELISA methods.
  • Methods for the preparation of genetic constructs, or expression vectors, comprising, or encoding, siNA targeted against nucleotide sequences of interest are detailed below.
  • binding agent refers to a molecule that specifically binds to a target antigen expressed on the surface of a target cells, and includes, but is not limited to, antibodies, including monoclonal antibodies and polyclonal antibodies; antigen-binding fragments thereof, such as F(ab) fragments, F(ab') 2 fragments, variable domain fragments (Fv) 5 small chain antibody variable domain fragments (scFv), and heavy chain variable domains (V HH ); small molecules; hormones; cytokines; ligands; peptides and viruses (either native or modified).
  • Antibodies, and fragments thereof may be derived from any species, including humans, or may be formed as chimeric proteins which employ sequences from more than one species.
  • binding agent as used herein thus encompasses humanized antibodies and veneered antibodies.
  • a binding agent is said to "specifically bind,” to a target antigen if it reacts at a detectable level (within, for example, an ELISA assay) with the target antigen, and does not react detectably with unrelated antigens under similar conditions.
  • Antibodies, and fragments thereof may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.
  • antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies as described, for example, by Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto, via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies, or by protein synthesis.
  • the binding agents employed in the inventive compositions and methods are preferably cell type-specific.
  • the binding agent may be specific for internalizable cell surface antigens found on metastatic cancer cells or mast cells, such as HER2neu, Fc ⁇ Rl or CXCR4 itself.
  • binding agents that may be usefully employed in the present invention thus include: anti- human HER2neu antibodies, anti-murine HER2neu antibodies, anti-human Fc ⁇ Rl antibodies, anti-murine Fc ⁇ Rl antibodies, anti-human CXCR4 antibodies, anti-murine CXCR4 antibodies, and antigen-binding fragments thereof.
  • an antibody that binds to HER2neu is the humanized monoclonal antibody trastuzumab (HerceptinTM).
  • trastuzumab as a cell-specific targeting agent have been described by Wei et al. ⁇ Int. J. Oncol. 23:1159-1165, 2003) and Chiu et al. (J. Control. Release 97:357-369, 2004).
  • binding agents that may be effectively used in the inventive compositions and methods include the CXCR4-specific chemokine ligand SDF-I (also known as CXCL 12), CXCR4-binding peptides derived from SDF-I, other peptides that specifically bind to CXCR4, and small molecules or drugs that bind to CXCR4.
  • NSC651016 One example of a drug that binds CXCR4 is the distamycin analog 2,2'[4,4'-[[aminocarbonyl]-amino]bis[N,4'-di[pyrrole-2-carboxamide-l,l '-dimethyl]] -6,8-napthalene disulfonic acid] hexasodium salt, also referred to as NSC651016.
  • NSC651016 has been shown to specifically inhibit binding of chemokines to the receptors CXCR4, CCR5, CCR3 and CCRl.
  • binding of NSC651016 to CXCR4 and to CCR5 has been demonstrated to induce receptor internalization and delayed recycling of the receptors (Howard et al., J. Leukoc. Biol. 64:6-13, 1998).
  • NSC651016 is also known to have anti-inflammatory and anti-angiogenesis activities.
  • T22 One example of a peptide that binds to CXCR4, and therefore may be usefully employed as a binding agent in the inventive compositions to target mast cells, is T22
  • T22 is a synthetic 18 amino acid peptide analog of polyphemusin II isolated from the hemocyte debris of American horseshoe crabs which has been shown to prevent infection by HIV-I isolates by blockage of CXCR4.
  • Another example of a binding agent that may be used to target mast cells by binding to CXCR4 is N- ⁇ -acetyl-nona- d-arginine (Arg) amide (ALX40-4C; Doranz et al, J. Exp. Med. 186:1395-1400, 1997) which has been shown to have a high degree of selectively for CXCR4 and to block infection by HIV-I strains at low, micromolar, concentrations.
  • compositions of the present invention comprise a binding agent, such as an antibody, connected to a genetic construct by means of a streptavidin-biotin linkage or via a peptide nuclear acid (PNA) linkage as described in Svahn et at, J. Gene Med. 6:S36-S44, 2004.
  • a binding agent such as an antibody
  • streptavidin encompasses both streptavidin and avidin, and derivatives or analogues thereof that are capable of high affinity, multivalent or univalent binding of biotin.
  • Techniques for the preparation of conjugates containing streptavidin-biotin linkages are well known in the art and include, for example, those described in US Patents 6,287,792 and 6,217,869, the disclosures of which are hereby incorporated by reference.
  • Biotin may be incorporated into the genetic construct using, for example Biotin-21-dUTP (BD Biosciences Clontech, Palo Alto, CA), which is a dTTP analog with biotin covalently attached to the pyrimidine ring through a 21 -atom spacer arm.
  • the biotin- labeled genetic construct is then linked to the streptavidin-antibody conjugate via biotin-streptavidin binding, using techniques well known to those of skill in the art.
  • Streptavidin-biotin linkers may, alternatively, be employed to link binding agent directly to "naked" siNA.
  • the present invention provides complexes that comprise a binding agent, such as an antibody, and a polynucleotide-binding component, such as a polycation, that is covalently bonded to the antibody through, for example, disulfide bonds.
  • a binding agent such as an antibody
  • a polynucleotide-binding component such as a polycation
  • Polycations that may be employed as polynucleotide- binding components include, for example, polylysine, polyarginine, polyornithine, polyethylenimine, chitosan and basic proteins, such as histones, avidin and protamines.
  • the polynucleotide-binding component is then attached to a genetic construct by means of electrostatic attraction between the opposite charges present on the genetic construct and the polynucleotide-binding component.
  • the antibody or other cell binding agent is thus bound to the genetic construct without functionally altering either the genetic construct or the antibody. Both the bond between the antibody and the polynucleotide-binding components and that between the polynucleotide-binding component and the genetic construct are cleaved following internalization of the complex into the target cell.
  • Such complexes may be prepared as described, for example, in US Patent 5,166,320.
  • helicases and other RNA-binding proteins may be linked to the binding agent, or antibody, and naked siNA is, in turn, linked to the helicase prior to administration.
  • Examples of such helicases and RNA-binding proteins are provided in Sasaki et ah, Genomics 82:323-330, 2003, Yan et ah, Nature 426:469-474, 2003 and Anderson et ah, MoI. Cell Proteomics, 3:311-326 Manuscript M300127- MCP200, Epub Jan 122004.
  • the genetic construct or siNA of the present invention is encapsulated in, or attached to a liposome, nanoparticle or polymer carrier, which in turn may be attached to a binding agent, such as an antibody directed against the target antigen.
  • a liposome protects the construct from degradation by endonucleases.
  • Methods for the encapsulation of biologically active molecules, such as nucleic acid molecules and proteins, within liposomes or polymers, and for the preparation of nucleic acid-lipid (lipoplex) and nucleic acid-polymer (polyplex) carrier complexes are well known in the art.
  • Liposome formulation, development and manufacturing services are available for example, from Gilead Liposome Technology Group (Foster City, CA).
  • Lipids for the preparation of liposomes are available, for example from Avanti Polar Lipids, Inc. (Alabaster, AL).
  • the resulting carrier containing the genetic construct of interest is then conjugated to the binding agent, using methods well known in the art, such as those taught, for example, in US patents 5,210,040, 4,925,661, 4,806,466 and 4,762,915.
  • Such methods include the use of linkers that fall into three major classes of functionality: conjugation through amide bond formation; disulfide or thioether formation or biotin-streptavidin binding.
  • the carrier is attached to the binding agent, such as an antibody, by means of a maleimide linker, as described, for example, in US Patent 6,372,250, the disclosure of which is hereby incorporated by reference.
  • the liposome employed in the inventive compositions is a pegylated liposome, wherein the surface of the liposome is conjugated with multiple (up to several thousand) strands of poly(ethylene glycol) (PEG) of approx. 2000 Da.
  • PEG poly(ethylene glycol)
  • the binding agent is then conjugated to the tips of some of the PEG strands.
  • the diameter of the liposome is preferably within the range of 100 nm to 10 ⁇ m.
  • the preparation of such pegylated liposomes and attachment of monoclonal antibodies to the liposomes is performed as described, for example, in Shi and Pardridge, Proc. Natl. Acad. Sd. USA 97:7567-7572, 2000; and Shi et al., Proc.
  • Pegylation of the liposome may increase the stability of the liposome and prevent non-specific attachment of cells, such as macrophages, and proteins to the liposome.
  • the preparation of pegylated liposomes, which encapsulate shRNA expression plasmids and are conjugated to monoclonal antibodies, and the use of such compositions in vivo in silencing gene expression in brain cancer is described in Zhang et al., J. Gene Med. 5:1039-1045, 2003.
  • the siNA or genetic construct of the present invention is packaged in an adenovirus or adeno-associated virus vector, which upon infection of the cell releases its genetic material enabling construct expression.
  • viral capsid proteins may act as the binding agent and target the siNA or genetic construct to specific cells.
  • Adenoviruses and adeno-associated viruses (AAV) do not integrate their genetic material into the host genome and do not require host replication for gene expression. AV and AAV vector delivery systems are thus well suited for rapid and efficient, transient expression of heterologous genes in a host cell.
  • AAV vector delivery systems have previously been shown to be effective in the treatment of cystic fibrosis (Aitken et al, Hum. Gene Ther. 12:1907-1916, 2001). Examples of AAV vector delivery systems which may be effectively employed in the present invention include, but are not limited to, those described in US Patent 6,642,051 and references cited therein.
  • Improvements have been made in the efficiency of targeting adenoviral vectors to specific cells by, for example, coupling adenovirus to DNA-polylysine complexes and by strategies that exploit receptor-mediated endocytosis for selective targeting. See, e.g., Curiel et al., Hum. Gene Ther. 3:147-154 (1992); and Cristiano and Curiel, Cancer Gene Ther. 3:49-57 (1996).
  • viral vectors that insert genetic material into a host cell's genome may be employed. Examples of such vectors include lentiviral, retroviral, plasmid and MLV vectors.
  • lentiviral vectors suitable for gene therapy are described, for example, in US Patents 6,531,123, 6,207,455 and 6,165,782, the disclosures of which are hereby incorporated by reference.
  • the use of lentivector- delivered RNA interference in silencing gene expression in transgenic mice is described by Rubinson et al. (Nat. Genet. 33:401-406, 2003).
  • the present invention further provides methods for the treatment or prevention of disorders in a patient by administration of a therapeutically effective amount of a composition disclosed herein.
  • a "patient” refers to any warm-blooded animal, including, but not limited to, a human. Such a patient may be afflicted with disease or may be free of detectable disease.
  • inventive methods may be employed for the treatment or prevention of disease.
  • inventive methods may also be employed in conjunction with other known therapies. For example, compositions directed against
  • HCLCAl may be employed to improve the efficacy of known therapeutic agents for disorders such as cystic fibrosis and other lung disorders by reducing mucus production and thereby facilitating increased delivery of the known therapeutic agent.
  • compositions of the present invention may be administered by injection (e.g., intradermal, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration), orally or epicutaneously (applied topically onto skin).
  • the compositions of the present invention are in a form suitable for delivery to the mucosal surfaces of the airways leading to or within the lungs.
  • the composition may be suspended in a liquid formulation for delivery to a patient in an aerosol form or by means of a nebulizer device similar to those currently employed in the treatment of asthma.
  • the inventive compositions may additionally contain a physiologically acceptable carrier.
  • a physiologically acceptable carrier any suitable carrier known to those of ordinary skill in the art may be employed in the compositions of this invention, the type of carrier will vary depending on the mode of administration.
  • the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer.
  • any of the above carriers or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, ultra-pure chitosan (Koping-Hoggard et al., Gene Ther.
  • Biodegradable microspheres e.g., polylactic galactide
  • Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075,109.
  • Other components, such as buffers, stabilizers, biocides, etc., may be included in the inventive compositions.
  • the preferred frequency of administration and effective dosage will vary from one individual to another and will depend upon the particular disease being treated, and may be determined by one skilled in the art.
  • the dosage is sufficient to provide siNA at a concentration of between 1 nM and 100 nM.
  • the inventive compositions may be administered in a single dosage, or in multiple, divided dosages, and may be employed in combination with one or more known therapeutic agents.
  • Preparation of pegylated liposomes, encapsulation of genetic constructs and conjugation with monoclonal antibody may be carried out as follows.
  • l-Pamiitoyl-2-oleoyl-5 «-glycerol-3-phosphocholine (POPC; Avanti Polar Lipids, Alabaster AL; 19.2 ⁇ mol), didodecyldimethylammonium bromide (DDAB; Avanti Polar Lipids; 0.2 ⁇ mol), distearolyphosphatidylethanolamine ((DSPE)-PEG 2000; Shearwater Polymers, Huntsville, AL; 0.6 ⁇ mol) and DSPE-PEG 2000- maleimide (30 nmol) are dissolved in chloroform/methanol (2:1, vol:vol) followed by evaporation.
  • POPC Avanti Polar Lipids, Alabaster AL
  • DDAB didodecyldimethylammonium bromide
  • DSPE-PEG 2000 distearolyphosphatid
  • the mean vesicle diameters may be determined using a Microtrac Ultrafine Particle Analyzer (Leeds-Northrup, St. Russia, FL). Plasmid attached to the exterior of the liposomes is removed by nuclease digestion as described by Monnard et al. (Biochim. Biophys. Acta 1329:39-50, 1997). For digestion of the unencapsulated DNA, 5 units of pancreatic endonuclease I and 5 units of exonuclease II are added in 5 mM MgCl 2 and 0.1 mM DTT to the liposome/DNA mixture after extrusion. After incubation at 37 0 C for 1 h, the reaction is stopped by adding 7 mM EDTA.
  • Monoclonal antibody specific for the target antigen is thiolated using a 40:1 molar excess of 2-iminothiolane (Traut's reagent) as described by Huwyler et al, Proc. Natl. Acad. Sci. USA 93:14164-14169, 1996. Thiolated antibody is then incubated with the liposomes overnight at room temperature, and the resulting immunoliposomes are separated from free monoclonal antibody by, for example, gel filtration chromatography.
  • Design OF SIRNA OLIGONUCLEOTIDES siNA molecules directed against HCLCAl, SDF-I or CXCR4 may be designed as described below.
  • the cDNA sequences for CXCR4 isoforms a and b are provided in SEQ ID NO: 1 and 2, respectively, with the cDNA sequences for SDF-I ⁇ and SDF-l ⁇ being provided in SEQ ID NO: 3 and 4, respectively.
  • the cDNA sequence for HCLCAl is provided in SEQ ID NO: 5.
  • thermodynamic stability of the siRNA duplex e.g., antisense siRNA binding energy, internal stability profiles, and differential stability of siRNA duplex ends
  • RNA interference Rosarz et al, Cell 115:199-208, 2003; Khvorova et al, Cell 115:209- 216, 2003.
  • Empirical rules such as those provided by the Tuschl laboratory (Elbashir et al, Nature 411:494-498, 2001; Elbashir et al, Genes Dev. 15:188-200, 2001) are also used.
  • Software and internet interactive services for siRNA design are available at the Ambion and Invitrogen websites.
  • Levenkova et al have described a software system for design and prioritization of siRNA oligos (Levenkova et al, Bioinformatics 20:430-432, 2004).
  • the Levenkova system is available on the internet and is downloadable freely for both academic and commercial purposes.
  • the siRNA molecules disclosed herein were based on the Invitrogen recommendations.
  • RNA target accessibility and secondary structure prediction can be carried out using, for example, Sfold software (Ding and Lawrence, CE. (2004) Rational design of siRNAs with Sfold software.
  • Sfold software Ding and Lawrence, CE. (2004) Rational design of siRNAs with Sfold software.
  • RNA secondary structure determination is also described in Current Protocols in Nucleic Acid Chemistry, Beaucage et al., ed, 2000, at 11.2.1-11.2.10.
  • the targeted region is selected from a cDNA sequence, such as the sequence of SDF-I.
  • nt nucleotide
  • Sense siRNA is used herein to mean a target sequence without the NN leader.
  • the sequence of the sense siRNA corresponds to (N19)TT of the Tuschl pattern AA(N19)TT (positions 3-23 if the 23 nt motif).
  • the siRNAs are preferably designed with symmetric 3' overhangs in order to form a symmetric duplex. For both sense and antisense siRNAs, either dTdT or UU are used as the 3' overhang.
  • siRNAs with an AA target motif leader the AA base pairs with the dTdT or UU overhang of the antisense siRNA.
  • the A pairs with the first dT or U of the overhang it is known however, that the overhang of the sense sequence can be modified without affecting targeted mRNA recognition.
  • the antisense siRNA is synthesized as the complement to position 1-21 of the 23 nt motif.
  • the 3' most nucleotide can be varied, but the nucleotide at position 2 of the 23 nt motif is selected to be complementary to the targeted sequence.
  • the first transcribed nt should be a purine.
  • the siRNA may be selected corresponding to the target motif NAR (N 17) YNN 5 where R is (A 3 G) and Y is (C 5 U).
  • the siRNAs are designed with symmetric 3 'TT overhangs (Elbashir et al., EMBO J. 20:6877-6888, 2001).
  • the target sequence motifs are selected to have about 30-70% GC content, preferably 40-60% GC content.
  • the "% GC” is calculated as: [the number of G or C nucleotides in the target sequence/ 21 for an AA target motif leader] x 100, [the number of G or C nucleotides in the target sequence/20 for a BA target motif leader] x 100, and [the number of G or C nucleotides in the target sequence/19 for an NB target motif leader] x 100.
  • thermodynamic properties of the sequences are determined, e.g., using the Sfold software referred to above.
  • DSSE refers to the differential stability of the siRNA duplex ends, i.e., the average difference between 5' antisense and 5' sense free energy values for the four nucleotide base pairs at the ends of the duplex. It has been shown that the 5 'AS region is less stable than the 5' S terminus in functional siRNA duplexes and vice versa for nonfunctional siRNA duplexes (Khvorova et ah, Cell 115:209-216, 2003). It is known that the siRNA duplex can be functionally asymmetric, in the sense that one of the two strands preferentially triggers RNAi (Schwartz et al., Cell 155:199-208, 2003).
  • AIS refers to the average internal stability of the duplex at positions 9-14 from the 5' end of the antisense strand. Comparisons between functional and nonfunctional siRNA duplexes indicate that the functional siRNA has lower internal stability in this region. It has been proposed that flexibility in this region may be important for target cleavage (the mRNA is cleaved between position 9 and 10) and/or release of cleaved products from RISC to regenerate RISC (Khvorova et al, Cell 115:209-216, 2003).
  • siRNA sequences and their thermodynamic properties are further selected according to the following criteria: (a) 40% ⁇ GC content ⁇ 60%; (b) antisense siRNA binding energy ⁇ -15 kcal/mol; and (c) exclusion of target sequence with at least one of AAAA, CCCC, GGGG or UUUU.
  • two additional criteria are used: (d) DSSE > 0 kcal/mol (Zamore asymmetry rule); and (e) AIS > -8.6 kcal/mol (cleavage site instability rule). This is the midpoint between the minimum of -3.6 and maximum of -13.6 (Khvorova et ah, 2003).
  • siRNA sequences are further checked for uniqueness against human and murine gene libraries (e.g., TIGR GI, ENSEMBL human genome), using Blast algorithms. Also, to increase the likelihood that the selected sequences will be active in all patients, sequences directed against targets having SNPs in the base pairing regions are excluded.
  • human and murine gene libraries e.g., TIGR GI, ENSEMBL human genome
  • siRNAs for use in the inventive compositions targeted against human HCLCAl are siRNA sequences corresponding to the target sequences provided in SEQ ID NO: 6-15.
  • SiRNA may be prepared by various methods, such as chemical synthesis, or from suitable templates using in vitro transcription, siRNA expression vectors or PCR generated siRNA expression cassettes. Preferably, chemical synthesis is used. Methods for chemical synthesis of RNA are well known in the art and are described, for example, in Usman et ah, J. Am. Chem. Soc. 109:7845, 1987; Scaringe et ah, Nucleic Acids Res. 18:5433, 1990; Wincott et ah, Nucleic Acids Res. 23:2677- 2684, 1995; and Wincott et ah, Methods MoI. Biol. 74:59, 1997.
  • siRNAs may be synthesized, for example, using protected ribonucleoside phosphoramidites and a DNA/RNA synthesizer, and are commercially available from a number of suppliers, such as Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO), Perbio Science (Rockford, IL), Glen Research (Sterling, VA), ChemGenes (Ashland, MA), and Ambion Inc. (Austin, TX). The siRNA strands can then be deprotected, annealed and purified before use, if necessary.
  • Annealing can be carried out, for example, by incubating single-stranded RNAs in 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM Mg acetate, 1 min at 90°C, then 1 hr at 37°C. The solution is then stored frozen at -20°C.
  • Useful protocols can be found in Elbashir et ah, Methods 26:199-213, 2002.
  • the ability of siRNAs to downregulate their target sequences may be tested in a model system by co-transfection of a cDNA encoding the target message and the siRNA to be tested.
  • Such a system comprises an easily transfectable cell line, such as HEK293.
  • siRNA sequences against endogenously expressed target genes in, for example, mast cells may be tested by transfecting primary mast cells, or cell lines derived from these cell types in vitro using commercially available transfection reagents, such as Lipofectamine 2000 (Invitrogen).
  • Expression vectors for generating siRNA fragments targeting CXCR4, SDF-I or HCLCAl are constructed by ligating annealed, chemically synthesized, oligonucleotide pairs into the appropriate vector (pSilencer, pSiren), or by PCR amplification of cDNA corresponding to siRNA sequences.
  • siRNA sequences should start with 5'G residues. Symmetric 3' overhangs and appropriate restriction sequences are added during amplification.
  • the amplified sequences are subcloned into, for example, pcDNA3 vectors (Invitrogen, San Diego, CA).
  • mast cell promoters such as the chymase promoter (SEQ ID NO: 19, NCBI Locus ID NM_001836, corresponding genomic contig NT_026437), tryptase promoters (tryptase alpha, SEQ ID NO: 16, NCBI Locus ID NM 003293 having corresponding genomic segment containing promoter areaNT_037887; tryptase beta 1, SEQ DD NO: 17, NCBI Locus ID NM_003294 having corresponding genomic segment containing promoter area NT_037887; tryptase beta 2, SEQ ED NO: 18, NCBI Locus ID NM_024164 having corresponding genomic segment containing promoter area NT_037887), or Fc ⁇ Rl promoters (SEQ ID NO: 20 and 21), are cloned into expression vectors containing a fluorescent reporter gene, such as EGFP, and tested in human and murine mast cell
  • a fluorescent reporter gene such as EGFP
  • RNAi vectors An exemplary RNAi vector is shown in Figure 1.
  • the vector can be constructed based on commercially available vectors such as pSilencer from Ambion and comparable vectors from other suppliers.
  • RNAi constructs that are expressed in the nucleus, and that contain promoter sequences directed against the CXCR4 promoter or the promoter of a transcription factor that activates the CXCR4 promoter.
  • RNAi-dependent chromatin silencing has been demonstrated in both fission yeast and plants (reviewed by Matzke and Matzke, Science 301:1060-1061, 2003). In plants, the synthesis of double-stranded RNA containing promoter sequences triggers transcriptional gene silencing and methylation of the target promoter (Mette et al, EMBOJ. 19:5194-5201, 2000).
  • Expression cassettes are designed to express siRNAs in the nucleus under the control of a human U6 snRNA promoter or tissue specific promoters such as the IgH, CD 19, CD20, CD21 or CD22 promoter. See, e.g., Miyagishi and Taira, Nature Biotechnology 20:497-500, 2002; Paul et al., ibid, 505-508.
  • the cassette also contains U6 termination sequences.
  • the desired CXCR4 promoter sequences or CXCR4 transcription factor sequences are subcloned into the cassette, e.g., a pU6 plasmid.
  • RNA expression cassettes may be found, for example, in the Ambion Technical Bulletin #506 (Ambion me, Austin, TX). Chromatin silencing in cells transfected with nuclear-targeted siRNA vectors is assessed by methods to detect gene-specific mRNA or protein expression such as quantitative PCR, Northern blotting, ELISA, flow cytometry and western blotting.
  • MC/9 cells (ATCC No. CRL-8306), an IL-3 dependent murine mast cell line derived from foetal liver, are cultured in Dulbecco's Modified Eagle's Medium with 4 mM L-glutamine supplemented with 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, 2 mM L-glutamine, 0.05 mM 2-mercaptoethanol, 10% v/v Rat T-STIM (Becton Dickinson #354115) or 10% v/v WEHI-3 supernatant as a source of IL-3 and 10% v/v foetal calf serum. Cells are maintained at a density of 2 x 10 5 - 2 x 10 6 cells/mL.
  • Cells are treated with immunoliposomes containing CXCR4-specific RNAi based vector conjugated to anti-human Transferrin Receptor (purchased from Biosource, Camarillo, CA) according to the procedure described in Example 1 above.
  • the effect of the immunoliposome treatment on CXCR4 expression is assessed by quantitative PCR, ELISA, flow cytometry and western blotting.
  • the appropriate antibody concentrations are predetermined in prior experiments with antibody- liposome complexes containing CMV-EGFP expression vectors.
  • the effects of treatment are monitored over a period of several days, by measuring total CXCR4 production (cells and medium) by, for example, Western blots, flow cytometry or ELISA, and CXCR4 mRNA by, for example, Northern blots or QC-PCR.
  • siRNA can produce nonspecific concentration-dependent effects on mammalian gene expression (Scherer and Rossi, Nature Biotechnology 21:1457-1465, 2003; Persengiev et al, RNA 10:12-18, 2004). These off-target effects can be minimized by selecting siRNAs with unique sequences, and using them at subnanomolar to nanomolar concentrations. In the above experiments, siRNA concentration is optimized for downregulation and nonspecific effects. Nonspecific effects are assessed by microarray-based expression profiling.
  • siRNA The therapeutic use of siRNA to, for example, knockdown IgE production in IgE-mediated diseases in humans and non-human animals may require rapid reversal when antigen (allergen) is no longer present.
  • Suppressor proteins from plant viruses are capable of reversing silencing in plant tissues where it is established, and preventing initiation of silencing in new tissues.
  • Plant virus genes encoding suppressor proteins include HC-Pro (Tobacco etch virus), P25 (Potato virus X), 2b (Cucumber mosaic virus), Turnip crinkle virus coat protein, and pi 9 (Cymbidium ringspot virus).
  • Vaccinia virus and human influenza A, B and C viruses each encode viral suppressors (E3L and NSI) which bind dsRNA and inhibit the mammalian IFN- regulated innate antiviral response (Li et ah, Proc. Natl. Acad. Sci. USA 101:1350-
  • siRNA sequences targeting murine Sdf-1 were transfected in two murine cell lines (HC-I l and 6AVS), and the knock-down in gene expression determined by Realtime PCR.
  • HC-Il cells mouse mammary epithelial cell line; American Type Culture Collection
  • cRPMI Invitrogen, Carlsbad CA
  • EGF Epidermal Growth Factor
  • 6AVS cells bone marrow stromal cell line; American Type Culture Collection
  • DMEM DMEM with 4 mM L-glutamine supplemented with 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, 2 mM L-glutamine and 0.05 mM 2- mercaptoethanol.
  • siRNA duplexes targeting murine Sdfl/Cxcll2 and corresponding to the DNA sequences given in SEQ ID NO: 22-26 were prepared by Invitrogen Corp. (Carlsbad, CA) using the StealthTM technology. These dsRNAs were blunt-ended 25-mers with a modified sense strand. The dsRNAs were transfected at concentrations of 1 nM to 40 nM using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. At 72 hours post transfection, cells were harvested and RNA isolated using the RNeasy96 kit (Qiagen, Venlo, The Netherlands) following the manufacturer's instructions.
  • First strand cDNA was prepared using Superscript II RT and Random Hexamers (Invitrogen).
  • TaqMan realtime RT-PCR was performed using Gene Expression Assays (containing the primers and probe) for SDFl and 18S (both from Applied Biosystems Inc., Foster City, CA) or RNA polymerase II (SEQ ID NO: 27 and 28; Universal Probe Library Probe #80 from Roche).
  • Message levels were determined by Real Time PCR using an Applied Biosystems Inc. 7900 (SDS Compendium 7900 Version 3.0 ABI) as directed by the manufacturer.
  • Fig. 2 shows analysis of Sdfl mRNA expression in transfected HC-I l cells, normalized to the scrambled control duplex (referred to as 81 cont; SEQ ID NO: 97) which is set at 1.
  • Three duplexes (330, 1246 and 2367; SEQ ID NO: 22, 24 and 26, respectively) showed good knockdown of Sdfl mRNA. At 40 nM concentration, the reductions were between 80-90%.
  • Fig. 3 shows analysis of Sdfl mRNA expression in transfected 6AVS cells, normalized to the scrambled control duplex (referred to as 81 cont; SEQ ID NO: 97) which is set at 1.
  • Two duplexes (330 and 2367; SEQ ID NO: 22 and 26, respectively) showed good knockdown of Sdfl mRNA. At 10 nM concentrations, reduction of up to 70% was achieved.
  • SEQ ID NO: 1-97 are set out in the attached Sequence Listing.
  • the codes for polynucleotide and polypeptide sequences used in the attached Sequence Listing confirm to WIPO Standard ST.25 (1988), Appendix 2.

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Abstract

La présente invention concerne des compositions pour le traitement et/ou la prévention d'un désordre chez les mammifères au moyen d'une interférence ARN, conjointement avec des procédés d'utilisation de telles compositions. Les compositions de l’invention comprennent une petite molécule d’acide nucléique d’interférence (pmANi) qui supprime l’expression d’un gène cible dans la cellule cible, ou un gène chimère qui exprime une telle pmANi, où le gène cible est CXCR4, SDF-1 et/ou HCLCA1.
PCT/NZ2006/000026 2005-02-22 2006-02-22 Compositions pour l’administration de molécules d’interférence arn et leur procédés d’utilisation Ceased WO2006091112A1 (fr)

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WO2006128141A3 (fr) * 2005-05-27 2007-07-12 Sirna Therapeutics Inc Inhibition, mediee par l'interference d'arn, de l'expression genique du facteur 1 derive des cellules stromales (sdf-1), au moyen d'acide nucleique interferant court (sina)
JP2010505428A (ja) * 2006-10-12 2010-02-25 ガニメド ファーマシューティカルズ アーゲー 癌及び癌転移の治療と診断のための組成物及び方法
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WO2011012897A1 (fr) 2009-07-31 2011-02-03 Astrazeneca Ab Nouvelle combinaison pour le traitement de l'asthme
WO2011061527A1 (fr) 2009-11-17 2011-05-26 Astrazeneca Ab Combinaisons qui comprennent un modulateur du récepteur glucocorticoïde, destinées au traitement de maladies respiratoires
WO2012085582A1 (fr) 2010-12-23 2012-06-28 Astrazeneca Ab Composé
WO2012085583A1 (fr) 2010-12-23 2012-06-28 Astrazeneca Ab Nouveau composé

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