WO2006119096A2 - Composition de particules infectieuses et methodes d'utilisation associees - Google Patents

Composition de particules infectieuses et methodes d'utilisation associees Download PDF

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WO2006119096A2
WO2006119096A2 PCT/US2006/016411 US2006016411W WO2006119096A2 WO 2006119096 A2 WO2006119096 A2 WO 2006119096A2 US 2006016411 W US2006016411 W US 2006016411W WO 2006119096 A2 WO2006119096 A2 WO 2006119096A2
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capsid protein
rna
papovavirus
infectious
short interfering
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WO2006119096A3 (fr
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Chava Kimchi-Sarfaty
Michael M. Gottesman
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The Government Of The United States Of America As Represented By The Secretary, Department Of Health And Human Services
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • 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
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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    • 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
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/22011—Polyomaviridae, e.g. polyoma, SV40, JC
    • C12N2710/22022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/22011—Polyomaviridae, e.g. polyoma, SV40, JC
    • C12N2710/22023—Virus like particles [VLP]
    • C—CHEMISTRY; METALLURGY
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/22011—Polyomaviridae, e.g. polyoma, SV40, JC
    • C12N2710/22032—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent

Definitions

  • the present invention relates generally to a infectious particle composition comprising a papovavirus capsid protein and a short interfering RNA.
  • the present invention further relates to methods of making the infectious particles and methods of use for the infectious particles.
  • RNA interference is a sequence-specific, naturally occurring gene- silencing mechanism mediated through the formation of an enzymatically active ribo- nucleoprotein termed the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • RNA component of RISC is a single-stranded RNA species 21-23 nucleotides in length, which acts as a guide for RISC such that RNA transcripts with a cognate sequence to the RNA within RISC are cleaved in a position-dependent manner. Sontheimer, Nat. Rev. MoI Cell Biol. 6: 127-138, 2005. A number of studies have shown that RISC formation and mRNA transcript cleavage occur primarily within the cytoplasm. Martinez et al., Cell 110: 563-574, 2002.
  • RNAi effector molecules are being actively applied to a very broad range of cell types including primary cells and are also being used in vivo as pre-clinical gene therapy studies have been reported. Caplen, Gene Ther. 11: 1241-1248, 2004.
  • siRNAs have been delivered using cationic lipid or polyplex delivery systems. These methods are not able to efficiently deliver siRNAs into cells in suspension, such as lymphoblastoids or erythroleukemia cells.
  • Short hairpin RNA expression cassettes have been adapted to be compatible with most plasmid and viral vector systems including retroviruses, adenoviruses, lentiviruses and adeno-associated viruses. As with the transfer of transgenes, all of these delivery systems require a significant degree of optimization and are often only useful in specific cell systems. Banan and Puri, Curr. Pharm. Biotechnol. 5: 441-450, 2004.
  • Some viral vectors also have the disadvantage of low titer, and also have a large genome size, which is difficult to manipulate. In addition, some are dependent on helper viruses or packaging cell lines, and some are not able to transduce non-dividing cells, or cells in suspension. Tiscornia et al., Proc. Natl. Acad. ScL U. S. A. 100: 1844-1848, 2003; Izquierdo, Cancer Gene Ther. 12: 217-227, 2005; Chromy et. al, Proc Natl Acad Sci USA, 100: 10477- 10482. 2003; Chen et. al., MoI Cell, 5: 557-67, 2000; Georgens et. al, Curr Pharm Biotechnol. 6: 49-55, 2005;.
  • siRNA for example, cationic lipid or polyplex delivery systems
  • cationic lipid or polyplex delivery systems do not efficiently deliver siRNA into a wide range of cell types.
  • the efficiency of DNA entry in to cells is very high using in vitro packaging pseudovirons.
  • some of the DNA does not enter the nucleus but remains in the cytoplasm. Therefore, the delivery and expression of shRNA in the cytoplasm from a DNA vector is limited.
  • the present invention provides compositions and methods for use of infectious particles, e.g., papovavirus pseudovirions, to deliver into mammalian cells a principal type of RNA interference (RNAi) effector molecule, e.g., RNA molecules encoding synthetic short interfering RNAs (siRNAs).
  • RNAi RNA interference
  • siRNA is encapsidated in a papovavirus capsid protein to form a papovavirus pseudoviral infectious particle.
  • the source of papovavirus capsid protein can be, for example, SV40 virus, polyoma vims, or papilloma virus.
  • the present invention further provides compositions and methods for papovavirus e.g., SV40, polyoma ,or papilloma pseudovirions, to deliver siRNAs.
  • siRNA is an example of an important mediator of a gene silencing mechanism by RNA interference (RNAi).
  • RNAi RNA interference
  • the compositions and methods further provide that S V40 pseudovirions can be used to deliver an RNAi effector, e.g., siRNAs.
  • infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA.
  • the infectious particle further comprises SV40 capsid protein VPl, papilloma virus capsid protein Ll, or polyoma virus capsid protein VPl.
  • the infectious particle comprises SV40 capsid protein VPl, VP2, and VP3.
  • the infectious particle comprises SV40 agno protein, SV40 E capsid protein, or polyoma E capsid protein.
  • the infectious particle comprises polyoma A capsid, B capsid, C capsid, D capsid, E capsid, or F capsid protein. In a further aspect, the infectious particle comprises SV40 A capsid, B capsid, C capsid, D capsid, E capsid, or F capsid protein. [0010] An infectious particle is provided comprising papilloma virus capsid protein Ll and L2 and an exogenous short interfering RNA. An infectious particle is provided comprising polyoma virus capsid protein VPl, VP2, and VP3 and an exogenous short interfering RNA.
  • a pharmaceutical composition comprising an infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA and a pharmaceutically acceptable carrrier.
  • a method for in vitro construction of an infectious particle comprising contacting a semi-purified or pure papovavirus capsid protein with an exogenous short interfering RNA, and allowing the semi-purified or pure papovavirus capsid protein to self assemble with the exogenous short interfering RNA into the infectious particle, so as to effect in vitro construction of the infectious particle.
  • the method comprises contacting the papovavirus capsid protein with the exogenous short interfering RNA in the presence of ATP, MgCl 2 , and CaCl 2 .
  • the ATP concentration is from about 1 mM to about 10 mM
  • MgCl 2 concentration is from about 1 mM to about 10 mM
  • CaCl 2 concentration is from about 0.1 mM to about 10 mM.
  • the ATP concentration is about 5 mM
  • MgCl 2 concentration is about 8 mM
  • CaCl 2 concentration is about 1 mM.
  • the semi-purified or pure papovavirus capsid protein is SV40 capsid protein VPl, papilloma virus capsid protein Ll, or polyoma virus capsid protein VPl.
  • the semi-purified or pure papovavirus capsid protein is SV40 capsid protein VPl, VP2, and VP3.
  • the semi-purified or pure papovavirus capsid protein is SV40 agno protein.
  • the semi-purified or pure papovavirus capsid protein is S V40 E capsid protein.
  • the semi-purified or pure papovavirus capsid protein is papilloma virus capsid protein Ll and L2.
  • the semi-purified or pure papovavirus capsid protein is polyoma virus capsid protein VPl, VP2, and VP3.
  • the semi-purified or pure papovavirus capsid protein is S V40 agno protein, S V40 E capsid protein, or polyoma E capsid protein.
  • the semi-purified or pure papovavirus capsid protein is polyoma A capsid, B capsid, C capsid, D capsid, E capsid, or F capsid protein.
  • the semi- purified or pure papovavirus capsid protein is SV40 A capsid, B capsid, C capsid, D capsid, E capsid, or F capsid protein.
  • an infectious complex comprising a semi-purified or pure papovavirus capsid protein and an exogenous short interfering RNA.
  • the papovavirus capsid protein is SV40 capsid protein VPl, papilloma virus capsid protein Ll, or polyoma virus capsid protein VPl .
  • the papovavirus capsid protein is SV40 capsid protein VPl, VP2, and VP3.
  • the papovavirus capsid protein is S V40 agno protein or SV40 E capsid protein.
  • the papovavirus capsid protein is papilloma virus capsid protein Ll and L2.
  • the papovavirus capsid protein is polyoma virus capsid protein VPl, VP2, and VP3.
  • the papovavirus capsid protein is SV40 agno protein, S V40 E capsid protein, or polyoma E capsid protein.
  • the papovavirus capsid protein is polyoma A capsid, B capsid, C capsid, D capsid, E capsid, or F capsid protein.
  • the papovavirus capsid protein is S V40 A capsid, B capsid, C capsid, D capsid, E capsid, or F capsid protein.
  • a method for inhibiting a sequence-specific cellular RNA activity in a mammalian cell comprising introducing an infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA to the mammalian cell, and contacting the short interfering RNA with the cellular RNA to inhibit the sequence-specific cellular RNA activity in the mammalian cell.
  • the method further comprises releasing the short interfering RNA in a cytoplasm of the cell; and contacting the short interfering RNA with a messenger RNA in the cytoplasm of the cell to inhibit protein synthesis from the messenger RNA.
  • the cellular RNA is a messenger RNA
  • the short interfering RNA inhibits protein synthesis from the messenger RNA.
  • the exogenous short interfering RNA is active in a cell cytoplasm.
  • the papovavirus capsid protein is SV40 capsid protein VPl, papilloma virus capsid protein Ll, or polyoma virus capsid protein VPl.
  • the papovavirus capsid protein is SV40 capsid protein VPl, VP2, and VP3.
  • the papovavirus capsid protein is SV40 agno protein.
  • the papovavirus capsid protein is S V40 E capsid protein.
  • the papovavirus capsid protein is papilloma virus capsid protein Ll and L2.
  • the papovavirus capsid protein is polyoma virus capsid protein VPl, VP2, and VP3.
  • the papovavirus capsid protein is SV40 agno protein, SV40 E capsid protein, or polyoma E capsid protein.
  • the papovavirus capsid protein is polyoma A capsid, B capsid, C capsid, D capsid, E capsid, or F capsid protein.
  • the papovavirus capsid protein is S V40 A capsid, B capsid, C capsid, D capsid, E capsid, or F capsid protein.
  • a method for in vivo transfer of a short interfering RNA into a host cell comprising introducing an infectious particle comprising a papovavirus capsid protein and the exogenous short interfering RNA to the host cell, and uncoating the pseudoviral particle in a cytoplasm of the cell to release the short interfering RNA.
  • the papovavirus capsid protein comprises SV40 capsid protein VPl, papilloma virus capsid protein Ll, polyoma virus capsid protein VPl, SV40 E capsid or polyoma E capsid.
  • a method for in vivo transfer of a short interfering RNA into a mammalian subject comprising administering an infectious particle comprising a papovavirus capsid protein and the exogenous short interfering RNA into the mammalian subject, introducing the infectious particle into a target cell, and contacting the short interfering RNA with a cellular RNA within the target cell to inhibit a sequence-specific cellular RNA activity in the mammalian cell.
  • the papovavirus capsid protein comprises SV40 capsid protein VPl, papilloma virus capsid protein Ll, or polyoma virus capsid protein VPl.
  • the method comprises administering the infectious particle orally, intranasally, intrapulmonarily, intramuscularly, intraperitoneally, or intravenously to the mammalian subject.
  • a method for preventing or treating an infectious disease in a mammalian subject comprises administering to the mammalian subject an infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA, contacting the short interfering RNA with a cellular RNA within a target cell of the mammalian subject, wherein the infectious particle is administered in an amount effective to inhibit a sequence-specific RNA activity to reduce or eliminate the infectious disease or to prevent its occurrence or recurrence in the mammalian subject.
  • the infectious disease is a viral disease, bacterial disease or parasite disease.
  • the viral disease includes, but is not limited to, human immunodeficiency virus infection, hepatitis B infection, or hepatitis C infection.
  • the sequence-specific RNA activity is a cellular RNA activity, viral RNA activity, bacterial RNA activity, or parasite RNA activity.
  • a method for preventing or treating a neoplastic disease in a mammalian subject comprises administering to the mammalian subject an infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA, contacting the short interfering RNA with a cellular RNA within a target cell of the mammalian subject, wherein the infectious particle is administered in an amount effective to inhibit a sequence- specific cellular RNA activity to reduce or eliminate the neoplastic disease or to prevent its occurrence or recurrence in the mammalian subject.
  • the cellular RNA is expressed from an oncogenic gene in the target cell.
  • the infectious particle is administered in combination with a chemotherapeutic agent to the mammalian subject.
  • the infectious particle is administered in combination with a chemotherapeutic agent
  • the cellular RNA can be expressed from an ABC transporter gene in the target cell.
  • a method for preventing or treating an autoimmune disease in a mammalian subject comprises administering to the mammalian subject an infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA, contacting the short interfering RNA with a cellular RNA within a target cell of the mammalian subject, wherein the infectious particle is administered in an amount effective to inhibit a sequence-specific cellular RNA activity to reduce or eliminate the autoimmune disease or to prevent its occurrence or recurrence in the mammalian subject.
  • the autoimmune disease includes, but is not limited to, thrombotic thrombocytopenic purpura disease, autoimmune hepatitis disease, rheumatoid arthritis, systemic lupus erythematosis, type 1 diabetes, multiple sclerosis, Sjogren's syndrome or inflammatory bowel disease.
  • a method for preventing or treating a disease caused by a genetic defect in a mammalian subject comprises administering to the mammalian subject an infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA, contacting the short interfering RNA with a cellular RNA within a target cell of the mammalian subject, wherein the infectious particle is administered in an amount effective to inhibit a sequence-specific cellular RNA activity to reduce or eliminate the disease caused by the genetic defect or to prevent its occurrence or recurrence in the mammalian subject.
  • the disease is Huntington's disease.
  • kits comprising an infectious particle comprising papovavirus capsid protein and an exogenous short interfering RNA.
  • a kit comprising a papovavirus capsid protein, a short interfering RNA molecule, a buffer, ATP, MgCl 2 , and CaCl 2 , in amounts suitable to reconstitute an infectious particle comprising papovavirus capsid protein and an exogenous short interfering RNA.
  • the ATP concentration is from about 1 mM to about 10 mM
  • MgCl 2 concentration is from about 1 mM to about 10 mM
  • CaCl 2 concentration is from about 0.1 mM to about 10 mM.
  • the ATP concentration is about 5 mM
  • MgCl 2 concentration is about 8 mM
  • CaCl 2 concentration is about 1 mM.
  • the papovavirus capsid protein is SV40 capsid protein VPl, papilloma virus capsid protein Ll, or polyoma virus capsid protein VPl.
  • the papovavirus capsid protein is SV40 capsid protein VPl, VP2, and VP3.
  • the papovavirus capsid protein is SV40 agno protein.
  • the papovavirus capsid protein is SV40 E capsid protein.
  • the papovavirus capsid protein is papilloma virus capsid protein Ll and L2.
  • the papovavirus capsid protein is polyoma virus capsid protein VPl, VP2, and VP3.
  • the papovavirus capsid protein is SV40 agno protein, SV40 E capsid protein, or polyoma E capsid protein.
  • the papovavirus capsid protein is polyoma A capsid, B capsid, C capsid, D capsid, E capsid, or F capsid protein.
  • the papovaviras capsid protein is SV40 A capsid, B capsid, C capsid, D capsid, E capsid, or F capsid protein.
  • a kit comprising an infectious particle comprising papovavirus capsid protein and an exogenous short interfering RNA and one or more of: packaging and instructions for use, a buffer, a papovavirus capsid protein, a polynucleotide encoding short interfering RNA molecule, a positive control sample, a negative control sample, and a negative control polynucleotide.
  • Figures IA and IB show the induction of RNAi against GFP in the human lymphoblastoid .45 cell line using the pseudovirion delivery of plasmids expressing short hairpin RNAs.
  • Figure 2 shows uptake studies of IVP-encapsulated fluorescent-tagged siRNAs in .45 human lymphoblastoid cells.
  • Figure 3 shows delivery of GFP siRNAs into human lymphoblastoid cells using pseudo virions.
  • Figure 4 shows dose response of siGFP packaged in vitro against HeLa cells stably expressing GFP.
  • Figure 5 shows expression of siRNA against HeLa cells stably expressing GFP delivered by S V40 pseudovirions and by lipid transduction.
  • RNA interference is a naturally occurring gene silencing mechanism mediated by small double-stranded RNA molecules (short interfering RNAs, siRNAs).
  • Papovavirus which include SV40, polyoma and papilloma, have the ability to self-assemble using either the major capsid protein VPl, all capsid proteins VPl, VP2 and VP3, or the E capsid proteins.
  • the capsid proteins are recombinant proteins prepared in insect, bacteria or mammalian cells. The assembled capsid proteins maintain their ability to transduce a wide variety of cells through natural receptors that make these pseudovirions infectious particles. No packaging cell line, and no viral DNA or RNA is required for packaging, which makes these pseudovirions safer than other delivery systems. Self-assembly in vitro does not require specific DNA sequences to signal the construction of the capsid envelope.
  • compositions and methods are provided which demonstrate the first use of infectious particles, e.g., papovavirus pseudovirions or SV40 pseudovirions, to deliver into human cells a principal type of RNAi effector molecule, siRNA.
  • infectious particles e.g., papovavirus pseudovirions or SV40 pseudovirions
  • Compositions and methods provide RNA molecules encoding synthetic siRNAs encapsidated in a papovims or S V40 pseudoviral particle.
  • siRNA-mediated RNA interference was observed in (1) human lymphoblastoid cells (.45 cells) following sequential transduction of in vzYro-packaged pseudoviral particles with green fluorescent protein (IVP-GFP) and IVP packaged siRNAs corresponding to GFP (IVP-siGFP), and (2) in HeLa cells stably expressing a GFP transduced with IVP-siGFP.
  • IVP-GFP green fluorescent protein
  • IVP-siGFP IVP packaged siRNAs corresponding to GFP
  • Infectious particles e.g., papovavirus pseudovirions or SV40 pseudovirions, are useful as a delivery system for the transfer of RNAi effector molecules, such as siRNA.
  • Papovavirus pseudovirions e.g., SV40 pseudovirions
  • the ability of human lymphoblastoid cells to support RNAi was further established using sequential transduction of .45 cells with packaged plasmid DNA expressing the green fluorescent protein (IVP-GFP), and an shRNA corresponding to the GFP (F/P-shGFP).
  • IVP-GFP green fluorescent protein
  • F/P-shGFP shRNA corresponding to the GFP
  • SV40 mediates DNA transfer of nucleic acid to the cytoplasm. A portion of the DNA reaches the nucleus where it is transcribed to form shRNA and transported back to the cytoplasm where RNAi-associated cleavage of mRNA principally occurs.
  • Infectious particle refers to infectious, non-replicative viral particles assembled from viral capsid , proteins and encapsidating a nucleic acid molecule, e.g., an siRNA molecule.
  • Infectious pseudoviral particles can be derived from viruses such as papovavirus, which include S V40, polyoma and papilloma, which have the ability to self-assemble using either the major viral capsid protein VPl, all capsid proteins VPl, VP2 and VP3, or the E capsid proteins.
  • the viral capsid proteins are recombinant proteins prepared in insect, bacteria or mammalian cells. The assembled capsid proteins maintain their ability to transduce a wide variety of cells through natural receptors that make these pseudovirions infectious particles.
  • An embodiment of the invention provides an infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA (siRNA) which can transfer siRNA to mammalian cells and is useful for inhibition of cellular RNAs, for example, messenger RNA, to inhibit expression of intracellular proteins.
  • the pseudoviral particles are targeted to the cytoplasmic compartment of a cell, e.g., HeLa cells or human lymphoblastoid cells, releasing an siRNA molecule which effectively targets an RNA or mRNA of interest and specifically inhibits protein synthesis of the targeted protein of interest.
  • the infectious particle is capable of delivering siRNA to the cytoplasmic compartment of the cell with high capacity, including no viral DNA, and with very high efficiency.
  • the infectious pseudoviral particles can infect cells including both dividing and non-dividing cells. The high efficiency and absence of viral DNA in the infectious pseudoviral particles is an important consideration for safety issues when using a gene therapy vector.
  • the infectious pseudoviral particles can infect cells, including both dividing and non-dividing cells.
  • non-dividing cells include, but are not limited to, differentiated cells from a tissue or organ of a mammalian subject including differentiated cells from the hematopoietic system such as lymphocytes, T lymphocytes, B lymphocytes, NK cells, macrophages, dendritic cells, granulocytes, neutrophil, eosinophil, basophil, mast cell precursors, monocytes, histiocyte, macrophages, dendritic cells, langerhans cells, microglia, kupffer cells, osteoclasts, megakaryoblast, megakaryocyte, platelets and erythrocytes.
  • differentiated cells from a tissue or organ of a mammalian subject including differentiated cells from the hematopoietic system such as lymphocytes, T lymphocytes, B lymphocytes, NK cells, macrophages, dendritic cells, granulocytes, neutr
  • the infectious pseudoviral particle is capable of delivering siRNA to the cytoplasmic compartment of the cell with high capacity, for example, to stem cells, including, but not limited to, bone marrow cells, dendritic cells, embryonic stem cells, somatic stem cells, umbilical cord blood stem cells, hematopoeitic stem cells, mesenchymal stem cells, neural stem cells, bone marrow stromal cells, fetal liver stromal cells, embryonic germ cells.
  • stem cells including, but not limited to, bone marrow cells, dendritic cells, embryonic stem cells, somatic stem cells, umbilical cord blood stem cells, hematopoeitic stem cells, mesenchymal stem cells, neural stem cells, bone marrow stromal cells, fetal liver stromal cells, embryonic germ cells.
  • an infectious particle comprises a papovavirus capsid protein and an exogenous short interfering RNA (siRNA) wherein the double-stranded siRNA molecule down-regulates expression of a target gene of interest, wherein the siRNA molecule comprises about 15 to about 28 base pairs.
  • siRNA short interfering RNA
  • the infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA further provides a double stranded short interfering ribonucleic acid (siRNA) molecule that directs cleavage of a target RNA of interest via RNA interference (RNAi), wherein the double stranded siRNA molecule comprises a first and a second strand, each strand of the siRNA molecule is about 18 to about 28 nucleotides in length, the first strand of the siRNA molecule comprises nucleotide sequence having sufficient complementarity to the target RNA for the siRNA molecule to direct cleavage of the target RNA via RNA interference, and the second strand of said siRNA molecule comprises nucleotide sequence that is complementary to the first strand.
  • siRNA ribonucleic acid
  • An infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA (siRNA) is useful in the treatment of disease, for example, neoplastic disease, autoimmune disease, genetic disease, or viral, bacterial, or parasite infectious disease
  • siRNA or shRNA sequences are known in the art and include, but are not limited to: CDH-I, p53, CDC20, Brummelkamp et ah, Science 296: 550-553, 2002; CYLD, Kovalenko et al, Nature 424: 801-5, 2003; Ras-Gap et al, Nature Biotechnology 21: 559-561, 2003; Tubulin et al., Proc. Natl.
  • siRNAs against cellular coreceptors CXCR4 and CCR5 can be of therapeutic value as an HIV/AIDS gene therapy; Anderson et al., AIDS Res Ther. 2: 1, 2005.
  • siRNA- directed TGF- ⁇ l silencing can be of therapeutic value in the prevention and treatment of fibrotic disease of the kidney; Takabatake, et al., Gene Ther. Feb 24, 2005, epub.
  • RNAi for Huntington's disease (HD) therapy.
  • Huntington's disease (HD) is a fatal, dominant neurogenetic disorder.
  • HD results from polyglutamine repeat expansion (CAG codon, Q) in exon 1 of HD, conferring a toxic gain of function on the protein huntingtin (htt).
  • siRNA directed against mutant human htt reduced htt mRNA and protein expression in cell culture and in HD mouse brain.
  • htt gene silencing improved behavioral and neuropathological abnormalities associated with HD. Harper et ah, Proc Natl Acad Sd USA. Apr 5, 2005; epub.
  • EGFR human epidermal growth factor receptor
  • Blocking CXCR4 expression at the mRNA level by a combination of two siRNAs impairs invasion of breast cancer cells in Matrigel invasion assay and inhibits breast cancer metastasis in an animal model. Targeting more than one site of the target gene may be important to overcome the functional redundancy of other variants of a single gene, especially in in vivo experiments. Moreover, our studies confirm the necessity of CXCR4 in breast cancer metastasis. Liang et al., Cancer Res. 65: 967-71, 2005.
  • Tpr-Met the oncogenic counterpart of the Met receptor, has been detected in gastric cancers, as well as in precursor lesions and in the adjacent normal gastric mucosa. This has prompted the suggestion that Tpr-Met may predispose to the development of gastric tumors.
  • oncogenes activated by point mutation or rearrangements can be targeted while sparing the product of the wild-type allele.
  • Suppression of Tpr-Met expression and inhibition of Tpr-Met-mediated transformation and tumorigenesis may occur by means of a pseudoviral particle comprising a short interfering RNA (siRNA) directed toward the Tpr-Met junction (anti-TM2). It is possible that pseudoviral particle mediated delivery of anti-TM2 siRNA may be developed into a powerful tool to treat Tpr-Met-positive cancers.
  • siRNA short interfering RNA
  • anti-TM2 siRNA Taulli et ah, Cancer Gene Titer. Feb 18, 2005; epub.
  • target gene siRNA or shRNA sequences include, but are not limited to: K-ras 12, K-ras 13, or K-ras 61; B-ras; Src; MMP-I, MMP-9, TIMP-I.
  • target gene siRNA or shRNA sequences include, but are not limited to: ⁇ -synuclein (Parkinson's disease); hepatitis B; hepatitis C; Huntington's disease; human immunodeficiency virus; hairless gene; IL-4, IL-13, IL-4 receptors, IL-13 receptors (asthma, respiratory diseases); NOGO and NOGO receptors (spinal cord injury); PTP-IB (diabetes, obesity); and VEGF (angiogenesis, age-related macular degeneration, diabetic retinopathy, cancer, kidney disease).
  • Sirna Therapeutics, Inc. San Francisco, CA.
  • An infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA (siRNA) is useful in the treatment of neoplastic disease in a mammalian subject, by providing a more effective chemotherapeutic treatment of neoplastic disease and overcoming the deleterious effects of multiple drug resistance on the effectiveness of chemotherapeutic treatment.
  • siRNA or shRNA sequences include, but are not limited to: genes encoding a superfamily of ATP-binding cassette (ABC) transporters, including about 48 target genes within the superfamily.
  • Exemplary target genes include, for example MDRl, MRPl, or MRP2. ABC proteins transport various molecules across extra- and intra-cellular membranes.
  • ABC genes are divided into seven distinct subfamilies (ABCl, MDR/TAP, MRP, ALD, OABP, GCN20, White).
  • ABCBl protein is an exemplary member of the MDR/TAP subfamily. Members of the MDR/TAP subfamily are involved in multidrug resistance.
  • the protein encoded by this gene is an ATP-dependent drug efflux pump for xenobiotic compounds with broad substrate specificity. It is responsible for decreased drug accumulation in multidrug-resistant cells and often mediates the development of resistance to anticancer drugs. This protein also functions as a transporter in the blood-brain barrier.
  • ATP binding cassette superfamily, subfamily B (MDR/TAP) member 1 putatively activated by random chromosomal rearrangements, and overexpressed in acute myeloid leukemia and cancer cell lines, by hypomethylation of the promoter and altered chromatin structure, up-regulated by P/CAF siRNA or shRNA sequences that inhibit the expression of these MDR or ABC transporter proteins are useful to treat multidrug resistance arising in chemotherapeutic treatment for cancer or neoplastic disease.
  • siRNA targeted to ABC transporter target genes for example, MDRl, MRPl, or MRP2
  • An infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA (siRNA) is useful in the treatment of autoimmune disease.
  • siRNA or shRNA sequences include, but are not limited to, autoantibodies or autoimmune proteins involved in autoimmune disease including, but not limited to, thrombotic thrombocytopenic purpura (TTP) disease, autoimmune hepatitis, rheumatoid arthritis, systemic lupus erythematosis (lupus), type 1 diabetes, multiple sclerosis (MS), Sjogren's syndrome and inflammatory bowel disease.
  • TTP thrombotic thrombocytopenic purpura
  • lupus systemic lupus erythematosis
  • MS multiple sclerosis
  • Sjogren's syndrome and inflammatory bowel disease.
  • an exemplary target for siRNA or shRNA sequences includes autoantibodies to Adam TS 13 protein, a protease of von Wildebrandt factor (vWF) involved in the clotting cascade.
  • an exemplary target for siRNA or shRNA sequences includes autoantibodies or T lymphocyte reactivity to myelin proteins such as myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), or myelin-associated glycoprotein (MAG), in MS patients.
  • myelin proteins such as myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), or myelin-associated glycoprotein (MAG), in MS patients.
  • MBP myelin basic protein
  • MOG myelin oligodendrocyte glycoprotein
  • MAG myelin-associated glycoprotein
  • an exemplary target for siRNA or shRNA sequences includes autoantibodies (i.e., nuclear, smooth muscle, thyroid, liver-kidney microsomal, soluble liver antigen, or hepatic lectin).
  • autoantibodies i.e., nuclear, smooth muscle, thyroid, liver-kidney microsomal, soluble liver antigen, or hepatic lectin.
  • RNA AND DNA INTERFERENCE METHODS A. Short Interfering RNAs (RNAi)
  • RNA interference is a mechanism of post-transcriptional gene silencing mediated by double-stranded RNA (dsRNA), which is distinct from antisense and ribozyme- based approaches (see Jain, Pharmaco genomics 5: 239-42, 2004 for a review of RNAi and siRNA).
  • RNA interference is useful in an infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA or in a method for in vitro construction of an infectious particle.
  • dsRNA molecules are believed to direct sequence-specific degradation of mRNA in cells of various types after first undergoing processing by an RNase EH-like enzyme called DICER (Bernstein et al, Nature 409: 363, 2001) into smaller dsRNA molecules comprised of two 21 nt strands, each of which has a 5' phosphate group and a 3' hydroxyl, and includes a 19 nt region precisely complementary with the other strand, so that there is a 19 nt duplex region flanked by 2 nt-3' overhangs.
  • DICER RNase EH-like enzyme
  • RNAi is thus mediated by short interfering RNAs (siRNA), which typically comprise a double-stranded region approximately 19 nucleotides in length with 1-2 nucleotide 3' overhangs on each strand, resulting in a total length of between approximately 21 and 23 nucleotides.
  • siRNA short interfering RNAs
  • dsRNA longer than approximately 30 nucleotides typically induces nonspecific mRNA degradation via the interferon response.
  • the presence of siRNA in mammalian cells rather than inducing the interferon response, results in sequence-specific gene silencing.
  • a short, interfering RNA comprises an RNA duplex that is preferably approximately 19 basepairs long and optionally further comprises one or two single- stranded overhangs or loops.
  • An siRNA may comprise two RNA strands hybridized together, or may alternatively comprise a single RNA strand that includes a self -hybridizing portion.
  • siRNAs may include one or more free strand ends, which may include phosphate and/or hydroxyl groups.
  • siRNAs typically include a portion that hybridizes under stringent conditions with a target transcript.
  • One strand of the siRNA (or, the self-hybridizing portion of the siRNA) is typically precisely complementary with a region of the target transcript, meaning that the siRNA hybridizes to the target transcript without a single mismatch. In certain embodiments of the invention in which perfect complementarity is not achieved, it is generally preferred that any mismatches be located at or near the siRNA termini.
  • siRNAs have been shown to downregulate gene expression when transferred into mammalian cells by an infectious pseudoviral particle.
  • the infectious particle comprises a papovavirus capsid protein and an exogenous siRNA.
  • RNA interference using siRNA is reviewed in, e.g., Tuschl, Nat. Biotechnol. 20: 446-448, 2002; See also Yu et al, Proc. Natl. Acad. ScL, 99: 6047-6052, 2002; Sui et al, Proc. Natl. Acad. Sci USA., 99: 5515-5520, 2002; Paddison et al, Genes and Dev.
  • the siRNA may consist of two individual nucleic acid strands or of a single strand with a self-complementary region capable of forming a hairpin (stem-loop) structure.
  • a hairpin stem-loop
  • siRNA capable of effectively mediating gene silencing.
  • intracellular processing e.g., by DICER
  • target exons rather than introns, and it may also be preferable to select sequences complementary to regions within the 3' portion of the target transcript.
  • sequences that contain approximately equimolar ratio of the different nucleotides and to avoid stretches in which a single residue is repeated multiple times.
  • siRNAs may thus comprise RNA molecules having a double-stranded region approximately 19 nucleotides in length with 1-2 nucleotide 3' overhangs on each strand, resulting in a total length of between approximately 21 and 23 nucleotides.
  • siRNAs also include various RNA structures that may be processed in vivo to generate such molecules. Such structures include RNA strands containing two complementary elements that hybridize to one another to form a stem, a loop, and optionally an overhang, preferably a 3' overhang.
  • the stem is approximately 19 bp long, the loop is about 1-20, more preferably about 4-10, and most preferably about 6-8 nt long and/or the overhang is about 1-20, and more preferably about 2-15 nt long.
  • the stem is minimally 19 nucleotides in length and may be up to approximately 29 nucleotides in length. Loops of 4 nucleotides or greater are less likely subject to steric constraints than are shorter loops and therefore may be preferred.
  • the overhang may include a 5' phosphate and a 3' hydroxyl. The overhang may but need not comprise a plurality of U residues, e.g., between 1 and 5 U residues.
  • RNAs are referred to as microRNAs (mRNAs) and are typically between approximately 20 and 26 nucleotides in length, e.g., 22 nt in length.
  • stRNAs small temporal RNAs
  • mRNA precursors typically approximately 70 nt long with an approximately 4-15 nt loop.
  • RNAs of this type have been identified in a number of organisms including mammals, suggesting that this mechanism of post- transcriptional gene silencing may be widespread (Lagos-Quintana et al, Science 294: 853-858, 2001; Pasquinelli, Trends in Genetics 18: 171-173, 2002, and references in the foregoing two articles). MicroRNAs have been shown to block translation of target transcripts containing target sites in mammalian cells (Zeng et al, Molecular Cell 9: 1-20, 2002).
  • siRNAs such as naturally occurring or artificial ⁇ i.e., designed by humans mRNAs that bind within the 3' UTR (or elsewhere in a target transcript) and inhibit translation may tolerate a larger number of mismatches in the siRNA/template duplex, and particularly may tolerate mismatches within the central region of the duplex.
  • some mismatches may be desirable or required as naturally occurring StRNAs frequently exhibit such mismatches as do mRNAs that have been shown to inhibit translation in vitro.
  • siRNAs when hybridized with the target transcript such siRNAs frequently include two stretches of perfect complementarity separated by a region of mismatch. A variety of structures are possible.
  • the mRNA may include multiple areas of nonidentity (mismatch).
  • the areas of nonidentity (mismatch) need not be symmetrical in the sense that both the target and the mRNA include nonpaired nucleotides.
  • the stretches of perfect complementarity are at least 5 nucleotides in length, e.g., 6, 7, or more nucleotides in length, while the regions of mismatch may be, for example, 1, 2, 3, or 4 nucleotides in length.
  • Hairpin structures designed to mimic siRNAs and mRNA precursors are processed intracellularly into molecules capable of reducing or inhibiting expression of target transcripts (McManus et al, RNA 8: 842-850, 2002). These hairpin structures, which are based on classical siRNAs consisting of two RNA strands forming a 19 bp duplex structure are classified as class I or class II hairpins. Class I hairpins incorporate a loop at the 5' or 3' end of the antisense siRNA strand (i.e., the strand complementary to the target transcript whose inhibition is desired) but are otherwise identical to classical siRNAs.
  • Class II hairpins resemble mRNA precursors in that they include a 19 nt duplex region and a loop at either the 3' or 5' end of the antisense strand of the duplex in addition to one or more nucleotide mismatches in the stem. These molecules are processed intracellularly into small RNA duplex structures capable of mediating silencing. They appear to exert their effects through degradation of the target mRNA rather than through translational repression as is thought to be the case for naturally occurring mRNAs and stRNAs.
  • RNA interference is useful in an or in a method for in vitro construction of the infectious particle.
  • siRNAs are useful both for therapeutic purposes, e.g., to modulate the expression of a protein in a subject at risk of or suffering from a neoplastic disease, infectious disease, or a disease caused by overexpression of a gene product or expression of a gene product in an inappropriate context or cell type.
  • the compositions and methods are useful in various cellular assays for the identification of compounds for treatment of a neoplastic . disease, infectious disease or genetic disease, that modulate the activity or level of the molecules described herein.
  • the therapeutic treatment, diagnostic assay, or cellular assay of a disease state with an infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA is provided.
  • An embodiment of the invention therefore provides a method for in vitro construction of an infectious particle comprising, contacting a semi-purified or pure papovavirus capsid protein with an exogenous nucleic acid encoding a short interfering RNA or a DNA encoding a short hairpin RNA, allowing the semi-purified or pure papovavirus capsid protein to self assemble with the exogenous nucleic acid into the infectious particle, so as to effect in vitro construction of the infectious particle.
  • the protein is encoded by a gene within or linked to a neoplastic disease susceptibility locus, or within which a functional mutation causing or contributing to susceptibility or development of a neoplastic disease, infectious disease, or genetic disease may exist.
  • the disease related proteins are inhibited.
  • the biological system comprises a cell, and the contacting step comprises expressing the siRNA in the cell.
  • the biological system comprises a subject, e.g., a mammalian subject such as a mouse or human, and the contacting step comprises administering the siRNA to the subject or comprises expressing the siRNA in the subject.
  • the siRNA is expressed inducibly and/or in a cell-type or tissue specific manner.
  • biological system refers to any vessel, well, or container in which biomolecules (e.g., nucleic acids, polypeptides, polysaccharides, lipids, and the like) are placed; a cell or population of cells; a tissue; an organ; an organism, and the like.
  • biomolecules e.g., nucleic acids, polypeptides, polysaccharides, lipids, and the like
  • the biological system is a cell or population of cells, but the method can also be performed in a vessel using purified or recombinant proteins.
  • An embodiment of the invention provides siRNA molecules targeted to a transcript encoding any target protein.
  • siRNA molecules are provided selectively or specifically targeted to a transcript encoding a polymorphic variant of such a transcript, wherein existence of the polymorphic variant in a subject is indicative of susceptibility to or presence of a neoplastic disease, infectious disease, or genetic disease.
  • Targeting more than one site of the target gene may be important to overcome the functional redundancy of other variants of a single gene, especially in in vivo experiments.
  • siRNA causes greater reduction in expression of the variant than of other variants (i.e., variants whose existence in a subject is not indicative of susceptibility to or presence of a neoplastic disease, infectious disease, or genetic disease).
  • the siRNA, or collections of siRNAs may be provided in the form of kits with additional components as appropriate.
  • RNA interference a mechanism of post-transcriptional gene silencing mediated by double-stranded RNA (dsRNA), is useful in a method for treating a disease state in a mammal by administering to the mammal a nucleic acid molecule (e.g., dsRNA) that hybridizes under stringent conditions to a target gene of the disease-related protein, e.g., neoplastic disease, infectious disease, or genetic disease, and attenuates expression of the target gene.
  • a nucleic acid molecule e.g., dsRNA
  • a target gene of the disease-related protein e.g., neoplastic disease, infectious disease, or genetic disease.
  • a plasmid containing a DNA sequence encoding for a particular desired siRNA sequence is delivered into a target cell via an infectious particle comprising a papovavirus capsid protein and an exogenous DNA encoding an shRNA.
  • the DNA sequence is continuously transcribed into RNA molecules that loop back on themselves and form hairpin structures through intramolecular base pairing.
  • These hairpin structures once processed by the cell, are equivalent to transfected siRNA molecules and are used by the cell to mediate RNAi of the desired protein.
  • shRNA has an advantage over siRNA transfection as the former can lead to stable, long-term inhibition of protein expression. Inhibition of protein expression by transfected siRNAs is a transient phenomenon that does not occur for times periods longer than several days. In some cases, this may be preferable and desired. In cases where longer periods of protein inhibition are necessary, shRNA mediated inhibition is preferable.
  • Antisense RNA transcripts have a base sequence complementary to part or all of any other RNA transcript in the same cell. Such transcripts have been shown to modulate gene expression through a variety of mechanisms including the modulation of RNA splicing, the modulation of RNA transport and the modulation of the translation of mRNA (Denhardt, Ann N Y Acad. ScL 660: 70, 1992; Nellen, Trends Biochem. ScL 18: 419, 1993; Baker and Monia, Biochim. Biophys. Acta 1489: 3, 1999; Xu et al, Gene Therapy 7: 438, 2000; French and Gerdes, Curr. Opin. Microbiol. 3: 159, 2000; Terryn and Rouze, Trends Plant ScL 5: 1360, 2000)
  • Antisense nucleic acids are generally single-stranded nucleic acids (DNA, RNA, modified DNA, or modified RNA) complementary to a portion of a target nucleic acid (e.g., an mRNA transcript) and therefore able to bind to the target to form a duplex.
  • a target nucleic acid e.g., an mRNA transcript
  • oligonucleotides that range from 15 to 35 nucleotides in length but may range from 10 up to approximately 50 nucleotides in length. Binding typically reduces or inhibits the function of the target nucleic acid.
  • antisense oligonucleotides may block transcription when bound to genomic DNA, inhibit translation when bound to mRNA, and/or lead to degradation of the nucleic acid.
  • Reduction in expression of a polypeptide related to neoplastic disease, infectious disease, or genetic disease may be achieved by the administration of antisense nucleic acids or peptide nucleic acids comprising sequences complementary to those of the mRNA that encodes the polypeptide.
  • Antisense technology and its applications are well known in the art and are described in Phillips, M. I. (ed.) Antisense Technology, Methods EnzymoL, 2000, Volumes 313 and 314, Academic Press, San Diego, and references mentioned therein. See also Crooke, S. (ed.) "AN ⁇ SENSE DRUG TECHNOLOGY: PRINCIPLES, STRATEGIES, AND APPLICATIONS” (1 st Edition) Marcel Dekker; and references cited therein.
  • Antisense oligonucleotides can be synthesized with a base sequence that is complementary to a portion of any RNA transcript in the cell. Antisense oligonucleotides may modulate gene expression through a variety of mechanisms including the modulation of RNA splicing, the modulation of RNA transport and the modulation of the translation of mRNA (Denhardt, 1992).
  • antisense oligonucleotides including stability, toxicity, tissue distribution, and cellular uptake and binding affinity may be altered through chemical modifications including (i) replacement of the phosphodiester backbone (e.g., peptide nucleic acid, phosphorothioate oligonucleotides, and phosphoramidate oligonucleotides), (ii) modification of the sugar base (e.g., 2'-O-propylribose and 2'-methoxyethoxyribose), and (iii) modification of the nucleoside (e.g., C-5 propynyl U, C-5 thiazole U, and phenoxazine C) W
  • the phosphodiester backbone e.g., peptide nucleic acid, phosphorothioate oligonucleotides, and phosphoramidate oligonucleotides
  • modification of the sugar base e.g., 2'-O-propy
  • An embodiment of the invention provides a method of inhibiting expression of a gene encoding a protein of interest comprising the step of (i) providing a biological system in which expression of a gene encoding a protein of interest is to be inhibited; and (ii) contacting the system with an infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA that hybridizes to a transcript encoding the protein of interest.
  • the protein of interest is encoded by a gene within or linked to a disease, e.g., chemotherapy-resistant neoplastic disease susceptibility locus, or within which a functional mutation causing or contributing to a chemotherapy-resistant neoplastic disease or development of a chemotherapy-resistant neoplastic disease may exist.
  • proteins of interest are inhibited.
  • the biological system comprises a cell, and the contacting step comprises expressing the siRNA molecule in the cell.
  • the biological system comprises a subject, e.g., a mammalian subject such as a mouse or human, and the contacting step comprises administering the siRNA molecule to the subject or comprises expressing the siRNA molecule in the subject.
  • the expression may be inducible and/or tissue or cell type-specific.
  • the siRNA molecule may be an oligonucleotide or a longer nucleic acid molecule.
  • RNA and DNA enzymes can be designed to cleave to any RNA molecule, thereby increasing its rate of degradation (Cotten and Birnstiel, EMBO J. 8: 3861-3866, 1989; Usman et al, Nucl Acids MoI. Biol. 10: 243, 1996; Usman et al, Curr. Opin. Struct. Biol. 1: 527, 1996; Sun et al, Pharmacol. Rev., 52: 325, 2000. See also e.g., Cotten and Birnstiel, EMBO J. 8: 3861-3866, 1989).
  • An embodiment of the invention provides a method for in vitro construction of an infectious particle comprising, contacting a semi-purified or pure papovavirus capsid protein with an exogenous nucleic acid encoding a short interfering RNA or a DNA encoding a short hairpin RNA, allowing the semi-purified or pure papovavirus capsid protein to self assemble with the exogenous nucleic acid into the infectious particle, so as to effect in vitro construction of the infectious particle.
  • An embodiment of the invention further provides an infectious particle comprising a papovav ⁇ rus capsid protein and an exogenous short interfering RNA.
  • the disease related protein is encoded by a gene within or linked to a neoplastic disease, infectious disease, or genetic disease susceptibility locus, or within which a functional mutation causing or contributing to susceptibility or development of neoplastic disease, infectious disease, or genetic disease may exist.
  • disease-related proteins are inhibited.
  • the biological system comprises a cell, and the contacting step comprises expressing the ribozyme in the cell.
  • the biological system comprises a subject, e.g., a mammalian subject such as a mouse or human, and the contacting step comprises administering the ribozyme to the subject or comprises expressing the ribozyme in the subject.
  • the expression may be inducible and/or tissue or cell-type specific according to certain embodiments of the invention.
  • An embodiment of the invention further provides ribozymes designed to cleave transcripts encoding disease-related proteins of neoplastic disease, infectious disease, or genetic disease, or polymorphic variants thereof, as described above. Targeting more than one site of the target gene may be important to overcome the functional redundancy of other variants of a single gene, especially in in vivo experiments, and in therapeutic applications of pseudoviral particles for treatment of a mammalian subject.
  • the infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA further provides a double stranded short interfering ribonucleic acid (siRNA) molecule that directs cleavage of a target RNA of interest via RNA interference (RNAi), wherein the double stranded siRNA molecule comprises a first and a second strand, each strand of the siRNA molecule is about 18 to about 28 nucleotides in length, the first strand of the siRNA molecule comprises nucleotide sequence having sufficient complementarity to the target RNA for the siRNA molecule to direct cleavage of the target RNA via RNA interference, and the second strand of said siRNA molecule comprises nucleotide sequence that is complementary to the first strand.
  • siRNA ribonucleic acid
  • the infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA further provides a siRNA used to inhibit the expression of a target gene of interest or a gene family, wherein the genes or gene family sequences share sequence homology.
  • a siRNA used to inhibit the expression of a target gene of interest or a gene family, wherein the genes or gene family sequences share sequence homology.
  • homologous sequences can be identified as is known in the art, for example using sequence alignments.
  • siRNA molecules can be designed to target such homologous sequences, for example using perfectly complementary sequences or by incorporating non-canonical base pairs, for example mismatches and/or wobble base pairs, that can provide additional target sequences.
  • non- canonical base pairs can be used to generate siRNA molecules that target more than one gene sequence.
  • non- canonical base pairs such as UU and CC base pairs are used to generate siRNA molecules that are capable of targeting sequences for differing targets that share sequence homology (e.g., other protein tyrosine phosphatase encoding sequences).
  • sequence homology e.g., other protein tyrosine phosphatase encoding sequences.
  • siRNAs one advantage of using siRNAs is that a single siRNA can be designed to include nucleic acid sequence that is complementary to the nucleotide sequence that is conserved between the homologous genes. In this approach, a single siRNA can be used to inhibit expression of more than one gene instead of using more than one siRNA molecule to target the different genes.
  • an infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA
  • the siRNA molecule comprises a sequence complementary to an RNA having variant target gene encoding sequence, for example other mutant target genes of interest but known in the art to be associated with the maintenance and/or development of a disease state.
  • Chemical modifications can be applied to any siRNA construct as a further embodiment of the invention.
  • a siRNA molecule in another embodiment, includes a nucleotide sequence that can interact with nucleotide sequence of a target gene and thereby mediate silencing of target gene expression, for example, wherein the siRNA mediates regulation of gene expression by cellular processes that modulate the chromatin structure or methylation patterns of the gene and prevent transcription of the gene.
  • siRNA molecules are used to down regulate or inhibit the expression of target proteins arising from haplotype polymorphisms that are associated with a disease or condition. Targeting more than one site of the target gene may be important to overcome the functional redundancy of other variants of a single gene, especially in in vivo experiments and for therapeutic treatment of a disease or condition in a mammalian subject.
  • an infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA
  • the siRNA molecule comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence encoding a target protein or a portion thereof.
  • the siRNA molecule further comprises a sense region, wherein said sense region comprises a nucleotide sequence of a target gene or a portion thereof.
  • the method of modulating the expression of a target gene of interest by RNA interference can be used in the following cells or tissues including but not limited to: in a cell or in a reconstituted in vitro system, on a tissue explant; in a subject or organism w
  • the infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA provides siRNA molecules that can be designed to down regulate or inhibit target gene expression through RNAi targeting of a variety of RNA molecules.
  • the siRNA molecules are used to target various RNAs corresponding to a target gene.
  • Non-limiting examples of such RNAs include messenger RNA (mRNA), alternate RNA splice variants of target gene(s), post-transcriptionally modified RNA of target gene(s), pre-mRNA of target gene(s), and/or RNA templates.
  • the infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA can be used to inhibit gene expression through the appropriate exons to specifically inhibit or to distinguish among the functions of gene family members.
  • a protein that contains an alternatively spliced transmembrane domain can be expressed in both membrane bound and secreted forms. Use of the infectious particle to target the exon containing the transmembrane domain can be used to determine the functional consequences of pharmaceutical targeting of membrane bound as opposed to the secreted form of the protein.
  • Non-limiting examples of applications relating to targeting these RNA molecules include therapeutic pharmaceutical applications, pharmaceutical discovery applications, molecular diagnostic and gene function applications, and gene mapping, for example using single nucleotide polymorphism mapping with siRNA molecules.
  • Such applications can be implemented using known gene sequences or from partial sequences available from an expressed sequence tag (EST).
  • EST expressed sequence tag
  • Target site refers to a sequence within a target RNA that is “targeted” for cleavage mediated by a siRNA construct which contains sequences within its antisense region that are complementary to the target sequence.
  • Detectable level of cleavage refers to cleavage of target RNA (and formation of cleaved product RNAs) to an extent sufficient to discern cleavage products above the background of RNAs produced by random degradation of the target RNA. Production of cleavage products from 1-5% of the target RNA is sufficient to detect above the background for most methods of detection.
  • a composition comprising a siRNA molecule of the invention can be chemically-modified in a pharmaceutically acceptable carrier or diluent.
  • a pharmaceutical composition comprising siRNA molecules of the invention can be chemically-modified to target one or more genes in a pharmaceutically acceptable carrier or diluent.
  • a method for diagnosing a disease or condition in a subject comprises administering to the subject a composition of the invention under conditions suitable for the diagnosis of the disease or condition in the subject.
  • a method for treating or preventing a disease or condition in a subject comprises administering to the subject a composition of the invention under conditions suitable for the treatment or prevention of the disease or condition in the subject, alone or in conjunction with one or more other therapeutic compounds.
  • a method for preventing or treating diabetes (e.g., type 1 and type T), obesity, and/or insulin resistance in a subject comprises administering to the subject an infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA, wherein the RNAi targets PTP-IB, under conditions suitable for the prevention or treatment of diabetes (e.g., type 1 and type T), obesity, and/or insulin resistance in the subject.
  • Bio system refers to, material, in a purified or unpurified form, from biological sources, including but not limited to human or animal, wherein the system comprises the components required for RNAi activity.
  • biological system includes, for example, a cell, tissue, subject, or organism, or extract thereof.
  • biological system also includes reconstituted RNAi systems that can be used in an in vitro setting.
  • Phenotypic change refers to any detectable change to a cell that occurs in response to contact or treatment with an infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA.
  • detectable changes include, but are not limited to, changes in shape, size, proliferation, motility, protein expression or RNA expression or other physical or chemical changes as can be assayed by methods known in the art.
  • the detectable change can also include expression of reporter genes/molecules such as Green Florescent Protein (GFP) or various tags that are used to identify an expressed protein or any other cellular component that can be assayed.
  • GFP Green Florescent Protein
  • ligand refers to any compound or molecule, such as a drug, peptide, hormone, or neurotransmitter, that is capable of interacting with another compound, such as a receptor, either directly or indirectly.
  • the receptor that interacts with a ligand can be present on the surface of a cell or can alternately be an intercullular receptor. Interaction of the ligand with the receptor can result in a biochemical reaction, or can simply be a physical interaction or association.
  • excipients include polymers such as cyclodextrins, lipids, cationic lipids, polyamines, phospholipids, nanoparticles, receptors, ligands, and others.
  • the infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA can be used alone or as a component of a kit having at least one of the reagents necessary to carry out the in vitro or in vivo introduction of RNA to test samples and/or subjects.
  • preferred components of the kit include a siRNA molecule and a infectious particle that promotes introduction of the siRNA into cells of interest as described herein ⁇ e.g., using lipids and other methods of transfection known in the art, see for example Beigelman et al, U.S. Pat. No. 6,395,713).
  • the kit can be used for target validation, such as in determining gene function and/or activity, or in drug optimization, and in drug discovery (see for example Usman et al., U.S. Ser. No. 60/402,996).
  • target validation such as in determining gene function and/or activity, or in drug optimization, and in drug discovery (see for example Usman et al., U.S. Ser. No. 60/402,996).
  • Such a kit can also include instructions to allow a user of the kit to practice the invention.
  • Short interfering ribonucleic acid refers to any nucleic acid molecule capable of inhibiting or down regulating gene expression or viral replication, for example by mediating RNA interference "RNAi” or gene silencing in a sequence-specific manner; see for example Zamore et al., Cell 101: 25-33, 2000; Bass, Nature 411: 428-429, 2001; Elbashir et al, Nature 411: 494-498, 2001; and Kreutzer et al., International PCT Publication No.
  • WO 00/44914 Allshire, Science 297: 1818- 1819, 2002; Volpe et al, Science 297: 1833-1837, 2002; Jenuwein, Science 297: 2215-2218, 2002; and Hall et al., Science 297: 2232-2237, 2002; Hutvagner and Zamore, Science 297: 2056- 60, 2002; McManus et al., RNA 8: 842-850, 2002; Reinhart et al., Gene & Dev. 16: 1616-1626, 2002; and Reinhart and Bartel, Science 297: 1831, 2002).
  • Non limiting examples of siRNA molecules are shown in "Therapeutic Targets of RNA Interference" herein.
  • the siRNA can be a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
  • the siRNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (Le.
  • each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure, for example wherein the double stranded region is about 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs; the antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof (e.g., about 15 to about 25 or more nucleotides of the siRNA molecule are complementary to the target nucleic acid or a portion thereof).
  • the siRNA is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siRNA are linked by means of a nucleic acid based or non-nucleic acid-based linker(s).
  • the siRNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
  • the siRNA can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNAi.
  • the siRNA can also comprise a single stranded polynucleotide having nucleotide sequence complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof (for example, where such siRNA molecule does not require the presence within the siRNA molecule of nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), wherein the single stranded polynucleotide can further comprise a terminal phosphate group, such as a 5'-phosphate (see for example Martinez et al., Cell 110: 563-574, 2002 and Schwarz et al., Molecular Cell 10: 537-568, 2002), or 5',3'- diphosphate.
  • a terminal phosphate group such as a 5'-phosphate (see for example Martinez et al., Cell 110: 563-574, 2002 and Schwarz et al., Molecular Cell 10: 537-568, 2002), or 5',3'- diphosphate.
  • the siRNA molecule comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non- covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic interactions, and/or stacking interactions.
  • the siRNA molecules comprise nucleotide sequence that is complementary to nucleotide sequence of a target gene.
  • the siRNA molecule interacts with nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene.
  • siRNA molecules need not be limited to those molecules containing only RNA, but further encompasses chemically-modified nucleotides and non-nucleotides.
  • the short interfering ribonucleic acid molecules lack 2'-hydroxy (2'-OH) containing nucleotides.
  • Applicant describes in certain embodiments short interfering ribonucleic acid s that do not require the presence of nucleotides having a 2'-hydroxy group for mediating RNAi and as such, short interfering ribonucleic acid molecules optionally do not include any ribonucleotides (e.g., nucleotides having a 2'-OH group).
  • siRNA molecules that do not require the presence of ribonucleotides within the siRNA molecule to support RNAi can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2'-OH groups.
  • siRNA molecules can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions.
  • the modified short interfering ribonucleic acid molecules can also be referred to as short interfering modified oligonucleotides "siMON.”
  • siRNA refers to to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (mRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering ribonucleic acid , short interfering modified oligonucleotide, chemically-modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others.
  • siRNA short interfering RNA
  • dsRNA double-stranded RNA
  • mRNA micro-RNA
  • shRNA short hairpin RNA
  • ptgsRNA post-transcriptional gene silencing RNA
  • RNAi refers to to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics.
  • siRNA molecules can be used to epigenetically silence genes at both the post-transcriptional level or the pre-transcriptional level.
  • epigenetic regulation of gene expression by siRNA molecules can result from siRNA mediated modification of chromatin structure or methylation pattern to alter gene expression (see, for example, Verdel et al, Science 303: 672- 676, 2004; Pal-Bhadra et al, Science 303: 669-672, 2004; Allshire, Science 297: 1818-1819, 2002; Volpe et al, Science 297: 833-1837, 2002; Jenuwein, Science 297: 2215-2218, 2002; and Hall et al, Science 297: 2232-2237, 2002).
  • Asymmetric hairpin refers to a linear siRNA molecule comprising an antisense region, a loop portion that can comprise nucleotides or non-nucleotides, and a sense region that comprises fewer nucleotides than the antisense region to the extent that the sense region " has enough " complementary nucleotides to base pair with the antisense region and form a duplex with loop.
  • an asymmetric hairpin siRNA molecule can comprise an antisense region having length sufficient to mediate RNAi in a cell or in vitro system (e.g.
  • asymmetric hairpin siRNA molecule can also comprise a 5'-terminal phosphate group that can be chemically modified.
  • the loop portion of the asymmetric hairpin siRNA molecule can comprise nucleotides, non-nucleotides, linker molecules, or conjugate molecules as described herein.
  • Asymmetric duplex refers to a siRNA molecule having two separate strands comprising a sense region and an antisense region, wherein the sense region comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to base pair with the antisense region and form a duplex.
  • an asymmetric duplex siRNA molecule can comprise an antisense region having length sufficient to mediate RNAi in a cell or in vitro system (e.g.
  • nucleotides about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides
  • a sense region having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides that are complementary to the antisense region.
  • Gene refers to a nucleic acid that encodes an RNA, for example, nucleic acid sequences including, but not limited to, structural genes encoding a polypeptide.
  • a “target gene” or a “target nucleic acid” refers to any nucleic acid sequence whose expression or activity is to be modulated.
  • the target nucleic acid can be DNA or RNA.
  • a gene or target gene can also encode a functional RNA (fRNA) or non-coding RNA (ncRNA), such as small temporal RNA (stRNA), micro RNA (mRNA), small nuclear RNA (snRNA), short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomal RNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof.
  • fRNA functional RNA
  • ncRNA non-coding RNA
  • stRNA small temporal RNA
  • mRNA micro RNA
  • snRNA small nuclear RNA
  • siRNA small interfering RNA
  • snRNA small nucleolar RNA
  • rRNA ribosomal RNA
  • tRNA transfer RNA
  • siRNA molecules targeting fRNA and ncRNA can also be used to manipulate or alter the genotype or phenotype of a subject, organism or cell, by intervening in cellular processes such as genetic imprinting, transcription, translation, or nucleic acid processing (e.g., transamination, methylation etc.).
  • the target gene can be a gene derived from a cell, an endogenous gene, a transgene, or exogenous genes such as genes of a pathogen, for example a virus, which is present in the cell after infection thereof.
  • the cell containing the target gene can be derived from or contained in any organism, for example a plant, animal, protozoan, virus, bacterium, or fungus.
  • the animal cell can be mammalian, such as humans, cows, sheep, apes, monkeys, swine, dogs, cats, rodents, rats and mice.
  • Non-limiting examples of plants include monocots, dicots, or gymnosperms.
  • Non-limiting examples of animals include vertebrates or invertebrates.
  • Non-limiting examples of fungi include molds or yeasts.
  • Target site refers to a sequence within a target RNA that is “targeted” for cleavage mediated by a siRNA construct which contains sequences within its antisense region that are complementary to the target sequence.
  • target or target gene refers to, a nucleic acid, protein, peptide, or polypeptide having a nucleic acid or protein activity.
  • target or target gene also refers to nucleic acid sequences encoding any protein, peptide, or polypeptide having an enzymatic, ligand, receptor, or other protein activity. The term is also meant to include other protein encoding sequence, such as other protein isoforms, mutant genes, splice variants of genes, and gene polymorphisms.
  • Non-canonical base pair refers to any non-Watson Crick base pair, such as mismatches and/or wobble base pairs, including flipped mismatches, single hydrogen bond mismatches, trans-type mismatches, triple base interactions, and quadruple base interactions.
  • Non-limiting examples of such non-canonical base pairs include, but are not limited to, AC reverse Hoogsteen, AC wobble, AU reverse Hoogsteen, GU wobble, AA N7 amino, CC 2- carbonyl-amino(Hl)-N-3-amino(H2), GA sheared, UC 4-carbonyl-amino, UU imino-carbonyl, AC reverse wobble, AU Hoogsteen, AU reverse Watson Crick, CG reverse Watson Crick, GC N3-amino-amino N3, AA Nl-amino symmetric, AA N7-amino symmetric, GA N7-N1 amino- carbonyl, GA+ carbonyl-amino N7-N1, GG Nl-carbonyl symmetric, GG N3-amino symmetric, CC carbonyl-amino symmetric, CC N3-amino symmetric, UU 2-carbonyl-imino symmetric, UU 4-
  • homologous sequence refers to, a nucleotide sequence that is shared by one or more polynucleotide sequences, such as genes, gene transcripts and/or non-coding polynucleotides.
  • a homologous sequence can be a nucleotide sequence that is shared by two or more genes encoding related but different proteins, such as different members of a gene family, different protein epitopes, different protein isoforms or completely divergent genes, such as a cytokine and its corresponding receptors.
  • a homologous sequence can be a nucleotide sequence that is shared by two or more non-coding polynucleotides, such as noncoding DNA or RNA, regulatory sequences, introns, and sites of transcriptional control or regulation. Homologous sequences can also include conserved sequence regions shared by more than one polynucleotide sequence. Homology does not need to be perfect homology (e.g., 100%), as partially homologous sequences are also contemplated by the instant invention (e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% etc.).
  • Consed sequence region refers to, a nucleotide sequence of one or more regions in a polynucleotide does not vary significantly between generations or from one biological system, subject, or organism to another biological system, subject, or organism.
  • the polynucleotide can include both coding and non-coding DNA and RNA.
  • Sense region refers to a nucleotide sequence of a siRNA molecule having complementarity to an antisense region of the siRNA molecule.
  • the sense region of a siRNA molecule can comprise a nucleic acid sequence having homology with a target nucleic acid sequence.
  • Antisense region refers to a nucleotide sequence of a siRNA molecule having complementarity to a target nucleic acid sequence.
  • the antisense region of a siRNA molecule can optionally comprise a nucleic acid sequence having complementarity to a sense region of the siRNA molecule.
  • Target nucleic acid refers to any nucleic acid sequence whose expression or activity is to be modulated.
  • the target nucleic acid can be DNA or RNA.
  • “Complementarity” refers to that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types.
  • the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi act ⁇ vit ' yr ⁇ )e ' terniination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et ah, CSHSymp. Quant. Biol.
  • a percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides in the first oligonucleotide being based paired to a second nucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100% complementary respectively).
  • Perfectly complementary means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • a siRNA molecule comprises about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides that are complementary to one or more target nucleic acid molecules or a portion thereof.
  • the infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA further provides each sequence of a siRNA molecule independently about 15 to about 30 nucleotides in length, in specific embodiments about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • the siRNA duplexes independently comprise about 15 to about 30 base pairs (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30).
  • one or more strands of the siRNA molecule independently comprises about 15 to about 30 nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) that are complementary to a target nucleic acid molecule.
  • siRNA molecules comprising hairpin or circular structures are about 35 to about 55 (e.g., about 35, 40, 45, 50 or 55) nucleotides in length, or about 38 to about 44 (e.g., about 38, 39, 40, 41, 42, 43, or 44) nucleotides in length and comprising about 15 to about 25 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs.
  • Exemplary siRNA molecules are shown in "Therapeutic Targets of RNA Interference" herein.
  • Cell is used in its usual biological sense, of a component of a multicellular organism.
  • the cell can be present in an organism, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats.
  • the cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell).
  • the cell can be of somatic or germ line origin, totipotent or pluripotent, dividing or non-dividing. Examples of non-dividing cells include, but are not limited to, differentiated cells from a tissue or organ of a mammalian subject including differentiated cells from the hematopoietic system.
  • the cell can also be derived from or can comprise a gamete or embryo, a stem cell, or a fully differentiated cell.
  • the infectious pseudoviral particle is capable of delivering siRNA to the cytoplasmic compartment of the cell with high capacity, for example, to stem cells, including, but not limited to, bone marrow cells, dendritic cells, embryonic stem cells, somatic stem cells, umbilical cord blood stem cells, hematopoeitic stem cells, mesenchymal stem cells, neural stem cells, bone marrow stromal cells, fetal liver stromal cells, embryonic germ cells.
  • mammalian cells containing one or more infectious particles comprising a papovavirus capsid protein and an exogenous short interfering RNA.
  • the one or more siRNA molecules can independently be targeted to the same or different sites.
  • RNA refers to a molecule comprising at least one ribonucleotide residue.
  • ribonucleotide refers to a nucleotide with a hydroxyl group at the 2' position of a .beta.-D- ribofuranose moiety.
  • the terms include double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides.
  • Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siRNA or internally, for example at one or more nucleotides of the RNA.
  • Nucleotides in the RNA molecules can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.
  • nucleic acid molecules e.g., siRNA or derivatives thereof, individually, or in combination or in conjunction with other drags, can be used to for preventing or treating disease in a subject or organism.
  • the infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA further provides the siRNA molecules to be administered to a subject or to be administered to other appropriate cells evident to those skilled in the art, individually or in combination with one or more drags under conditions suitable for the treatment.
  • the infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA can be used in combination with other known treatments to prevent or treat disease in a subject or organism.
  • the described molecules could be used in combination with one or more known compounds, treatments, or procedures to prevent or treat disease in a subject or organism as are known in the art.
  • an expression vector is provided comprising a nucleic acid sequence encoding at least one siRNA molecule , in a manner which allows expression of the siRNA molecule.
  • the vector can contain sequence(s) encoding both strands of a siRNA molecule comprising a duplex. The vector can.
  • Vectors refers to any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.
  • each component can be administered at the same time or sequentially in any order at different points in time. Thus, each component can be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect.
  • Treating" or "treatment” of neoplastic disease, infectious disease, or genetic disease administering to a mammalian subject an infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA includes preventing the onset of symptoms in a subject that may be at increased risk of disease or infection but does not yet experience or exhibit symptoms of disease or infection, inhibiting the symptoms of disease or infection (slowing or arresting its development), providing relief from the symptoms or side- effects of disease or infection (including palliative treatment), and relieving the symptoms of disease or infection (causing regression).
  • treating refers to any indicia of success in the treatment or amelioration or prevention of an neoplastic disease, infectious disease, or genetic disease, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating.
  • the treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of an examination by a physician.
  • treating includes the administration of the infectious particle compounds or agents to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with ocular disease.
  • therapeutic effect refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.
  • Dosage unit refers to physically discrete units suited as unitary dosages for the particular individual to be treated. Each unit can contain a predetermined quantity of active compound(s) calculated to produce the desired therapeutic effect(s) in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms can be dictated by (a) the unique characteristics of the active compound(s) and the particular therapeutic effect(s) to be achieved, and (b) the limitations inherent in the art of compounding such active compound(s).
  • Disease-related genes and proteins are targets of a pharmaceutical composition which an infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA in a mammalian subject in need of treatment for a neoplastic disease, an infectious disease, or a genetic disease. Determination of a DNA or RNA sequence for a gene target of siRNA can be determined by a variety of sequence comparison methods.
  • nucleic acids or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region (e.g., nucleotide sequence encoding an RNA oligonucleotide or dsRNA oligonucleotide, or a nucleotide sequence encoding a protein of interest described herein or amino acid sequence of a gene or protein target described herein), when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by
  • sequences are then said to be “substantially identical.”
  • This term also refers to, or can be applied to, the compliment of a test sequence.
  • the term also includes sequences that have deletions and/or additions, as well as those that have substitutions.
  • the preferred algorithms can account for gaps and the like.
  • identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence algorithm program parameters Preferably, default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well- known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2: 482, 1981, by the homology alignment algorithm of Needleman and Wunsch, J. MoI. Biol.
  • a preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et ah, Nuc. Acids Res. 25: 3389-3402, 1977 and Altschul et ah, J. MoI. Biol. 215: 403-410, 1990, respectively.
  • BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence.
  • T is referred to as the neighborhood word score threshold (Altschul et ah, supra).
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end " of " either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • polypeptide peptide
  • protein protein
  • amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymer.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ - carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ⁇ carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups ⁇ e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU " all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
  • the following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
  • Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For a general discussion of this organization, see, e.g., Alberts et ah, Molecular Biology of the Cell (3rd ed., 1994) and Cantor and Schimmel, Biophysical Chemistry Part I: The Conformation of Biological Macromolecules (1980).
  • Primary structure refers to the amino acid sequence of a particular peptide.
  • “Secondary structure” refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains, e.g., enzymatic domains, extracellular domains, transmembrane domains, pore domains, and cytoplasmic tail domains.
  • Domains are portions of a polypeptide that form a compact unit of the polypeptide and are typically 15 to 350 amino acids long. Exemplary domains include domains with enzymatic activity, e.g., a. kinase domain. Typical domains are made up of sections of lesser organization such as stretches of ⁇ -sheet and ⁇ -helices. "Tertiary structure” refers to the complete three dimensional structure of a polypeptide monomer. “Quaternary structure” refers to the three dimensional structure formed by the noncovalent association of independent tertiary units. Anisotropic terms are also known as energy terms.
  • a particular nucleic acid sequence also implicitly encompasses "splice variants.”
  • a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant of that nucleic acid.
  • "Splice variants,” as the name suggests, are products of alternative splicing of a gene. After transcription, an initial nucleic acid transcript can be spliced such that different (alternate) nucleic acid splice products encode different polypeptides.
  • Mechanisms for the production of splice variants vary, but include alternate splicing of exons. Alternate polypeptides derived from the same nucleic acid by read- through transcription are also encompassed by this definition. Any products of a splicing reaction, including recombinant forms of the splice products, are included in this definition.
  • recombinant when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
  • stringent hybridization conditions refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-1O 0 C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength pH.
  • T m thermal melting point
  • the T m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T n ,, 50% of the probes are occupied at equilibrium).
  • Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times background, preferably 10 times background hybridization.
  • Exemplary stringent hybridization conditions can be as following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42 0 C, or, 5x SSC, 1% SDS, incubating at 65 0 C, with wash in 0.2x SSC, and 0.1% SDS at 65 0 C.
  • nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
  • Exemplary "moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37 0 C, and a wash in IX SSC at 45 0 C. A positive hybridization is at least twice background.
  • Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in numerous reference, e.g., Ausubel et al, supra.
  • a temperature of about 36°C is typical for low stringency amplification, although annealing temperatures can vary between about 32°C and 48°C depending on primer length.
  • a temperature of about 62°C is typical, although high stringency annealing temperatures can range from about 50 0 C to about 65°C, depending on the primer length and specificity.
  • Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 90 0 C - 95°C for 30 sec - 2 min., an annealing phase lasting 30 sec. - 2 min., and an extension phase of about 72°C for 1 - 2 min. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g., in Innis et al. PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N. Y. (1990).
  • Antibody refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • the antigen-binding region of an antibody will be most critical in specificity and affinity of binding.
  • “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.
  • “Pharmaceutically acceptable salts and esters” means salts and esters that are pharmaceutically acceptable and have the desired pharmacological properties. Such salts include salts that can be formed where acidic protons present in the compounds are capable of reacting with inorganic or organic bases. Suitable inorganic salts include those formed with the alkali metals, e.g. sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as the amine bases, e.g. ethanolamine, diethanolamine, triethanolamine, tromethamine, N methylglucamine, and the like. Such salts also include acid addition salts formed with inorganic acids (e.g.
  • esters include esters formed from carboxy, sulfonyloxy, and phosphonoxy groups present in the compounds, e.g. C 1-6 alkyl esters.
  • a pharmaceutically acceptable salt or ester can be a mono-acid-mono-salt or ester or a di-salt or ester; and similarly where there are more than two acidic groups present, some or all of such groups can be salified or esterified.
  • Compounds named in this invention can be present in unsalified or unesterified form, or in salified and/or esterified form, and the naming of such compounds is intended to include both the original (unsalified and unesterified) compound and its pharmaceutically acceptable salts and esters. Also, certain compounds named in this invention may be present in more than one stereoisomeric form, and the naming of such compounds is intended to include all single stereoisomers and all mixtures (whether racemic or otherwise) of such stereoisomers.
  • compositions, carriers, diluents and reagents are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of undesirable physiological effects to a degree that would prohibit administration of the composition.
  • a “therapeutically effective amount” means the amount that, when administered to a subject for treating a disease, is sufficient to effect treatment for that disease.
  • the terms “subject” or “patient” are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, and other animals. Accordingly, the term “subject” or “patient” as used herein means any mammalian patient or subject to which the infectious particle compositions can be administered. In some embodiments of the present invention, the patient will be suffering from a condition that causes lowered resistance to disease, e.g., HIV.
  • a pharmaceutical composition comprising one or more antibodies or ds RNA compounds to proteins of interest according to the methods described herein
  • accepted screening methods are employed to determine the status of an existing disease or condition in a subject or risk factors associated with a targeted or suspected disease or condition.
  • Subject also refers to an organism, which is a donor or recipient of explanted cells or the cells themselves.
  • Subject also refers to an organism to which the infectious particle compositions can be administered.
  • a subject can be a mammal or mammalian cells, including a human or human cells.
  • Concomitant administration of a known cancer therapeutic drug with a pharmaceutical composition means administration of the drug and composition comprising an infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA at such time that both the known drug and the composition will have a therapeutic effect.
  • Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of the antimicrobial drug with respect to the administration of a compound.
  • a person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and compositions of the present invention.
  • either or both the heavy and light chain variable regions are produced by grafting the CDRs from the originating species into the hybrid framework regions.
  • Assembly of hybrid antibodies or hybrid antibody fragments having hybrid variable chain regions with regard to either of the above aspects can be accomplished using conventional methods known to those skilled in the art.
  • DNA sequences encoding the hybrid variable domains described herein i.e., frameworks based on the target species and CDRs from the originating species
  • the nucleic acid encoding CDR regions may also be isolated from the originating species antibodies using suitable restriction enzymes and ligated into the target species framework by ligating with suitable ligation enzymes.
  • suitable restriction enzymes e.g., restriction enzymes for ligating the target species framework
  • suitable ligation enzymes e.g., ligation enzymes for ligation the framework regions of the variable chains of the originating species antibody may be changed by site-directed mutagenesis.
  • hybrids are constructed from choices among multiple candidates corresponding to each framework region, there exist many combinations of sequences which are amenable to construction in accordance with the principles described herein. Accordingly, libraries of hybrids can be assembled having members with different combinations of individual framework regions. Such libraries can be electronic database collections of sequences or physical collections of hybrids.
  • oligonucleotides are designed to have overlapping regions so that they could anneal and be filled in by a polymerase, such as with polymerase chain reaction (PCR). Multiple steps of overlap extension are performed in order to generate the V L and V H gene inserts. Those fragments are designed with regions of overlap with human constant domains so that they could be fused by overlap extension to produce full length light chains and Fd heavy chain fragments. The light and heavy Fd chain regions may be linked together by overlap extension to create a single Fab library insert to be cloned into a display vector.
  • Alternative methods for the assembly of the humanized library genes can also be used .
  • the library may be assembled from overlapping oligonucleotides using a Ligase Chain Reaction (LCR) approach. Chalmers et al., Biotechniques 30-2: 249-252, 2001.
  • LCR Ligase Chain Reaction
  • variable genes can be cloned into a vector that contains, in-frame, the remaining portion of the necessary constant domain.
  • additional fragments that can be cloned include whole light chains, the Fd portion of heavy chains, or fragments that contain both light chain and heavy chain Fd coding sequence.
  • the antibody fragments used for humanization may be single chain antibodies (scFv).
  • Any selection display system may be used in conjunction with a library according to the present disclosure.
  • Selection protocols for isolating desired members of large libraries are known in the art, as typified by phage display techniques.
  • Such systems in which diverse peptide sequences are displayed on the surface of filamentous bacteriophage have proven useful for creating libraries of antibody fragments (and the nucleotide sequences that encode them) for the in vitro selection and amplification of specific antibody fragments that bind a target antigen.
  • Scott et ah Science 249: 386, 1990.
  • the nucleotide sequences encoding the V H and V L regions are linked to gene fragments which encode leader signals that direct them to the periplasmic space of E.
  • phage-based display systems An advantage of phage-based display systems is that, because they are . biological systems, selected library members can be amplified simply by growing the phage containing the selected library member in bacterial cells. Furthermore, since the nucleotide sequence that encode the polypeptide library member is contained on a phage or phagemid vector, sequencing, expression and subsequent genetic manipulation is relatively straightforward.
  • Module includes inhibitors and activators.
  • Inhibitors are agents that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of disease-related genes, e.g., neoplastic disease, infectious disease, or genetic disease.
  • Activators are agents that, e.g., bind to, stimulate, increase, open, activate, facilitate, enhance activation, sensitize or up regulate the activity of disease- related genes, e.g., neoplastic disease, infectious disease, or genetic disease.
  • Modulators include agents that, e.g., alter the interaction of disease-related genes with: proteins that bind activators or inhibitors, receptors, including proteins, peptides, lipids, carbohydrates, polysaccharides, or combinations of the above, e.g., lipoproteins, glycoproteins, and the like.
  • Modulators include genetically modified versions of naturally-occurring disease-related genes, e.g., with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like.
  • Such assays for inhibitors and activators include, e.g., applying putative modulator compounds to a cell expressing disease-related gene and then determining the functional effects on tumor metastasis, as described herein.
  • Samples or assays comprising a protein of interest that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition.
  • Control samples (untreated with inhibitors) can be assigned a relative tumor cell growth activity value of 100%.
  • Inhibition of a disease-related gene is achieved when the neoplastic disease-related cell, infectious-related disease cell, or genetic disease-related cell growth activity value relative to the control is about 80%, optionally 50% or 25-0%.
  • Activation of disease-related gene is achieved when the tumor cell growth activity value relative to the control is 110%, optionally 150%, optionally 200-500%, or 1000-3000% higher.
  • Modulate refers to that the expression of the gene, or level of RNA molecule or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the modulator.
  • modulate can mean “inhibit,” but the use of the word “modulate” is not limited to this definition.
  • “Inhibit”, “down-regulate”, or “reduce” refers to that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, is reduced below that observed in the absence of the nucleic acid molecules (e.g., siRNA) .
  • inhibition, down-regulation or reduction with an siRNA molecule is below that level observed in the presence of an inactive or attenuated molecule.
  • inhibition, down- regulation, or reduction with siRNA molecules is below that level observed in the presence of, for example, an siRNA molecule with scrambled sequence or with mismatches.
  • inhibition, down-regulation, or reduction of gene expression with an infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA is greater in the presence of the infectious particle than in its absence.
  • inhibition, down regulation, or reduction of gene expression is associated with post transcriptional silencing, such as RNAi mediated cleavage of a target nucleic acid molecule (e.g. siRNA) or inhibition of translation.
  • inhibition, down regulation, or reduction of gene expression is associated with pretranscriptional silencing.
  • Inhibitors are used to refer to inhibitory, activating, or modulating molecules, respectively, identified using in vitro and in vivo assays for gene expression, e.g., siRNA, shRNA, or antisense RNA.
  • a composition comprising an infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA or in a method for in vitro construction of an infectious particle, including a nucleic acid compositions, e.g., antisense oligonucleotides or double stranded RNA oligonucleotides, short interfereing RNA (siRNA), or short hairpin RNA (shRNA), useful in the present compositions and methods can be administered to a human patient per se, in the form of a stereoisomer, prodrug, pharmaceutically acceptable salt, hydrate, solvate, acid salt hydrate, N-oxide or isomorphic crystalline form thereof, or in the form of a pharmaceutical composition where the compound is mixed with suitable carriers or excipient(s) in a therapeutically effective amount, for example, to treat neoplastic disease, infectious disease, or genetic disease.
  • a nucleic acid compositions e.g., antisense oligonucleotides or
  • compositions for administering the infectious pseudo viral particle compositions (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 18 th ed., 1990, incorporated herein by reference).
  • the pharmaceutical compositions generally comprise an infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA in a form suitable for administration to a patient.
  • the pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
  • GMP Good Manufacturing Practice
  • the particular label or detectable group used in the assay is not a critical aspect of the invention, so long as it does not significantly interfere with the specific binding of the siRNA used in the assay.
  • the detectable group can be any material having a detectable physical or chemical property.
  • detectable labels have been well-developed in the field of immunoassays and, in general, most any label useful in such methods can be applied to the present invention.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels include, but are not limited to, magnetic beads (e.g.
  • DynabeadsTM DynabeadsTM
  • fluorescent dyes e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like
  • radiolabels e.g., 3 H 5 14 C, 35 S, 125 1, 121 1, 112 In, "mTc
  • other imaging agents such as microbubbles (for ultrasound imaging), 18 F, 11 C, 15 O, (for Positron emission tomography), 99m TC, 111 In (for Single photon emission tomography)
  • enzymes e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA
  • calorimetric labels such as colloidal gold or colored glass or plastic (e.g.
  • the label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.
  • Non-radioactive labels are often attached by indirect means.
  • a ligand molecule e.g., biotin
  • the ligand then binds to an anti- ligand (e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • a signal system such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • a number of ligands and anti-ligands can be used.
  • a ligand has a natural anti- ligand, for example, biotin, thyroxine, and Cortisol, it can be used in conjunction with the labeled, naturally occurring anti-ligands.
  • any haptenic or antigenic compound can be used in combination with an infectious pseudoviral particle composition.
  • the molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore.
  • Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases.
  • Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, and the like
  • Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol.
  • Means of detecting labels are well known to those of skill in the art.
  • means for detection include a scintillation counter or photographic film as in autoradiography.
  • the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like.
  • CCDs charge coupled devices
  • enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product.
  • simple calorimetric labels may be detected simply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.
  • agglutination assays can be used to detect the presence of the target antibodies.
  • antigen-coated particles are agglutinated by samples comprising the target antibodies.
  • none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.
  • the siRNA will be labeled by joining, either covalently or non- covalently, a substance which provides for a detectable signal.
  • compositions comprising one or a combination of infectious particles comprising a papovavirus capsid protein and an exogenous short interfering RNA or comprising nucleic acid compositions, e.g., antisense oligonucleotides, double stranded RNA oligonucleotides (RNAi), short interfering RNA (siRNA), or DNA oligonucleotides (vectors) containing nucleotide sequences encoding for the transcription of shRNA molecules, formulated together with a pharmaceutically acceptable carrier.
  • Some compositions include a combination of multiple (e.g., two or more) infectious pseudoviral particles. In some compositions, each of the infectious pseudoviral particles of the composition binds to a distinct, pre-selected region of a target nucleic acid.
  • compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of a disease or condition (e.g., neoplastic disease, infectious disease, or genetic disease) in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the outset of the disease, including biochemical, histologic and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease.
  • a disease or condition e.g., neoplastic disease, infectious disease, or genetic disease
  • compositions or medicants are administered to a patient suspected of, or already suffering from such a disease in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease (biochemical, histologic and/or behavioral), including its complications and intermediate pathological phenotypes in development of the disease.
  • An amount adequate to accomplish therapeutic or prophylactic treatment is defined as a therapeutically- or prophylactically-effective dose.
  • agents are usually administered in several dosages until a sufficient immune response has been achieved.
  • the effect of siRNA on levels of a disease related polypeptide is monitored and repeated dosages are given if the levels of a disease related polypeptide starts to increase.
  • Effective doses of an infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA or other nucleic acid compositions e.g., antisense oligonucleotides, double stranded RNA oligonucleotides (RNAi), short interfering RNA (siRNA), or DNA oligonucleotides (vectors) containing nucleotide sequences encoding for the transcription of shRNA molecules
  • RNAi double stranded RNA oligonucleotides
  • siRNA short interfering RNA
  • vectors DNA oligonucleotides containing nucleotide sequences encoding for the transcription of shRNA molecules
  • neoplastic disease, infectious disease, or genetic disease vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic.
  • the patient is a human but nonhuman mammals including
  • the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.
  • dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg.
  • An exemplary treatment regime entails administration once per every two weeks or once a month or once every 3 to 6 months.
  • two or more infectious pseudoviral particles with different binding specificities are administered simultaneously, in which case the dosage of each infectious pseudoviral particle composition administered falls within the ranges indicated.
  • Infectious pseudoviral particle composition is usually administered on multiple occasions.
  • Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of infectious pseudoviral particles in the patient. In some methods, dosage is adjusted to achieve a plasma infectious pseudoviral particle concentration of 1-1000 ⁇ g/ml and in some methods 25-300 ⁇ g/ml.
  • infectious pseudoviral particle composition can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the infectious pseudoviral particle composition in the patient. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patent can be administered a prophylactic regime.
  • Doses for infectious pseudoviral particles or nucleic acids derived therefrom can range from about 10 ng to 1 g, 100 ng to 100 mg, 1 ⁇ g to 10 mg, or 30-300 ⁇ g DNA per patient. Doses for infectious pseudoviral particles vary from 10-100, or more, virions per dose.
  • An infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA, or other nucleic acid compositions, e.g., antisense oligonucleotides, double stranded RNA oligonucleotides (RNAi), short interfering RNA (siRNA),or DNA oligonucleotides (vectors) containing nucleotide sequences encoding for the transcription of shRNA molecules, for the treatment of chemotherapy-resistant neoplastic disease, e.g., glioma, can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal or intramuscular means for prophylactic as inhalants for infectious pseudoviral particle preparations targeting neoplastic disease, infectious disease, or genetic disease, and/or other therapeutic treatment.
  • RNAi double stranded RNA oligonucleotides
  • siRNA short interfer
  • infectious pseudoviral particle agent The most typical route of administration of an infectious pseudoviral particle agent is subcutaneous although other routes can be equally effective.
  • the next most common route is intramuscular injection. This type of injection is most typically performed in the arm or leg muscles.
  • agents are injected directly into a particular tissue where a tumor is found, for example intracranial injection or convection enhanced delivery. Intramuscular injection or intravenous infusion are preferred for administration of infectious pseudoviral particle composition.
  • particular therapeutic infectious pseudoviral particle composition are delivered directly into the cranium.
  • infectious pseudoviral particle composition are administered as a sustained release composition or device, such as a MedipadTM device.
  • Infectious particles can optionally be administered in combination with other agents that are at least partly effective in treating various diseases including various immune-related diseases.
  • both primary and metastatic, infectious particle can also be administered in conjunction with other agents that increase passage of the infectious particle across the blood-brain barrier (BBB).
  • BBB blood-brain barrier
  • An infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA or other nucleic acid compositions, e.g., antisense oligonucleotides, double stranded RNA oligonucleotides (RNAi), short interfering RNA (siRNA), or DNA oligonucleotides (vectors) containing nucleotide sequences encoding for the transcription of shRNA molecules, for the treatment of neoplastic disease, infectious disease, or genetic disease, are often administered as pharmaceutical compositions comprising an active therapeutic agent, i.e., and a variety of other pharmaceutically acceptable components.
  • an active therapeutic agent i.e., and a variety of other pharmaceutically acceptable components.
  • compositions can also include, depending on the formulation desired, pharmaceutically- acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration.
  • diluents are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration.
  • the diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution.
  • compositions may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
  • Pharmaceutical compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized SepharoseTM, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes). Additionally, these carriers can function as immunostimulating agents (i.e., adjuvants).
  • infectious pseudoviral particle compositions can be administered as injectable dosages of a solution or suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier that can be a sterile liquid such as water oils, saline, glycerol, or ethanol.
  • a pharmaceutical carrier that can be a sterile liquid such as water oils, saline, glycerol, or ethanol.
  • auxiliary substances such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be present in compositions.
  • Other components of pharmaceutical compositions are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil.
  • glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.
  • Infectious pseudoviral particle composition can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained release of the active ingredient.
  • An exemplary composition comprises infectious pseudoviral particles at 5 mg/mL, formulated in aqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl, adjusted to pH 6.0 with HCl.
  • compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
  • the preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997.
  • the infectious particles can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.
  • Additional formulations suitable for other modes of administration include oral, intranasal, and pulmonary formulations, suppositories, and transdermal applications.
  • binders and carriers include, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably l%-2%.
  • Oral formulations include excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%- 95% of active ingredient, preferably 25%-70%.
  • Topical application can result in transdermal or intradermal delivery. Topical administration can be facilitated by co-administration of the agent with cholera toxin or detoxified derivatives or subunits thereof or other similar bacterial toxins. Glenn et al., Nature 391: 851, 1998. Co-administration can be achieved by using the components as a mixture or as linked molecules obtained by chemical crosslinking or expression as a fusion protein.
  • transdermal delivery can be achieved using a skin patch or using transferosomes. Paul et al, Eur. J. Immunol. 25: 3521-24, 1995; Cevc et al., Biochem. Biophys. Acta 1368: 201-15, 1998.
  • compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
  • GMP Good Manufacturing Practice
  • a therapeutically effective dose of an infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA or other nucleic acid compositions e.g., antisense oligonucleotides, double stranded RNA oligonucleotides (RNAi), short interfering RNA (siRNA), or DNA oligonucleotides (vectors) containing nucleotide sequences encoding for the transcription of shRNA molecules, described herein will provide therapeutic benefit without causing substantial toxicity.
  • RNAi double stranded RNA oligonucleotides
  • siRNA short interfering RNA
  • vectors DNA oligonucleotides
  • Toxicity of the proteins described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD 50 (the dose lethal to 50% of the population) or the LD 100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human.
  • the dosage of the proteins described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1, KITS
  • kits comprising the compositions (e.g., an infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA or other nucleic acid compositions, e.g., antisense oligonucleotides, double stranded RNA oligonucleotides (RNAi), short interfering RNA (siRNA), or DNA oligonucleotides (vectors) containing nucleotide sequences encoding for the transcription of shRNA molecules) and instructions for use.
  • compositions e.g., an infectious particle comprising a papovavirus capsid protein and an exogenous short interfering RNA or other nucleic acid compositions, e.g., antisense oligonucleotides, double stranded RNA oligonucleotides (RNAi), short interfering RNA (siRNA), or DNA oligonucleotides (vectors) containing nucleotide sequences en
  • the kit can further contain a least one additional reagent, or one or more additional infectious pseudoviral particle compositions (e.g., an infectious particle having a complementary activity which binds to a region of the target gene distinct from the first infectious pseudoviral particle).
  • Kits typically include a label indicating the intended use of the contents of the kit.
  • the term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.
  • Nuclei were extracted using 20 mM Hepes pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, ImM DTT and a protease inhibitor cocktail tablet (Roche). DTT, PMSF and the protease inhibitor cocktail were added to the buffer immediately before use.
  • Nuclear extract concentration was measured using the BCA Protein Assay protocol (Pierce, Rockford, Illinois). The nuclear extracts were stored for more than a year at -20°C.
  • RNAi effectors RNAi effectors, plasmids and preparation of in vitro packaging vectors.
  • Commercial or previously characterized RNAi effectors were utilized in this study.
  • a short hairpin RNA corresponding to GFP expressed in pSilencer 3.1 -H (Ambion, Austin, TX) - shGFP (under the promoter of pol II) was packaged into in vitro pseudovirions (IVPs) as previously described to generate F/P-shGFP.
  • IVPs in vitro pseudovirions
  • packaging was performed using 100 ⁇ g of DNA, 100 ⁇ g of nuclear extract from Sf9 cells, in the presence of 5 mM ATP and 8 mM MgCl 2 at 37°C for 6 hours, and 1 mM CaCl 2 on ice for 1 hour (defined as one reaction). Mock reactions were empty in vitro packaging, or cells only.
  • pEGFP-Cl (Clontech, Palo Alto, CA) was utilized, also encapsidated into IVPs (IVP-GFP).
  • siRNA 5'- A AGCTG ACCCTG A AGTTC ATC-3 1 ; SEQ ID NO: 1, and 5'-AAGCAGCACGACTTCTTCAAG-S'; SEQ ID NO: 2).
  • the siRNA was labeled at the 3' end which has previously been shown to have no effect on its silencing efficiency (IVP- siRNA.3'.Fl).
  • si-GFP-1 sense 5'-AAGCTGACCCTGAAGTTCATCT-S'; SEQ ID NO: 4, (induces over 80% decrease in eGFP expression in HeIa cells expressing an eGFP transgene with a 2-hour half -life when assayed 48 hours after transfection with a cationic lipid), and siGFP-2, sense 5'- GCAAGCTGACCCTGAAGTTCAT-3'; SEQ ID NO: 5, which has been previously described and induces a 70% decrease in eGFP expression in HeIa cells expressing an eGFP transgene with a 2-hour half -life when assayed 48 hours after transfection with a cationic lipid.
  • siRNA were used for the in vitro packaging (IVP-siGFP-1; IVP-siGFP-2).
  • transduced cells were washed twice with PBS supplemented with 0.1% BSA, and fixed for 0.5 hour with 4% paraformaldehyde (PFA) (Sigma-Aldrich) and with additional fixation for 0.5 hour with 70% ethanol at room temperature. Then the cells were washed with PBS/ 0.1% BSA as before, dropped onto lysine-coated microscope slides (Erie Scientific Co., Portsmouth, NH), and allowed to dry. Fluorescent mounting medium (DAKO Corp., Carpinteria, CA) was used to affix a glass coverslip to the microscope slide, and the slides were stored in the absence of light at 4°C.
  • PFA paraformaldehyde
  • RNAi in human lymphoblastoid .45 cells using the delivery of a plasmid expressed short hairpin RNA (shRNA)
  • SV40-m vitro pseudovirions can efficiently transfer plasmid DNA into a wide variety of cell lines in vitro and in vivo. It was previously reported that human lymphoblastoid .45 cells are efficiently transduced with in vz ' tr ⁇ -packaged plasmid DNA carrying the fluorescent marker gene GFP (IVP-GFP). To determine if these cells support RNAi,.45 cells were transduced with IVP-GFP and then subsequently treated these cells with WPs encapsulating plasmid DNA expressing an shRNA corresponding to GFP (IVP-shGFP).
  • IVP-GFP fluorescent marker gene
  • FIG. 1 shows the induction of RNAi against GFP in the human lymphoblastoid .45 cell line, using pseudovirion delivery of plasmids expressing short hairpin RNAs.
  • Human lymphoblastoid .45 cells were transduced with a half-reaction (IA: 1:1 ratio of IVP-GFP to rVP-shGFP) or a 1:7 reaction (IB: 1:7 ratio of IVP-GFP to IVP-shGFP) of GFP plasmid DNA packaged in vitro only (green).
  • IA 1:1 ratio of IVP-GFP to rVP-shGFP
  • IB 1:7 ratio of IVP-GFP to IVP-shGFP
  • GFP plasmid DNA packaged in vitro only green
  • the same cells were transduced with GFP plasmid DNA packaged in vitro and then immediately with a half reaction (A) or a 1:7 reaction (B) of IVP-shGFP pSilencer 3.1-H1 plasmid DNA packaged in vitro (blue), and were compared with mock, empty virion-transduced cells (red).
  • the blue curve in IA appears shifted to the left
  • SV40 pseudovirions are a partial cellular localization within the cystoplasm that can reduce the expression from plasmid expression vectors that require nuclear localization. Kimchi-Sarfaty and Gottesman, Curr. Pharm. Biotechnol. 5: 451-458, 2004.
  • cytoplasmic localization can be advantageous for synthetic siRNAs as this is a critical functional cellular compartment for RISC-mediated mRNA cleavage.
  • S V40 pseudovirions the delivery of fluorescent- tagged siRNA to cells in suspension was studied. These cells are very difficult to transduce using lipid-based transfection reagents.
  • FIG. 2 shows uptake studies of IVP-encapsulated fluorescent-tagged siRNAs in .45 human lymphoblastoid cells.
  • .45 human lymphoblastoid cells were transduced with fluorescent-tagged siRNA packaged in vitro (IVP-siRNA.3'.Fl) (green) or empty particles (red). Cells were harvested three days post-transduction. The same cells were washed and fixed and were scanned by confocal microscopy.
  • At the upper right is an example of a human lymphoblastoid cell expressing the fluorescent-tagged siRNA (X600).
  • SV40 pseudovirion-delivered siRNAs can mediate RNAi
  • siRNAs To determine if the in w ⁇ r ⁇ -packaged siRNAs can mediate RNAi, established sequences of siGFP (TVP-siGFPs) were packaged, and half of the in vitro-packaged reaction were delivered into 10 5 .45 cells following IVP-GFP transduction using half a reaction. Transduction with IVP-siGFP was performed either immediately or 24 hours after IVP-GFP transduction and revealed similar results. Two different sequences for siRNA were encapsidated and tested (TVP-siGFP-1 and IVP-GFP-2). The use of each of the individual siRNAs reduced GFP expression by 50%.
  • TVP-siGFPs siGFP-1 and IVP-GFP-2
  • Figure 3 shows delivery of GFP siRNAs into human lymphoblastoid cells using pseudovirions.
  • Human lymphoblastoid .45 cells were transduced with a half reaction of GFP plasmid DNA packaged in vitro only (green). The same cells were then immediately transduced with a half reaction of IVPs mix containing two synthetic siRNAs corresponding to GFP (blue), and were compared to mock, non-transduced cells (red). Cells were harvested three days post transduction.
  • RNAi in HeLa-GFP cells transduced with SV40 in vitro -packaged plasmid expressed shRNAs or synthetic siRNAs.
  • RNAi as measured by a reduction in GFP expression was observed with both RNAi effector molecules.
  • IVP-siGFP was prepared with 1, 10, 25, 50 and 100 ⁇ g of siGFP. Only a limited dose response was seen ( Figure 4) and so 1 ⁇ g was used routinely. These results indicate that an even lower concentration than 1 ⁇ g of siGFP packaged and delivered by the SV40 pseudovirions would be sufficient to silence the reporter gene.
  • the same experiment was performed on .45 cells and similar results were obtained—the packaging reaction included 1 ⁇ g of siGFP with 100 ⁇ g of nuclear extracts.
  • Figure 4 shows dose response of siGFP packaged in vitro against HeLa cells stably expressing GFP.
  • HeLa cells stably expressing GFP red
  • the in vitro packaging reaction was prepared with different concentrations of the siGFP: 1 ⁇ g (green), 10 ⁇ g (light blue), 25 ⁇ g (orange), 50 ⁇ g (dark blue), 100 ⁇ g (yellow).
  • Transducing with IVP containing these various concentrations resulted in complete elimination of GFP expression.
  • Cells were analyzed three days post-transduction. The right side of the FACS figure is enlarged to better display the slight differences in efficiency of the various siGFP concentrations.
  • Figure 5 shows expression of siRNA against HeLa cells stably expressing GFP delivered by SV40 pseudovirions and by lipid transduction.
  • HeLa cells stably expressing GFP red
  • the majority of the cells ceased GFP expression, while lipid- transfected siRNAs - siGFP- 1 and siGFP-2 cells showed much higher expression (blue).
  • Cells were analyzed three days post-transduction.
  • Cordelier et al Oligonucleotides 13: 281-294, 2003. They designed a DNA vector that expresses two different anti-CCR5 siRNAs, driven by the adenoviral VAl polymerase III (pol III) promoter.
  • the infectious particles comprising a papovavirus capsid protein and an exogenous short interfering RNA provides the first demonstration that the SV40 vectors are not limited to delivery of DNA; they are also capable of carrying RNA sequences.
  • Papovavirus and polyomavirus pseudoviral particles package siRNA to produce RNA interference
  • Viruses such as the papova and polyoma have the ability to self-assemble using either the major capsid protein VPl, all capsid proteins VPl, VP2 and VP3, or the E capsid proteins.
  • the capsid proteins are recombinant proteins prepared in insect, bacteria or mammalian cells.
  • the assembled capsid proteins maintain their ability to transduce a wide variety of cells through natural receptors that make these pseudovirions infectious particles. No packaging cell line, and no viral DNA or RNA is required for packaging, which makes these pseudovirions safer than other delivery systems. Self-assembly in vitro does not require specific DNA sequences to signal the construction of the capsid envelope.
  • RNA interference is a naturally occurring gene silencing mechanism mediated by small double-stranded RNA molecules. It is sequence-specific and is mediated through the formation of an enzymatically active ribo-nucleoprotein termed the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • the RISC formation and mRNA transcript cleavage occur primarily within the cytoplasm. Because the pathway of these pseudoviruses is through the cytoplasm, there is no need for nuclear targeting sequences. The capacity of these pseudovirions is larger than the capacity of viral DNA; therefore, a large number of siRNA molecules can be packaged and delivered into a single cell.

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L'invention concerne une composition de particules infectieuses comprenant une protéine capside du papovavirus et un ARN interférant court. L'invention concerne également des méthodes de préparation des particules infectieuses et d'utilisation des particules infectieuses.
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WO2017072303A1 (fr) * 2015-10-28 2017-05-04 Life Science Inkubator Gmbh Utilisation de vlp pour la détection des acides nucléiques
CN108841822A (zh) * 2018-05-04 2018-11-20 广州市妇女儿童医疗中心 纳米硒负载VP1基因siRNA及其制备方法与应用

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2036980A1 (fr) * 2007-09-14 2009-03-18 Gruber, Jens Dérégulation de l'expression génétique à l'aide de particules semblables à un virus chargées d'acide nucléique
WO2009036933A2 (fr) * 2007-09-14 2009-03-26 Jens Gruber Régulation à la baisse de l'expression génique à l'aide de particules pseudovirales chargées d'acide nucléique
WO2009036933A3 (fr) * 2007-09-14 2009-08-13 Jens Gruber Régulation à la baisse de l'expression génique à l'aide de particules pseudovirales chargées d'acide nucléique
JP2010538625A (ja) * 2007-09-14 2010-12-16 グルベル，ジェンス 核酸担持ウイルス様粒子を用いた遺伝子発現の下方制御
US8729038B2 (en) 2007-09-14 2014-05-20 Jens Gruber Down regulation of the gene expression by means of nucleic acid-loaded virus-like particles
US20140205632A1 (en) * 2007-09-14 2014-07-24 Gabriele Jansen Down regulation of the gene expression by means of nucleic acid-loaded virus-like particles
EP3199633A1 (fr) * 2007-09-14 2017-08-02 Deutsches Primatenzentrum GmbH Régulation à la baisse de l'expression génique à l'aide de particules pseudo-virales chargées d'acide nucléique
US9951329B2 (en) 2007-09-14 2018-04-24 Gabriele Jansen Down regulation of the gene expression by means of nucleic acid-loaded virus-like particles
WO2009120883A2 (fr) * 2008-03-26 2009-10-01 Life Technologies Corporation Administration cellulaire médiée par une particule de type virus
WO2009120883A3 (fr) * 2008-03-26 2010-03-11 Life Technologies Corporation Administration cellulaire médiée par une particule de type virus
WO2017072303A1 (fr) * 2015-10-28 2017-05-04 Life Science Inkubator Gmbh Utilisation de vlp pour la détection des acides nucléiques
CN108841822A (zh) * 2018-05-04 2018-11-20 广州市妇女儿童医疗中心 纳米硒负载VP1基因siRNA及其制备方法与应用

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