US20090162365A1 - Novel siRNAS and methods of use thereof - Google Patents

Novel siRNAS and methods of use thereof Download PDF

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US20090162365A1
US20090162365A1 US11/978,089 US97808907A US2009162365A1 US 20090162365 A1 US20090162365 A1 US 20090162365A1 US 97808907 A US97808907 A US 97808907A US 2009162365 A1 US2009162365 A1 US 2009162365A1
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set forth
nos
compound
antisense
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Elena Feinstein
Rami Skaliter
Igor Mett
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Quark Pharmaceuticals Inc
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Quark Pharmaceuticals Inc
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Priority to US11/978,089 priority Critical patent/US20090162365A1/en
Priority to US12/008,409 priority patent/US20090156524A1/en
Assigned to QUARK PHARMACEUTICALS, INC. reassignment QUARK PHARMACEUTICALS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SKALITER, RAMI, KALINSKI, HAGAR, METT, IGOR, IDELSON, GREGORY HIRSH, FEINSTEIN, ELENA
Publication of US20090162365A1 publication Critical patent/US20090162365A1/en
Priority to US13/082,244 priority patent/US20110251260A1/en
Priority to US14/592,386 priority patent/US9446062B2/en
Priority to US15/267,651 priority patent/US20170226519A1/en
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Definitions

  • the present invention relates to compounds, pharmaceutical compositions comprising same and methods of use thereof for the inhibition of certain genes, including pro-apoptotic genes.
  • the compounds and compositions are thus useful in the treatment of subjects suffering from diseases or conditions and or symptoms associated with such diseases or conditions in which gene expression has adverse consequences.
  • the invention provides siRNA oligonucleotides, compositions comprising same and methods of use thereof in the treatment of hearing loss including acoustic trauma and presbycusis; acute renal failure (ARF); glaucoma; acute respiratory distress syndrome (ARDS) and other acute lung and respiratory injuries; ischemia-reperfusion (I/R) injury following lung transplantation, organ transplantation including lung, liver, heart, pancreas, and kidney transplantation; nephro- and neurotoxicity; spinal cord injury; pressure sores; age-related macular degeneration (AMD); dry eye syndrome; oral mucositis, and chronic obstructive pulmonary disease (COPD).
  • APD age-related macular degeneration
  • COPD chronic obstructive pulmonary disease
  • RNA interference is a phenomenon involving double-stranded (ds) RNA-dependent gene-specific posttranscriptional silencing.
  • ds double-stranded
  • ds RNA-dependent gene-specific posttranscriptional silencing
  • synthetic duplexes of 21 nucleotide RNAs could mediate gene specific RNAi in mammalian cells, without stimulating the generic antiviral defense mechanisms Elbashir et al. Nature 2001, 411:494-498 and Caplen et al. PNAS 2001, 98:9742-9747).
  • siRNAs small interfering RNAs
  • RNA interference is mediated by small interfering RNAs (siRNAs) (Fire et al, Nature 1998, 391:806) or microRNAs (miRNAs) (Ambros V. Nature 2004, 431:350-355); and Bartel D P. Cell. 2004 116(2):281-97).
  • siRNAs small interfering RNAs
  • miRNAs microRNAs
  • the corresponding process is commonly referred to as specific post-transcriptional gene silencing when observed in plants and as quelling when observed in fungi.
  • siRNA is a double-stranded RNA which down-regulates or silences (i.e. fully or partially inhibits) the expression of an endogenous or exogenous gene/mRNA.
  • RNA interference is based on the ability of certain dsRNA species to enter a specific protein complex, where they are then targeted to complementary cellular RNAs and specifically degrades them.
  • the RNA interference response features an endonuclease complex containing an siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having a sequence complementary to the antisense strand of the siRNA duplex.
  • RISC RNA-induced silencing complex
  • Cleavage of the target RNA may take place in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir, et al., Genes Dev., 2001, 15:188).
  • longer dsRNAs are digested into short (17-29 bp) dsRNA fragments (also referred to as short inhibitory RNAs or “siRNAs”) by type III RNAses (DICER, DROSHA, etc., (see Bernstein et al., Nature, 2001, 409:363-6 and Lee et al., Nature, 2003, 425:415-9).
  • DIER type III RNAses
  • the RISC protein complex recognizes these fragments and complementary mRNA.
  • siRNA can be effective in vivo in both mammals and humans. Specifically, Bitko et al., showed that specific siRNAs directed against the respiratory syncytial virus (RSV) nucleocapsid N gene are effective in treating mice when administered intranasally (Bitko et al., Nat. Med. 2005, 11(1):50-55). For reviews of therapeutic applications of siRNAs see Barik (Mol. Med. 2005, 83: 764-773) and Chakraborty (Current Drug Targets 2007 8(3):469-82). In addition, clinical studies with short siRNAs that target the VEGFR1 receptor in order to treat age-related macular degeneration (AMD) have been conducted in human patients. In studies such siRNA administered by intravitreal (intraocular) injection was found effective and safe in 14 patients tested (Kaiser, Am J Opthalmol. 2006 142(4):660-8).
  • RSV respiratory syncytial virus
  • Pro-apoptotic genes are generally defined as genes that play a role in apoptotic cell death.
  • a non-limiting list of pro-apoptotic genes, useful in the present invention is as follows: tumor protein p53 binding protein 2 (TP53BP2); leucine-rich repeats and death domain containing (LRDD); cytochrome b-245, alpha polypeptide (CYBA, p22phox); activating transcription factor 3 (ATF3); caspase 2, apoptosis-related cysteine peptidase (CASP2); NADPH oxidase 3 (NOX3); harakiri, BCL2 interacting protein (HRK, BID3); complement component 1, q subcomponent binding protein (C1QBP); BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3); mitogen-activated protein kinase 8 (MAPK8, JNK1); mitogen-activated protein kinase 14 (MAPK14, p38);
  • ototoxic effects of various therapeutic drugs on auditory cells and spiral ganglion neurons are often the factor limiting their therapeutic usefulness.
  • Commonly used ototoxic drugs include the widely used chemotherapeutic agent cisplatin and its analogs, aminoglycoside antibiotics, e.g. gentamycin, quinine and its analogs, salicylate and its analogs, and loop-diuretics.
  • antibacterial aminoglycosides such as gentamycin, streptomycin, kanamycin, tobramycin, and the like are known to have serious toxic side effects, particularly ototoxicity and nephrotoxicity, which reduce their value as therapeutic agents (see Goodman and Gilman's The Pharmacological Basis of Therapeutics, 6th ed., A. Goodman Gilman et al., eds; Macmillan Publishing Co., Inc., 1980. NY, pp. 1169-71).
  • ototoxicity is a recognized dose-limiting side-effect of antibiotic administration. Studies have shown that from 4 to 15% of patients receiving one gram per day for greater than one week develop measurable hearing loss, which gradually worsens and can lead to permanent deafness if treatment continues.
  • Ototoxicity is also a serious dose-limiting side-effect for cisplatin, a platinum coordination complex, that has proven effective on a variety of human cancers including testicular, ovarian, bladder, and head and neck cancer.
  • Cisplatin (Platinol®) damages auditory and vestibular systems.
  • Salicylates such as aspirin, are the drugs most commonly used because of their anti-inflammatory, analgesic, anti-pyretic and anti-thrombotic effects. Unfortunately, they too have ototoxic side effects including tinnitus (“ringing in the ears”) and temporary hearing loss. Moreover, if the drug is used at high doses for a prolonged time, hearing impairment can become persistent and irreversible.
  • patent application Ser. No. 11/655,610 assigned to the applicant of the present invention relates to methods for treating hearing impairment in a subject comprising administering to the subject a composition comprising an effective amount of a p53 polynucleotide inhibitor, and optionally an inhibitor of a pro-apoptotic gene.
  • presbycusis Another type of hearing loss is presbycusis, which is hearing loss that gradually occurs in most individuals as they age. About 30-35 percent of adults between the ages of 65 and 75 years and 40-50 percent of people 75 and older experience hearing loss. Accordingly, there exists a need for means to prevent, reduce or treat the incidence and/or severity of inner ear disorders and hearing impairments involving inner ear tissue, particularly inner ear hair cells.
  • Acoustic trauma is a type of hearing loss that is caused by prolonged exposure to loud noises. Without wishing to be bound to theory, exposure to loud noise causes the hair cells on the cochlea to become less sensitive. With more severe exposure, injury can proceed from a loss of adjacent supporting cells to complete disruption of the organ of Corti. Death of the sensory cell can lead to progressive Wallerian degeneration and loss of primary auditory nerve fibers.
  • compositions and methods are needed that provide a safe, effective, and prolonged means for prophylactic or curative treatment of hearing impairments related to inner ear tissue damage, loss, or degeneration, particularly ototoxin-induced and particularly involving inner ear hair cells.
  • auditory hair cells are produced only during embryonic development and do not regenerate if lost during postnatal life, therefore, a loss of hair cells will result in profound and irreversible deafness.
  • therapies to treat the cochlea and reverse this condition.
  • an effective therapy to prevent cell death of auditory hair cells would be of great therapeutic value.
  • Acute renal failure is a clinical syndrome characterized by rapid deterioration of renal function that occurs within days.
  • the principal feature of ARF is an abrupt decline in glomerular filtration rate (GFR), resulting in the retention of nitrogenous wastes (urea, creatinine).
  • GFR glomerular filtration rate
  • severe ARF occurs in about 170-200 persons per million of population annually.
  • drugs have been found to ameliorate toxic and ischemic experimental ARF, as manifested by lower serum creatinine levels, reduced histological damage and faster recovery of renal function in animal models. These include anti-oxidants, calcium channel blockers, diuretics, vasoactive substances, growth factors, anti-inflammatory agents and more. However, when these drugs were tested in clinical trials no benefit was shown and their use for treating ARF has not been approved.
  • ARF acute tubular necrosis
  • Renal hypoperfusion is caused by hypovolemic, cardiogenic and septic shock, by administration of vasoconstrictive drugs or renovascular injury.
  • Nephrotoxins include exogenous toxins such as contrast media and aminoglycosides as well as endogenous toxin such as myoglobin. Recent studies suggest that apoptosis in renal tissues is prominent in most human cases of ARF. The principal site of apoptotic cell death is the distal nephron.
  • DGF Delayed graft function
  • graft rejection has been categorized into three subsets depending on the onset of graft destruction: Hyperacute rejection is the term applied to very early graft destruction, usually within the first 48 hours. Acute rejection has an onset of several days days to months or even years after transplantation and can involve humoral and/or cellular mechanisms. Chronic rejection relates to chronic alloreactive immune response.
  • Glaucoma is one of the leading causes of blindness in the world. It affects approximately 66.8 million people worldwide. At least 12,000 Americans are blinded by this disease each year (Kahn and Milton, Am J Epidemiol. 1980, 111(6):769-76). Glaucoma is characterized by the degeneration of axons in the optic nerve head, primarily due to elevated intraocular pressure (IOP).
  • IOP intraocular pressure
  • POAG primary open-angle glaucoma
  • POAG primary open-angle glaucoma
  • TM trabecular meshwork
  • IOP elevation and eventual optic nerve damage trabecular meshwork
  • ARDS Acute respiratory distress syndrome
  • RDS respiratory distress syndrome
  • IRDS adult respiratory distress syndrome
  • ARDS is a severe lung disease caused by a variety of direct and indirect insults. It is characterized by inflammation of the lung parenchyma leading to impaired gas exchange with concomitant systemic release of inflammatory mediators which cause inflammation, hypoxemia and frequently result in failure of multiple organs. This condition is life threatening and often lethal, usually requiring mechanical ventilation and admission to an intensive care unit. A less severe form is called acute lung injury (ALI).
  • ALI acute lung injury
  • Acute allograft rejection remains a significant problem in lung transplantation despite advances in immunosuppressive medication. Rejection, and ultimately early morbidity and mortality may result from ischemia-reperfusion (I/R) injury and hypoxic injury.
  • I/R ischemia-reperfusion
  • Spinal cord injury or myelopathy is a disturbance of the spinal cord that results in loss of sensation and/or mobility.
  • the two most common types of spinal cord injury are due to trauma and disease. Traumatic injuries are often due to automobile accidents, falls, gunshots diving accidents, and the like. Diseases which can affect the spinal cord include polio, spina bifida, tumors, and Friedreich's ataxia.
  • Ischemia reperfusion injury is one of the leading causes of death in organ allograft recipients. Significant IRI occurs in every organ transplant from a deceased donor and in some from live donors. It contributes to increased acute rejection and impaired long-term allograft function. Lung transplantation, the only definitive therapy for many patients with end stage lung disease, has poor survival rates in all solid allograft recipients.
  • Pressure sores are areas of damaged skin and tissue.
  • tissue ischemia can develop resulting in the accumulation of metabolic waste in the interstitial tissue, resulting in anoxia and cellular death.
  • This pressure-induced ischemia also leads to excessive tissue hypoxia, further promoting bacterial proliferation and tissue destruction.
  • AMD age-related macular degeneration
  • choroidal neovascularization CNV
  • CNV choroidal neovascularization
  • Diabetic retinopathy is recognized as a retinal vascular disorder exhibiting excess capillary permeability, vascular closure, and proliferation of new vessels. DR occurs in two stages: nonproliferative and proliferative. In the nonproliferative stage the disease is characterized by a loss of retinal capillary pericytes, thickening of the basement membrane and development of microaneurysms, dot-blot hemorrhages, and hard exudates. In the proliferative stage the disease is characterized by extensive neovascularization, vessel intrusion into the vitreous, bleeding and fibrosis with subsequent retinal traction, which leads to severe vision impairment.
  • U.S. Pat. No. 6,740,738 and related patents and applications to the assignee of the present invention are directed to inhibition of RTP801 gene and protein, involved in retinopathy.
  • Oral mucositis also referred to as a stomatitis
  • a stomatitis is a common and debilitating side effect of chemotherapy and radiotherapy regimens, which manifests itself as erythema and painful ulcerative lesions of the mouth and throat. Routine activities such as eating, drinking, swallowing, and talking may be difficult or impossible for subjects with severe oral mucositis.
  • Palliative therapy includes administration of analgesics and topical rinses.
  • Dry eye syndrome is a common problem usually resulting from a decrease in the production of tear film that lubricates the eyes.
  • Most patients with dry eye experience discomfort, and no vision loss; although in severe cases, the cornea may become damaged or infected.
  • Wetting drops artificial tears may be used for treatment while lubricating ointments may help more severe cases.
  • the present invention provides inhibitors of a pro-apoptotic gene selected from the group consisting of TP53BP2, LRDD, CYBA, ATF3, CASP2, NOX3, HRK, CIQBP, BNIP3, MAPK8, MAPK14, RAC1, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, CX43, TYROBP, CTGF, SPP1, RTN4R, ANXA2, RHOA, and DUOX1 (See Table A, infra, for genes' details).
  • the inhibitor is selected from the group consisting of siRNA, shRNA, an aptamer, an antisense molecule, miRNA, a ribozyme, and an antibody.
  • the inhibitor is siRNA.
  • the present invention provides novel double stranded oligoribonucleotides that inhibit expression of a pro-apoptotic gene selected from the group consisting of TP53BP2, LRDD, CYBA, ATF3, CASP2, NOX3, HRK, CIQBP, BNIP3, MAPK8, MAPK14, RAC1, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, CX43, TYROBP, CTGF, SPP1, RHOA, and DUOX1.
  • the gene is one of TP53BP2, CASP2, NOX3, RAC1, RHOA, or DUOX1.
  • the gene is one of LRDD, CYBA, HRK, BNIP3, CD38, BMP2, or SPP1.
  • the invention also provides pharmaceutical compositions comprising one or more such oligoribonucleotides or a vector capable of expressing the oligoribonucleotide.
  • the present invention further relates to methods for treating or preventing the incidence or severity of various diseases or conditions in a subject in need thereof wherein the disease or condition and/or symptoms associated therewith is selected from the group consisting of hearing loss, acute renal failure (ARF), glaucoma, acute respiratory distress syndrome (ARDS) and other acute lung and respiratory injuries, ischemia-reperfusion injury following lung transplantation, organ transplantation including lung, liver, heart, pancreas, and kidney transplantation, nephro- and neurotoxicity, spinal cord injury, pressure sores, age-related macular degeneration (AMD), dry eye syndrome, oral mucositis and chronic obstructive pulmonary disease (COPD).
  • Such methods involve administering to a mammal in need of such treatment a prophylactically or therapeutically effective amount of one or more such compounds which inhibit or reduce expression or activity of at least one such gene.
  • the present invention provides a compound having the structure:
  • the covalent bond joining each consecutive N or N′ is a phosphodiester bond. In various embodiments all the covalent bonds are phosphodiester bonds.
  • the compound is blunt ended, for example wherein both Z and Z′ are absent.
  • the compound comprises at least one 3′ overhang, wherein at least one of Z or Z′ is present.
  • Z and Z′ can independently comprise one or more covalently linked modified or non-modified nucleotides, for example inverted dT or dA; dT, LNA, mirror nucleotide and the like.
  • each of Z and Z′ are independently selected from dT and dTdT.
  • N or N′ comprises a modification in the sugar residue of one or more ribonucleotides.
  • the compound comprises at least one ribonucleotide modified in the sugar residue.
  • the compound comprises a modification at the 2′ position of the sugar residue.
  • the modification in the 2′ position comprises the presence of an amino, a fluoro, an alkoxy or an alkyl moiety.
  • the 2′ modification comprises methoxy moiety.
  • a presently preferred modification is a 2′ methoxy of the sugar residue (2′-O-methyl; 2′-O-Me; 2′-O—CH 3 ).
  • the compound comprises modified alternating ribonucleotides in one or both of the antisense and the sense strands. In certain embodiments the compound comprises modified alternating ribonucleotides in the antisense and the sense strands. In other embodiments the compound comprises modified alternating ribonucleotides in the antisense strand only. In certain embodiments the middle ribonucleotide of the antisense strand is not modified; e.g. ribonucleotide in position 10 in a 19-mer strand or position 12 in a 23-mer strand.
  • the compound comprises modified ribonucleotides in alternating positions wherein each N at the 5′ and 3′ termini of (N) x are modified in their sugar residues, and each N′ at the 5′ and 3′ termini of (N′) y are unmodified in their sugar residues.
  • neither (N) x nor (N′) y are phosphorylated at the 3′ and 5′ termini.
  • either or both (N) x and (N′) y are phosphorylated at the 3′ termini.
  • the compound comprises an antisense sequence present in Tables B1-B76 (SEQ ID NOS:277 to 50970 and 50993-68654).
  • the present invention provides a mammalian expression vector comprising an antisense sequence present in Tables B1-B76 (SEQ ID NOS:277 to 50970 and 50993-68654).
  • N and N′ are selected from the oligomers set forth in any one of Tables C1, C2 or C3 (SEQ ID NOS: 97-276 and SEQ ID NOS: 50971-50992).
  • each of (N) x and (N′) y is set forth in any one of SEQ ID NOS: 97-276 (Tables C1, C2) and SEQ ID NOS: 50971-50992 (Table C3).
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising one or more compounds of the present invention, in an amount effective to inhibit human gene expression wherein the gene is selected from the group consisting of TP53BP2, LRDD, CYBA, ATF3, CASP2, NOX3, HRK, CIQBP, BNIP3, MAPK8, MAPK14, RAC1, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, CX43, TYROBP, CTGF, SPP1, RTN4R, ANXA2 RHOA, and DUOX1; and a pharmaceutically acceptable carrier.
  • the gene is selected from the group consisting of TP53BP2, LRDD, CYBA, ATF3, CASP2, NOX3, HRK, CIQBP, BNIP3, MAPK8, MAPK14, RAC1, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, CX43, TYROBP, CTGF,
  • the present invention relates to a method for the treatment of a subject in need of treatment for a disease or disorder or symptoms associated with the disease or disorder, associated with the expression of a gene selected from TP53BP2, LRDD, CYBA, ATF3, CASP2, NOX3, HRK, CIQBP, BNIP3, MAPK8, MAPK14, RAC1, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, CX43, TYROBP, CTGF, SPP1, RTN4R, ANXA2, RHOA, and DUOX1, comprising administering to the subject an amount of an siRNA which reduces or inhibits expression of at least one of those pro-apoptotic genes.
  • a gene selected from TP53BP2, LRDD, CYBA, ATF3, CASP2, NOX3, HRK, CIQBP, BNIP3, MAPK8, MAPK14, RAC1, GSK3B, P2RX7, TRPM2, PARG, CD38,
  • the present invention provides methods and compositions useful in treating a subject suffering from acute renal failure (ARF), hearing loss, glaucoma, acute respiratory distress syndrome (ARDS) and other acute lung and respiratory injuries, injury (e.g. ischemia-reperfusion injury) in organ transplant including lung, kidney, bone marrow, heart, pancreas, cornea or liver transplantation, nephrotoxicity, spinal cord injury, pressure sores, dry eye syndrome, oral mucositis and chronic obstructive pulmonary disease (COPD).
  • APF acute renal failure
  • ARDS acute respiratory distress syndrome
  • COPD chronic obstructive pulmonary disease
  • the methods of the invention comprise administering to the subject one or more inhibitory compounds which reduces, inhibits or down-regulate expression of a gene selected from the group consisting of TP53BP2, LRDD, CYBA, ATF3, CASP2, NOX3, HRK, CIQBP, BNIP3, MAPK8, MAPK14, RAC1, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, CX43, TYROBP, CTGF, SPP1, RTN4R, ANXA2, RHOA, and DUOX1; and in particular siRNA in a therapeutically effective dose so as to thereby treat the patient.
  • a gene selected from the group consisting of TP53BP2, LRDD, CYBA, ATF3, CASP2, NOX3, HRK, CIQBP, BNIP3, MAPK8, MAPK14, RAC1, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, CX43,
  • the present invention provides methods of treating a disease or condition selected from hearing loss, acute renal failure, glaucoma, acute respiratory distress syndrome, an acute lung injury, organ transplantation rejection, ischemia-reperfusion injury, nephrotoxicity, neurotoxicity, spinal cord injury, pressure sores, osteoarthritis, dry eye syndrome and chronic obstructive pulmonary disease (COPD), in a subject in need thereof, comprising administering to the subject an oligonucleotide which inhibits expression of a gene whose mRNA sequence is set forth in any one of SEQ ID NOS:1-41 or 46-48, in an amount effective to treat the disease or condition.
  • a disease or condition selected from hearing loss, acute renal failure, glaucoma, acute respiratory distress syndrome, an acute lung injury, organ transplantation rejection, ischemia-reperfusion injury, nephrotoxicity, neurotoxicity, spinal cord injury, pressure sores, osteoarthritis, dry eye syndrome and chronic obstructive pulmonary disease (COPD), in a subject in need thereof,
  • the present invention provides methods of treating acute renal failure in a subject in need thereof, comprising administering to the subject an oligonucleotide which inhibits expression of any one of TP53BP (whose mRNA sequence is set forth in SEQ ID NOS: 1-2); LRDD (whose mRNA sequence is set forth in SEQ ID NO:3-5); CYBA (whose mRNA sequence is set forth in SEQ ID NO:6), CASP2 (whose mRNA sequence is set forth in SEQ ID NO:10-11), BNIP3 (whose mRNA sequence is set forth in SEQ ID NO:15), or RAC1 (whose mRNA sequence is set forth in SEQ ID NO:24-26) in an amount effective to treat the acute renal failure.
  • TP53BP whose mRNA sequence is set forth in SEQ ID NOS: 1-2
  • LRDD whose mRNA sequence is set forth in SEQ ID NO:3-5
  • CYBA whose mRNA sequence is set forth in SEQ ID NO:6
  • CASP2 whose
  • the present invention provides methods of treating spinal-cord injury in a subject in need thereof, comprising administering to the subject an oligonucleotide which inhibits expression of any one of RHOA (whose mRNA sequence is set forth in SEQ ID NO:46); TP53BP (whose mRNA sequence is set forth in SEQ ID NOS:1-2); LRDD (whose mRNA sequence is set forth in SEQ ID NO:3-5); CYBA (whose mRNA sequence is set forth in SEQ ID NO:6), CASP2 (whose mRNA sequence is set forth in SEQ ID NO: 10-11), BNIP3 (whose mRNA sequence is set forth in SEQ ID NO:15), RAC1 (whose mRNA sequence is set forth in SEQ ID NO:24-26), CD38 (whose mRNA sequence is set forth in SEQ ID NO:32) or BMP2 (whose mRNA sequence is set forth in SEQ ID NO:34) in an amount effective to treat the spinal cord injury.
  • RHOA whose mRNA sequence is set forth
  • the present invention provides methods of treating hearing loss in a subject in need thereof, comprising administering to the subject an oligonucleotide which inhibits expression of any one of TP53BP (whose mRNA sequence is set forth in SEQ ID NOS:1-2); LRDD (whose mRNA sequence is set forth in SEQ ID NO:3-5); CYBA (whose mRNA sequence is set forth in SEQ ID NO:6), CASP2 (whose mRNA sequence is set forth in SEQ ID NO:10-11), NOX3 (whose mRNA sequence is set forth in SEQ ID NO:12), BNIP3 (whose mRNA sequence is set forth in SEQ ID NO:15), RAC1 (whose mRNA sequence is set forth in SEQ ID NO:24-26), CD38 (whose mRNA sequence is set forth in SEQ ID NO:32) or BMP2 (whose mRNA sequence is set forth in SEQ ID NO:34) in an amount effective to treat the hearing loss.
  • TP53BP whose mRNA sequence is set forth in S
  • the present invention provides methods of treating a disease or condition selected from chronic obstructive pulmonary disease, acute respiratory distress syndrome and acute lung injury in a subject in need thereof, comprising administering to the subject an oligonucleotide which inhibits expression of any one of LRDD (whose mRNA sequence is set forth in SEQ ID NO:3-5); CYBA (whose mRNA sequence is set forth in SEQ ID NO:6), CASP2 (whose mRNA sequence is set forth in SEQ ID NO:10-11), BNIP3 (whose mRNA sequence is set forth in SEQ ID NO: 15), RAC1 (whose mRNA sequence is set forth in SEQ ID NO:24-26), CD38 (whose mRNA sequence is set forth in SEQ ID NO:32), BMP2 (whose mRNA sequence is set forth in SEQ ID NO:34), SPP1 (whose mRNA sequence is set forth in SEQ ID NOS:39-41) or DUOX (whose mRNA sequence is set forth in SEQ ID NOS:47
  • the present invention provides methods of treating a subject who is an organ transplant recipient or organ transplant donor comprising administering to the subject an oligonucleotide which inhibits expression of any one of TP53BP (whose mRNA sequence is set forth in SEQ ID NOS:1-2); LRDD (whose mRNA sequence is set forth in SEQ ID NO:3-5); CYBA (whose mRNA sequence is set forth in SEQ ID NO:6), CASP2 (whose mRNA sequence is set forth in SEQ ID NO: 10-11), BNIP3 (whose mRNA sequence is set forth in SEQ ID NO:15), RAC1 (whose mRNA sequence is set forth in SEQ ID NO:24-26), GSK3B (whose mRNA sequence is set forth in SEQ ID NO:27), P2RX7 (whose mRNA sequence is set forth in SEQ ID NO:28), TRPM2 (whose mRNA sequence is set forth in SEQ ID NO:30) or PARG (whose mRNA sequence is set forth in SEQ ID NO:
  • the present invention provides methods of treating glaucoma in a subject in need thereof, comprising administering to the subject an oligonucleotide which inhibits expression of any one of TP53BP (whose mRNA sequence is set forth in. SEQ ID NOS: 1-2); LRDD (whose mRNA sequence is set forth in SEQ ID NO:3-5); CYBA (whose mRNA sequence is set forth in SEQ ID NO:6), CASP2 (whose mRNA sequence is set forth in SEQ ID NO:10-11), BNIP3 (whose mRNA sequence is set forth in SEQ ID NO:15), RAC1 (whose mRNA sequence is set forth in SEQ ID NO:24-26), SPP1 (whose mRNA sequence is set forth in SEQ ID NOS:39-41), or RHOA (whose mRNA sequence is set forth in SEQ ID NO:46) in an amount effective to treat glaucoma.
  • TP53BP whose mRNA sequence is set forth in. SEQ ID NOS: 1-2
  • the present invention provides methods of treating oral mucositis in a subject in need thereof, comprising administering to the subject an oligonucleotide which inhibits expression of any one of TP53BP (whose mRNA sequence is set forth in SEQ ID NOS:1-2); LRDD (whose mRNA sequence is set forth in SEQ ID NOS:3-5); CASP2 (whose mRNA sequence is set forth in SEQ ID NOS: 10-11) or ATF3 (whose mRNA sequence is set forth in SEQ ID NOS:7-9) in an amount effective to treat oral mucositis.
  • TP53BP whose mRNA sequence is set forth in SEQ ID NOS:1-2
  • LRDD whose mRNA sequence is set forth in SEQ ID NOS:3-5
  • CASP2 whose mRNA sequence is set forth in SEQ ID NOS: 10-11
  • ATF3 whose mRNA sequence is set forth in SEQ ID NOS:7-9
  • the present invention provides methods of treating osteoarthritis in a subject in need thereof, comprising administering to the subject an oligonucleotide which inhibits expression of SPP1 (whose mRNA sequence is set forth in SEQ ID NOS:39-41), in an amount effective to treat osteoarthritis.
  • the present invention provides methods of treating dry eye syndrome in a subject in need thereof, comprising administering to the subject an oligonucleotide which inhibits expression of any one of TP53BP (whose mRNA sequence is set forth in SEQ ID NOS:1-2); LRDD (whose mRNA sequence is set forth in SEQ ID NO:3-5); CYBA (whose mRNA sequence is set forth in SEQ ID NO:6), CASP2 (whose mRNA sequence is set forth in SEQ ID NO:10-11), BNIP3 (whose mRNA sequence is set forth in SEQ ID NO:15), or RAC1 (whose mRNA sequence is set forth in SEQ ID NO:24-26) in an amount effective to treat the syndrome.
  • TP53BP whose mRNA sequence is set forth in SEQ ID NOS:1-2
  • LRDD whose mRNA sequence is set forth in SEQ ID NO:3-5
  • CYBA whose mRNA sequence is set forth in SEQ ID NO:6
  • CASP2 whose mRNA sequence
  • the present invention provides methods of treating a pressure sore in a subject in need thereof, comprising administering to the subject an oligonucleotide which inhibits expression of any one of CIQBP (whose mRNA sequence is set forth in SEQ ID NO: 14), RAC1 (whose mRNA sequence is set forth in SEQ ID NOS:24-26), GSK3B (whose mRNA sequence is set forth in SEQ ID NO:27), P2RX7 (whose mRNA sequence is set forth in SEQ ID NO:28), TRPM2 (whose mRNA sequence is set forth in SEQ ID NO:30), PARG (whose mRNA sequence is set forth in SEQ ID NO:31), CD38 (whose mRNA sequence is set forth in SEQ ID NO:32), STEAP4 (whose mRNA sequence is set forth in SEQ ID NO:33), BMP2 (whose mRNA sequence is set forth in SEQ ID NO:34), GJA1 (whose mRNA sequence is set forth in SEQ ID NO:35), or
  • the present invention provides methods of treating a disease or condition selected from hearing loss, acute renal failure, glaucoma, acute respiratory distress syndrome, an acute lung injury, organ transplantation rejection, ischemia-reperfusion injury, nephrotoxicity, neurotoxicity, spinal cord injury, pressure sores, osteoarthritis and chronic obstructive pulmonary disease (COPD), in a subject in need thereof, comprising administering to the subject an antibody which inhibits a polypeptide whose sequence is set forth in any one of SEQ ID NOS: 90-93 in an amount effective to treat the disease or condition.
  • a disease or condition selected from hearing loss, acute renal failure, glaucoma, acute respiratory distress syndrome, an acute lung injury, organ transplantation rejection, ischemia-reperfusion injury, nephrotoxicity, neurotoxicity, spinal cord injury, pressure sores, osteoarthritis and chronic obstructive pulmonary disease (COPD), in a subject in need thereof, comprising administering to the subject an antibody which inhibits a polypeptide whose sequence is set
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody which inhibits a polypeptide whose sequence is set forth in any one of SEQ ID NOS: 90-93, in an amount effective to inhibit the polypeptide, and a pharmaceutically acceptable carrier.
  • the present invention relates to the use of a therapeutically effective dose of an oligonucleotide for the preparation of a composition for treating a subject suffering from a disease or condition selected from hearing loss, acute renal failure, glaucoma, acute respiratory distress syndrome, an acute lung injury, organ transplantation rejection, ischemia-reperfusion injury, nephrotoxicity, neurotoxicity, spinal cord injury, pressure sores, osteoarthritis and chronic obstructive pulmonary disease (COPD), wherein the oligonucleotide inhibits expression of a gene whose mRNA sequence is set forth in any one of SEQ ID NOS: 1-41 or 46-48.
  • a disease or condition selected from hearing loss, acute renal failure, glaucoma, acute respiratory distress syndrome, an acute lung injury, organ transplantation rejection, ischemia-reperfusion injury, nephrotoxicity, neurotoxicity, spinal cord injury, pressure sores, osteoarthritis and chronic obstructive pulmonary disease (COPD), wherein the oligonucleo
  • the present invention relates to the use of a therapeutically effective dose of an antibody for the preparation of a composition for treating a subject suffering from a disease or condition selected from hearing loss, acute renal failure, glaucoma, acute respiratory distress syndrome, an acute lung injury, organ transplantation rejection, ischemia-reperfusion injury, nephrotoxicity, neurotoxicity, spinal cord injury, pressure sores, osteoarthritis and chronic obstructive pulmonary disease (COPD), wherein the antibody inhibits a polypeptide whose sequence is set forth in any one of SEQ ID NOS: 90-93.
  • a disease or condition selected from hearing loss, acute renal failure, glaucoma, acute respiratory distress syndrome, an acute lung injury, organ transplantation rejection, ischemia-reperfusion injury, nephrotoxicity, neurotoxicity, spinal cord injury, pressure sores, osteoarthritis and chronic obstructive pulmonary disease (COPD), wherein the antibody inhibits a polypeptide whose sequence is set forth in any one of SEQ ID NOS: 90-
  • the present invention relates generally to compounds which down-regulate expression of various genes including pro-apoptotic genes, particularly to novel small interfering RNAs (siRNAs), and to the use of these novel siRNAs in the treatment of various diseases and medical conditions.
  • diseases and conditions to be treated are hearing loss, acute renal failure (ARF), glaucoma, acute respiratory distress syndrome (ARDS) and other acute lung and respiratory injuries, ischemia-reperfusion injury following lung transplantation, organ transplantation including lung, liver, heart, bone marrow, pancreas, cornea and kidney transplantation, spinal cord injury, pressure sores, age-related macular degeneration (AMD), dry eye syndrome, oral mucositis and chronic obstructive pulmonary disease (COPD).
  • ALD age-related macular degeneration
  • COPD chronic obstructive pulmonary disease
  • siRNA to be used in the present invention are provided in Table B, SEQ ID NOS:277 to 50970 and 50993-68654.
  • 21- or 23-mer siRNA sequences can also be generated by 5′ and/or 3′ extension of the 19-mer sequences disclosed herein. Such extension is preferably complementary to the corresponding mRNA sequence.
  • Certain 23-mer oligomers were devised by this method where the order of the prioritization is the order of the corresponding 19-mer.
  • pro-apoptotic gene is generally defined as a gene that plays a positive role in apoptotic cell death.
  • preferred pro-apoptotic genes and the preferred uses of siRNA or other inhibitors of these pro-apoptotic genes are listed in Table A below. It should be noted that whereas the compounds of the present invention are useful in treating the listed indications, certain compounds may be more effective in a particular tissue than in another. Those preferred indications are listed in Table A, hereinbelow.
  • Preferred diseases/ No. Gene Full name and Human Gene ID conditions 1 TP53BP2 tumor protein p53 binding protein, 2 ARF, nephrotoxicity, gi
  • Table A comprises the polynucleotide SEQ ID NOS of the mRNA of the genes targeted by the compounds of the present invention (set forth as SEQ ID NOS: 1-48).
  • the corresponding polypeptides are set forth in SEQ ID NOS:49-96.
  • the genes listed in Table A, supra, are described in more detail as follows:
  • T53BP2 Tumor Protein p53 Binding Protein, 2
  • This gene encodes a member of the ASPP (apoptosis-stimulating protein of p53) family of p53 interacting proteins.
  • the corresponding protein contains four ankyrin repeats and an SH3 domain involved in protein-protein interactions. It is localized to the perinuclear region of the cytoplasm, and regulates apoptosis and cell growth through interactions with other regulatory molecules including members of the p53 family. Multiple transcript variants encoding different isoforms have been found for this gene.
  • the polynucleotide sequences of human TP53BP2 mRNA transcriptional variants 1 and 2 are SEQ ID NOS:1 and 2, respectively, and the corresponding polypeptide sequence are set forth in SEQ ID NOS:49-50, respectively.
  • the protein encoded by this gene contains a leucine-rich repeat and a death domain. This protein has been shown to interact with other death domain proteins, such as Fas (TNFRSF6)-associated via death domain (FADD) and MAP-kinase activating death domain-containing protein (MADD), and thus may function as an adaptor protein in cell death-related signaling processes.
  • the expression of the mouse counterpart of this gene has been found to be positively regulated by the tumor suppressor p53 and to induce cell apoptosis in response to DNA damage, which suggests a role for this gene as an effector of p53-dependent apoptosis.
  • Three alternatively spliced transcript variants encoding distinct isoforms have been reported.
  • the polynucleotide sequence of human LRDD transcriptional variants 2, 1 and 3 are set forth in SEQ ID NOS: 3-5, respectively, and the corresponding polypeptide sequence are set forth in SEQ ID NOS:51-53, respectively.
  • cytochrome b light chain cytochrome b(558) alpha-subunit
  • cytochrome b alpha polypeptide
  • flavocytochrome b-558 alpha polypeptide p22-phox.
  • Cytochrome b is comprised of a light chain (alpha) and a heavy chain (beta).
  • This gene encodes the light, alpha subunit which has been proposed as a primary component of the microbicidal oxidase system of phagocytes. Mutations in this gene are associated with autosomal recessive chronic granulomatous disease (CGD), that is characterized by the failure of activated phagocytes to generate superoxide, which is important for the microbicidal activity of these cells.
  • CCD autosomal recessive chronic granulomatous disease
  • the polynucleotide sequence of human CYBA mRNA is depicted as SEQ ID NO:6, and the corresponding polypeptide sequence is set forth in SEQ ID NO:54.
  • ATF3 is a member of the mammalian activation transcription factor/cAMP responsive element-binding (CREB) protein family of transcription factors. Multiple transcript variants encoding two different isoforms have been found for this gene. The longer isoform represses rather than activates transcription from promoters with ATF binding elements. The shorter isoform (deltaZip2) lacks the leucine zipper protein-dimerization motif and does not bind to DNA, and it stimulates transcription presumably by sequestering inhibitory co-factors away from the promoter. It is possible that alternative splicing of the ATF3 gene may be physiologically important in the regulation of target genes.
  • the polynucleotide sequences of human ATF3 transcriptional variants 3, 1 and 2 are set forth in SEQ ID NOS: 7-9, respectively, and the corresponding polypeptide sequences are set forth in SEQ ID NOS:55-57, respectively.
  • This gene encodes a protein, which is a member of the cysteine-aspartic acid protease (caspase) family. Sequential activation of caspases plays a central role in the execution-phase of cell apoptosis.
  • Caspases exist as inactive proenzymes, which undergo proteolytic processing at conserved aspartic residues to produce two subunits, large and small, that dimerize to form the active enzyme. The proteolytic cleavage of this protein is induced by a variety of apoptotic stimuli.
  • Alternative splicing of this gene results in multiple transcript variants that encode different isoforms.
  • the polynucleotide sequences of human CASP2 transcriptional variants 1 and 3 are set forth in SEQ ID NOS:10-11, respectively, and the corresponding polypeptide sequences are set forth in SEQ ID NOS:58-59, respectively.
  • U.S. Pat. No. 6,083,735 relates to the alternative splicing products of Casp2.
  • U.S. Pat. Nos. 5,929,042 and 7,223,856 disclose specific Casp2 antisense compounds for the treatment of neurodegenerative disorders.
  • International Patent Publication WO 02/024720 teaches Casp2 antisense.
  • International Patent Publication WO 02/034201 discloses methods of treating diabetic retinopathy; WO 03/05821 relates to the inhibition of apoptosis related genes; WO 2004/009797 teaches Casp2 antisense; and WO 2004/103389 relates to methods for preventing cell death.
  • NADPH oxidases such as NOX3, are plasma membrane-associated enzymes found in many cell types. They catalyze the production of superoxide by a 1-electron reduction of oxygen, using NADPH as the electron donor.
  • the polynucleotide sequence of human NOX3 mRNA is set forth in SEQ ID NO: 12, and the corresponding polypeptide sequence is set forth in SEQ ID NO:60.
  • Gene aliases DP5, Bid3; BCL2-interacting protein; activator of apoptosis Hrk; BH3 interacting (with BCL2 family) domain, apoptosis agonist.
  • Hrk regulates apoptosis through interaction with death-repressor proteins Bcl-2 and Bcl-X(L).
  • the HRK protein lacks significant homology to other BCL2 family members except for an 8-amino acid region that was similar to the BCL2 homology domain-3 (BH3) motif of BIK.
  • HRK interacts with BCL2 and BCLXL via the BH3 domain, but not with the death-promoting BCL2-related proteins BAX, BAK, or BCLXS.
  • HRK localizes to membranes of intracellular organelles in a pattern similar to that previously reported for BCL2 and BCLXL.
  • the polynucleotide sequence of human HRK mRNA is set forth in SEQ ID NO: 13 and the corresponding polypeptide sequence is set forth in SEQ ID NO.61.
  • Gene aliases GC1QBP, HABP1, SF2p32, gC1Q-R, gC1qR, p32, RP23-83113.1, AA407365, AA986492, D11Wsu182e, MGC91723; C1q globular domain-binding protein; hyaluronan-binding protein 1; splicing factor SF2-associated protein.
  • the human complement subcomponent C1q associates with C1r and C1s in order to yield the first component of the serum complement system.
  • the protein encoded by this gene is known to bind to the globular heads of C1q molecules and inhibit C1 activation. This protein has also been identified as the p32 subunit of pre-mRNA splicing factor SF2, as well as a hyaluronic acid-binding protein.
  • the polynucleotide sequence of human C1QBP mRNA is set forth in SEQ ID NO: 14 and the corresponding polypeptide sequence is set forth in SEQ ID NO:62.
  • This gene is a member of the BCL2/adenovirus E1B 19 kd-interacting protein (BNIP) family. It interacts with the E1B 19 kDa protein, which is responsible for the protection of virally-induced cell death, as well as E1B 19 kDa-like sequences of BCL2, also an apoptotic protector.
  • This gene contains a BH3 domain and a transmembrane domain, which have been associated with pro-apoptotic function.
  • the dimeric mitochondrial protein encoded by this gene is known to induce apoptosis, even in the presence of BCL2.
  • the polynucleotide sequence of human BNIP3 mRNA is set forth in SEQ ID NO: 15 and the corresponding polypeptide sequence is set forth in SEQ ID NO:63.
  • U.S. Pat. No. 5,858,678 relates to the BNIP3 polynucleotide and polypeptide sequences.
  • International Patent Publication WO 2004/009780 discloses methods of preventing ischemia induced cell damage.
  • MAP kinases act as an integration point for multiple biochemical signals, and are involved in a wide variety of cellular processes such as proliferation, differentiation, transcription regulation and development.
  • This kinase is activated by various cell stimuli, and targets specific transcription factors, and thus mediates immediate-early gene expression in response to cell stimuli.
  • TNF- ⁇ tumor-necrosis factor alpha
  • This kinase is also involved in UV radiation induced apoptosis, which is thought to be related to cytochrome c-mediated cell death pathway.
  • Gene aliases CSBP1; CSBP2; CSPB1; EXIP; Mxi2; PRKM14; PRKM15; RK; SAPK2A; p38; p38ALPHA; MGC102436; p38MAPK; CSBP; Exip; Hog; MGC105413; p38Hog; Csaids binding protein; MAP kinase Mxi2; MAX-interacting protein 2; cytokine suppressive anti-inflammatory drug binding protein; p38 MAP kinase; p38 mitogen activated protein kinase; p38alpha Exip; stress-activated protein kinase 2A, tRNA synthetase cofactor p38.
  • the protein encoded by this gene is a member of the MAP kinase family, which act as an integration point for multiple biochemical signals, and are involved in a wide variety of cellular processes such as proliferation, differentiation, transcription regulation and development.
  • This kinase is activated by various environmental stresses and proinflammatory cytokines. The activation requires its phosphorylation by MAP kinase kinases (MKKs), or its autophosphorylation triggered by its interaction with MAP3K7IP1/TAB1 protein.
  • MKKs MAP kinase kinases
  • the substrates of this kinase include transcription regulator ATF2, MEF2C, and MAX, cell cycle regulator CDC25B, and tumor suppressor p53, which suggest its role in stress related transcription and cell cycle regulation, as well as in genotoxic stress response.
  • transcription regulator ATF2 MEF2C
  • MAX cell cycle regulator CDC25B
  • tumor suppressor p53 tumor suppressor p53
  • the protein encoded by this gene is a GTPase, which belongs to the RAS superfamily of small GTP-binding proteins. Members of this superfamily regulate a diverse array of cellular events, including the control of cell growth, cytoskeletal reorganization, and the activation of protein kinases.
  • GTPase belongs to the RAS superfamily of small GTP-binding proteins. Members of this superfamily regulate a diverse array of cellular events, including the control of cell growth, cytoskeletal reorganization, and the activation of protein kinases.
  • Several alternatively spliced transcript variants of this gene have been described, but the full-length nature of some of these variants has not been determined.
  • the polynucleotide sequences of human RAC1 transcriptional variants 1c, 1b and 1 are set forth in SEQ ID NOS:24-26, respectively and the corresponding polypeptide sequences are set forth in SEQ ID NO:72-74.
  • U.S. Pat. No. 6,180,597 relates to rho GTPase inhibitors that increase endothelial cell nitric oxide synthase levels.
  • International Patent Publication WO 01/15739 teaches antisense modulation of Rho family members.
  • International Patent Publication WO 2004/042052 teaches methods of suppressing TNF- ⁇ secretion.
  • Glycogen synthase kinase-3 is a proline-directed serine-threonine kinase that was initially identified as a phosphorylating and inactivating glycogen synthase.
  • GSK3B is involved in energy metabolism, neuronal cell development, and body pattern formation (Plyte et al., Biochim Biophys Acta. 1992. 1114(2-3):147-62).
  • the polynucleotide sequence of human GSK3B mRNA is set forth in SEQ ID NO:27, and the corresponding polypeptide sequence is set forth in SEQ ID NO:75.
  • U.S. Pat. No. 6,323,029 relates to antisense inhibition of GSK3B.
  • the product of this gene belongs to the family of purinoceptors for ATP.
  • This receptor functions as a ligand-gated ion channel and is responsible for ATP-dependent lysis of macrophages through the formation of membrane pores permeable to large molecules.
  • Activation of this nuclear receptor by ATP in the cytoplasm may be a mechanism by which cellular activity can be coupled to changes in gene expression.
  • Multiple alternatively spliced variants which would encode different isoforms have been identified although some fit nonsense-mediated decay (NMD) criteria.
  • NMD nonsense-mediated decay
  • Gene aliases EREG1, KNP3, LTRPC2, MGC133383, NUDT9H, NUDT9L1, TRPC7, 9830168K16Rik, C79133, Trp7, Trrp7; estrogen responsive element associated gene 1; long transient receptor potential channel 2; transient receptor potential channel 7, transient receptor potential cation channel, subfamily M, member 2 (Trpm2); transient receptor potential channel 7; transient receptor protein 7.
  • the protein encoded by this gene is a calcium-permeable cation channel that is regulated by free intracellular ADP-ribose.
  • the encoded protein is activated by oxidative stress and confers susceptibility to cell death.
  • the polynucleotide sequences of the human TRPM2 is set forth in SEQ ID NO:29 and the corresponding polypeptide sequences is set forth in SEQ ID NO:77. (Two transcript variants encoding different isoforms S and L had been found for this gene. The S variant was removed by NCBI since it contains a sequencing error and does not exist).
  • Poly(ADP-ribose) glycohydrolase is the major enzyme responsible for the catabolism of poly(ADP-ribose), a reversible covalent-modifier of chromosomal proteins.
  • the protein is found in many tissues and may be subject to proteolysis generating smaller, active products.
  • the polynucleotide sequence of human PARG mRNA is set forth in SEQ ID NO:31, and the corresponding polypeptide sequence is set forth in SEQ ID NO:79.
  • Gene aliases T10, Cd38-rs1; ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase; CD38 antigen; CD38 antigen (p45); cyclic ADP-ribose hydrolase.
  • CD38 is a novel multifunctional ectoenzyme widely expressed in cells and tissues; especially in leukocytes. CD38 also functions in cell adhesion, signal transduction and calcium signaling.
  • the polynucleotide sequence of human CD38 mRNA is set forth in SEQ ID NO: 32, and the corresponding polypeptide sequences are set forth in SEQ ID NO:80.
  • a membrane protein induced by TNF- ⁇ and IL-6 in adipose tissues Both IL-6 and TNF- ⁇ were shown to be unregulated in a spinal cord injury model (Ahn, et al., BBRC 2006 348(2):560-70) and are thought to promote apoptotic events.
  • the polynucleotide sequence of human STEAP4 mRNA is set forth in SEQ ID NO:33, and the corresponding polypeptide sequence is set forth in SEQ ID NO:81.
  • BMP2 Bone Morphogenetic Protein 2
  • the protein encoded by this gene belongs to the transforming growth factor-beta (TGFB) superfamily.
  • the encoded protein acts as a disulfide-linked homodimer and induces bone and cartilage formation.
  • the polynucleotide sequence of human BMP2 mRNA is set forth in SEQ ID NO:34, and the corresponding polypeptide sequence is set forth in SEQ ID NO:82.
  • Gap junction protein, alpha 1 is a member of the connexin gene family of proteins and is a component of gap junctions in the heart, and is believed to have a crucial role in the synchronized contraction of the heart and in embryonic development.
  • Connexin 43 is targeted by several protein kinases that regulate myocardial cell-cell coupling.
  • a related intron-less connexin 43 pseudogene, GJA1P has been mapped to chromosome 5.
  • the polynucleotide sequence of human GJA1 mRNA is set forth in SEQ ID NO:35, and the corresponding polypeptide sequence is set forth in SEQ ID NO:83.
  • U.S. Pat. No. 7,098,190 teaches antisense compounds for treating, inter alia, wounds and spinal cord injury.
  • TYRO Protein Tyrosine Kinase Binding Protein (21) TYRO Protein Tyrosine Kinase Binding Protein (TYROBP)
  • This gene encodes a transmembrane signaling polypeptide that contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
  • ITAM immunoreceptor tyrosine-based activation motif
  • the protein may associate with the killer-cell inhibitory receptor (KIR) family of membrane glycoproteins and may act as an activating signal transduction element.
  • KIR killer-cell inhibitory receptor
  • This protein may bind zeta-chain (TCR) associated protein kinase 70 kDa (ZAP-70) and spleen tyrosine kinase (SYK) and play a role in signal transduction, bone modeling, brain myelination, and inflammation.
  • CTGF Connective Tissue Growth Factor
  • Gene aliases CCN2, HCS24, IGFBP8, MGC102839, NOV2, CTGRP, Fisp12, Hcs24, fisp-12; hypertrophic chondrocyte-specific protein 24; insulin-like growth factor-binding protein 8, FISP-12 protein; fibroblast inducible secreted protein; fibroblast inducible secreted protein; hypertrophic chondrocyte-specific gene product 24.
  • a major connective tissue mitoattractant secreted by vascular endothelial cells promotes proliferation and differentiation of chondrocytes.
  • the polynucleotide sequence of human CTGF mRNA is set forth in SEQ ID NO:38 and the corresponding polypeptide sequence is set forth in SEQ ID NO:86.
  • SSP1 is a secreted protein which acts as a cytokine involved in enhancing production of interferon-gamma and interleukin-12 and reducing production of interleukin-10 and which is essential in the pathway that leads to type I immunity.
  • the polynucleotide sequences of human SPP1 transcriptional variants 1, 2 and 3 are set forth in SEQ ID NOS:39-41, and the corresponding polypeptide sequences are set forth in SEQ ID NOS:87-89.
  • This gene encodes the receptor for reticulon 4, oligodendrocyte myelin glycoprotein and myelin-associated glycoprotein. This receptor mediates axonal growth inhibition and may play a role in regulating axonal regeneration and plasticity in the adult central nervous system.
  • the polynucleotide sequence of human RTN4R mRNA is set forth in SEQ ID NO:42 and the corresponding polypeptide sequence is set forth in SEQ ID NO:90.
  • Gene aliases ANX2, ANX2L4, CAL1H, LIP2, LPC2, LPC2D, P36, PAP-IV; annexin II; calpactin I heavy polypeptide; chromobindin 8; lipocortin II; placental anticoagulant protein IV.
  • This gene encodes a member of the annexin family.
  • Members of this calcium-dependent phospholipid-binding protein family play a role in the regulation of cellular growth and in signal transduction pathways.
  • This protein functions as an autocrine factor, which heightens osteoclast formation and bone resorption.
  • This gene has three pseudogenes located on chromosomes 4, 9 and 10, respectively. Multiple alternatively spliced transcript variants encoding different isoforms have been found for this gene.
  • the polynucleotide sequences of human ANXA2 transcriptional variants 1, 3 and 2 are set forth in SEQ ID NOS:43-45 and the corresponding polypeptide sequences are set forth in SEQ ID NOS:91-93.
  • RHOA is a small GTPase protein known to regulate the actin cytoskeleton in the formation of stress fibers. It acts upon the effector proteins: Rho kinase (ROCK) culminating in the inhibition of axonal regeneration.
  • Rho kinase RI-1
  • C3 transferase enhances axonal growth on myelin substrates while in vivo studies have not been effective.
  • the polynucleotide sequence of human RHOA mRNA is set forth in SEQ ID NO:46 and the corresponding polypeptide sequence is set forth in SEQ ID NO:94.
  • the protein encoded by this gene is a glycoprotein and a member of the NADPH oxidase family.
  • the synthesis of thyroid hormone is catalyzed by a protein complex located at the apical membrane of thyroid follicular cells.
  • This complex contains an iodide transporter, thyroperoxidase, and a peroxide generating system that includes this encoded protein and DUOX2.
  • This protein has both a peroxidase homology domain and a gp91phox domain.
  • Two alternatively spliced transcript variants encoding the same protein have been described for this gene.
  • the polynucleotide sequence of human DUOX1 transcriptional variants 1 and 2 are set forth in SEQ ID NOS:47-48 and the corresponding polypeptide sequences are set forth in SEQ ID NOS:95-96.
  • pro-apoptotic polypeptide refers to a polypeptide encoded by any of the above listed genes, including splice variants, isoforms, orthologs, or paralogs and the like.
  • an “inhibitor” is a compound which is capable of inhibiting the expression of a gene or the activity of the product of such gene to an extent sufficient to achieve a desired biological or physiological effect.
  • the term “inhibitor” as used herein refers to one or more of an oligonucleotide inhibitor, including siRNA, shRNA, aptamers, antisense molecules, miRNA and ribozymes, as well as antibodies The inhibitor may cause complete or partial inhibition.
  • inhibitor refers to reducing the expression of a gene or the activity of the product of such gene to an extent sufficient to achieve a desired biological or physiological effect. Inhibition may be complete or partial.
  • polynucleotide and “nucleic acid” may be used interchangeably and refer to nucleotide sequences comprising deoxyribonucleic acid (DNA), and ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the terms should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs.
  • mRNA sequences are set forth as representing the corresponding genes.
  • Oligonucleotide refers to a compound comprising deoxyribonucleotides and/or ribonucleotides from about 2 to about 50 nucleotides. Each DNA or RNA nucleotide may be independently natural or synthetic, and or modified or unmodified. Modifications include changes to the sugar moiety, the base moiety and or the linkages between nucleotides in the oligonucleotide.
  • the present invention provides methods and compositions for inhibiting expression of a target pro-apoptotic gene in vivo.
  • the method includes administering oligoribonucleotides, in particular small interfering RNAs (i.e., siRNAs) or a nucleic acid material that can produce siRNA in a cell, that target an mRNA transcribed from a pro-apoptotic gene in an amount sufficient to down-regulate expression of a target gene by an RNA interference mechanism.
  • siRNAs small interfering RNAs
  • the subject method can be used to inhibit expression of the pro-apoptotic gene for treatment of a disease.
  • the siRNA molecules or inhibitors of the pro-apoptotic gene are used as drugs to treat various pathologies.
  • Table B (B1-B76) comprises nucleic acid sequences of sense and corresponding antisense oligomers, useful in preparing corresponding siRNA compounds.
  • Tables C1, C2 and C3 comprise certain currently preferred nucleic acid sequences of sense and corresponding antisense oligomers, useful in preparing the corresponding siRNA compounds.
  • siRNA corresponding to known genes has been widely reported; see for example Ui-Tei et al., J Biomed Biotechnol. 2006; 65052; Chalk et al., BBRC. 2004, 319(1):264-74; Sioud and Leirdal, Met. Mol Biol.; 2004, 252:457-69; Levenkova et al., Bioinform. 2004, 20(3):430-2; Ui-Tei et al., NAR 2004, 32(3):936-48.
  • the present invention provides double-stranded oligoribonucleotides (eg. siRNAs), which down-regulate the expression of the pro-apoptotic gene according to the present invention.
  • siRNAs double-stranded oligoribonucleotides
  • An siRNA of the invention is a duplex oligoribonucleotide in which the sense strand is derived from the mRNA sequence of the pro-apoptotic gene, and the antisense strand is complementary to the sense strand. In general, some deviation from the target mRNA sequence is tolerated without compromising the siRNA activity (see e.g. Czauderna et al., 2003, NAR 31(11), 2705-2716).
  • An siRNA of the invention inhibits gene expression on a post-transcriptional level with or without destroying the mRNA. Without being bound by theory, siRNA may target the mRNA for specific cleavage and degradation and/or may inhibit translation from the targeted message.
  • ribonucleotide encompasses natural and synthetic, unmodified and modified ribonucleotides. Modifications include changes to the sugar moiety, to the base moiety and/or to the linkages between ribonucleotides in the oligonucleotide.
  • the oligoribonucleotide according to the present invention comprises modified siRNA.
  • the siRNA comprises an RNA duplex comprising a first strand and a second strand, whereby the first strand comprises a ribonucleotide sequence at least partially complementary to about 18 to about 40 consecutive nucleotides of a target nucleic acid, and the second strand comprises ribonucleotide sequence at least partially complementary to the first strand and wherein said first strand and/or said second strand comprises a plurality of groups of modified ribonucleotides having a modification at the 2′-position of the sugar moiety whereby within each strand each group of modified ribonucleotides is flanked on one or both sides by a group of flanking ribonucleotides whereby each ribonucleotide forming the group of flanking ribonucleotides is selected from an unmodified ribonucleotide or a ribonucle
  • the group of modified ribonucleotides and/or the group of flanking ribonucleotides comprise a number of ribonucleotides selected from the group consisting of an integer from 1 to 12. Accordingly, the group thus comprises one nucleotide, two nucleotides, three nucleotides, four nucleotides, five nucleotides, six nucleotides, seven nucleotides, eight nucleotides, nine nucleotides, ten nucleotides, eleven nucleotides or twelve nucleotides.
  • the groups of modified nucleotides and flanking nucleotides may be organized in a pattern on at least one of the strands.
  • the first and second strands comprise a pattern of modified nucleotides.
  • only one strand comprises a pattern of modified nucleotides.
  • the pattern of modified nucleotides of said first strand is identical relative to the pattern of modified nucleotides of the second strand.
  • the pattern of modified nucleotides of said first strand is shifted by one or more nucleotides relative to the pattern of modified nucleotides of the second strand.
  • the middle ribonucleotide in the antisense strand is an unmodified nucleotide.
  • ribonucleotide number 10 is unmodified; in a 21-oligomer antisense strand, ribonucleotide number 11 is unmodified; and in a 23-oligomer antisense strand, ribonucleotide number 12 is unmodified.
  • the modifications or pattern of modification, if any, of the siRNA must be planned to allow for this.
  • the modifications on the 2′ moiety of the sugar residue include amino, fluoro, alkoxy e.g. methoxy, alkyl, amino, fluoro, chloro, bromo, CN, CF, imidazole, carboxylate, thioate, C 1 to C 10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl, OCF 3 , OCN, O-, S-, or N-alkyl; O-, S, or N-alkenyl; SOCH 3 ; SO 2 CH 3 ; ONO 2 ; NO 2 , N 3 ; heterocycloalkyl; heterozycloalkaryl; aminoalkylamino; polyalkylamino or substituted silyl, as, among others, described in European patents EP 0 586 520 B1 or EP 0 618 925 B1.
  • the siRNA is blunt ended, at one or both ends. More specifically, the siRNA may be blunt ended on the end defined by the 5′-terminus of the first strand and the 3′-terminus of the second strand, or the end defined by the 3′-terminus of the first strand and the 5′-terminus of the second strand. In other embodiments at least one of the two strands may have an overhang of at least one nucleotide at the 5′-terminus. At least one of the strands may also optionally have an overhang of at least one nucleotide at the 3′-terminus. The overhang may consist of from about 1 to about 5 consecutive nucleotides. A nucleotide of the overhang may be a modified or unmodified ribonucleotide or deoxyribonucleotide.
  • RNA duplex is from about 18 to about 40 ribonucleotides, preferably 19, 21 or 23 ribonucleotides. Further, the length of each strand may independently have a length selected from the group consisting of about 15 to about 40 bases, preferably 18 to 23 bases and more preferably 19, 21 or 23 ribonucleotides.
  • the complementarity between said first strand and the target nucleic acid may be perfect.
  • the strands are substantially complementary, i.e. having one, two or up to three mismatches between said first strand and the target nucleic acid.
  • substantially complementary refers to complementarity of greater than about 84%, to another sequence.
  • substantially identical refers to identity of greater than about 84%, to another sequence.
  • the first strand and the second strand each comprise at least one group of modified ribonucleotides and at least one group of flanking ribonucleotides, whereby each group of modified ribonucleotides comprises at least one ribonucleotide and whereby each group of flanking ribonucleotides comprises at least one ribonucleotide, wherein each group of modified ribonucleotides of the first strand is aligned with a group of flanking ribonucleotides on the second strand, and wherein the 5′ most terminal ribonucleotide is selected from a group of modified ribonucleotides, and the 3′ most terminal ribonucleotide of the second strand is a selected from the group of flanking ribonucleotide.
  • each group of modified ribonucleotides consists of a single ribonucleotide and each group of flanking ribon
  • the ribonucleotide forming the group of flanking ribonucleotides on the first strand is an unmodified ribonucleotide arranged in a 3′ direction relative to the ribonucleotide forming the group of modified ribonucleotides
  • the ribonucleotide forming the group of modified ribonucleotides on the second strand is a modified ribonucleotide which is arranged in 5′ direction relative to the ribonucleotide forming the group of flanking ribonucleotides.
  • the first strand of the siRNA comprises five to about twenty, eight to twelve, preferably nine to twelve, groups of modified ribonucleotides, and the second strand comprises seven to eleven, preferably eight to eleven, groups of modified ribonucleotides.
  • the first strand and the second strand may be linked by a loop structure, which may be comprised of a non-nucleic acid polymer such as, inter alia, polyethylene glycol.
  • the loop structure may be comprised of a nucleic acid, including modified and non-modified ribonucleotides and modified and non-modified deoxyribonucleotides.
  • the 5′-terminus of the first strand of the siRNA may be linked to the 3′-terminus of the second strand, or the 3′-terminus of the first strand may be linked to the 5′-terminus of the second strand, said linkage being via a nucleic acid linker or a non-nucleic acid linker.
  • a nucleic acid linker has a length of between about 2-100 nucleic acids, preferably about 2 to about 30 nucleic acids.
  • the present invention provides a compound having the structure:
  • the compound comprises a phosphodiester bond.
  • the compound is blunt ended, for example wherein Z and Z′ are both absent.
  • the compound comprises at least one 3′ overhang, wherein at least one of Z or Z′ is present.
  • Z and Z′ can be independently comprise one or more covalently linked modified or non-modified nucleotides, as described infra, for example inverted dT or dA; dT, LNA (locked nucleic acids), mirror nucleotide and the like.
  • each of Z and Z′ are independently selected from dT and dTdT.
  • the compound comprises one or more ribonucleotides unmodified in their sugar residues. In other embodiments the compound comprises at least one ribonucleotide modified in the sugar residue. In some embodiments the compound comprises a modification at the 2′ position of the sugar residue. Modifications in the 2′ position of the sugar residue include amino, fluoro, alkoxy and alkyl moieties. In certain preferred embodiments the alkoxy modification is a methoxy moiety at the 2′ position of the sugar residue (2′-O-methyl; 2′-O-Me; 2′-O—CH 3 ).
  • the compound comprises modified alternating ribonucleotides in one or both of the antisense and the sense strands. In certain embodiments the compound comprises modified alternating ribonucleotides in the antisense and the sense strands. In some preferred embodiments the middle ribonucleotide of the antisense strand is not modified; e.g. ribonucleotide in position 10 in a 19-mer strand.
  • the compound comprises an antisense sequence present in Table B SEQ ID NOS:277 to 50970 and 50993-68654).
  • the present invention provides a mammalian expression vector comprising an antisense sequence present in Table B (SEQ ID NOS:277 to 50970 and 50993-68654).
  • Certain presently preferred compounds are listed in Tables C1, C2 and C3, and their sequences are set forth in SEQ ID NOS: 97-276 and SEQ ID NOS 50971-50992.
  • (N) x and (N′) y may be phosphorylated or non-phosphorylated at the 3′ and 5′ termini.
  • alternating ribonucleotides are modified in the 2′ position of the sugar residue in both the antisense and the sense strands of the compound.
  • the exemplified siRNA has been modified such that a 2′-O-methyl (Me) group was present on the first, third, fifth, seventh, ninth, eleventh, thirteenth, fifteenth, seventeenth and nineteenth nucleotide of the antisense strand, whereby the very same modification, i.e. a 2′-O-Me group, was present at the second, fourth, sixth, eighth, tenth, twelfth, fourteenth, sixteenth and eighteenth nucleotide of the sense strand.
  • these particular siRNA compounds are also blunt ended.
  • alternating ribonucleotides are modified in both the antisense and the sense strands of the compound.
  • the exemplified siRNA has been modified such that a 2′-O-methyl (2′-OMe) group was present on the first, third, fifth, seventh, ninth, eleventh, thirteenth, fifteenth, seventeenth, nineteenth, twenty-first and twenty-third nucleotide of the antisense strand (N) x , and whereby the very same modification, i.e.
  • a 2′-OMe group was present at the second, fourth, sixth, eighth, tenth, twelfth, fourteenth, sixteenth, eighteenth, twentieth and twenty-second nucleotide of the sense strand (N′) y . Additionally, it is to be noted that these particular siRNA compounds are also blunt ended.
  • the compounds of the invention having alternating ribonucleotides modified in one or both of the antisense and the sense strands of the compound; for 19-mers and 23-mers the ribonucleotides at the 5′ and 3′ termini of the antisense strand are modified in their sugar residues, and the ribonucleotides at the 5′ and 3′ termini of the sense strand are unmodified in their sugar residues.
  • the ribonucleotides at the 5′ and 3′ termini of the sense strand are modified in their sugar residues, and the ribonucleotides at the 5′ and 3′ termini of the antisense strand are unmodified in their sugar residues.
  • it is preferred that the middle nucleotide of the antisense strand is unmodified.
  • the invention provides siRNA comprising a nucleic acid sequence set forth in Table B (B1-B76; SEQ ID NOS:277-50970 and 50993-68654) wherein 1, 2, or 3 of the nucleotides in one strand or both strands are substituted thereby providing at least one base pair mismatch.
  • the substituted nucleotides in each strand are preferably in the terminal region of one strand or both strands.
  • the ribonucleic acid sequences of the siRNA are SEQ ID NOS:97-276 and SEQ ID NOS: 50971-50992 of Tables C1, C2 and C3.
  • the ribonucleic acid sequences of the siRNA are set forth in SEQ ID NOS:99-100; SEQ ID NOS:133-134; SEQ ID NOS:137-138; SEQ ID NOS:211-212; SEQ ID NOS:213-214 as shown in Table C1.
  • the antisense and the sense strands of the siRNA are phosphorylated only at the 3′-terminus and not at the 5′-terminus.
  • the antisense and the sense strands are non-phosphorylated.
  • the 5′ most ribonucleotide in the sense strand is modified to abolish any possibility of in vivo 5′-phosphorylation.
  • the invention further provides a vector capable of expressing any of the aforementioned oligoribonucleotides in unmodified form in a cell after which appropriate modification may be made.
  • the cell is a mammalian cell, preferably a human cell.
  • the present invention provides a pharmaceutical composition comprising one or more of the compounds of the invention; and a pharmaceutically acceptable carrier.
  • This composition may comprise a mixture of two or more different siRNAs.
  • this composition may comprise a mixture of siRNA to RhoA and siRNA to one or more of the other pro-apoptotic genes of the invention.
  • this composition may comprise a mixture of siRNA to RhoA and siRNA to Casp2.
  • RhoA is a small GTPase that when activated inhibits neurite outgrowth. Its inhibition is relevant for spinal cord injury and it can be combined for this indication with anti-apoptotic siRNAs of the invention. The latter will protect, and siRNA to RhoA will promote regeneration, and so a combined or even synergistic effect is produced.
  • the invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising at least one compound of the invention covalently or non-covalently bound to one or more compounds of the invention in an amount effective to inhibit the pro-apoptotic genes of the present invention; and a pharmaceutically acceptable carrier.
  • the compound may be processed intracellularly by endogenous cellular complexes to produce one or more oligoribonucleotides of the invention.
  • the invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more of the compounds of the invention in an amount effective to inhibit expression in a cell of a human pro-apoptotic gene of the present invention, the compound comprising a sequence which is substantially complementary to the sequence of (N) x .
  • Substantially complementary refers to complementarity of greater than about 84%, to another sequence.
  • one mismatch results in 94.7% complementarity
  • two mismatches results in about 89.5% complementarity
  • 3 mismatches results in about 84.2% complementarity, rendering the duplex region substantially complementary.
  • substantially identical refers to identity of greater than about 84%, to another sequence.
  • the invention provides a method of inhibiting the expression of the pro-apoptotic genes of the present invention by at least 20%, preferably 30%, even more preferably 40% or even 50% as compared to a control comprising contacting an mRNA transcript of the pro-apoptotic gene of the present invention with one or more of the compounds of the invention.
  • the oligoribonucleotide is inhibiting one or more of the pro-apoptotic genes of the present invention, whereby the inhibition is selected from the group comprising inhibition of gene function, inhibition of polypeptide and inhibition of mRNA expression.
  • the compound is inhibiting a pro-apoptotic polypeptide, whereby the inhibition is selected from the group comprising inhibition of function (which may be examined by an enzymatic assay or a binding assay with a known interactor of the native gene/polypeptide, inter alia), inhibition of protein (which may be examined by Western blotting, ELISA or immuno-precipitation, inter alia) and inhibition of mRNA expression (which may be examined by Northern blotting, quantitative RT-PCR, in-situ hybridisation or microarray hybridisation, inter alia).
  • inhibition of function which may be examined by an enzymatic assay or a binding assay with a known interactor of the native gene/polypeptide, inter alia
  • inhibition of protein which may be examined by Western blotting, ELISA or immuno-precipitation, inter alia
  • inhibition of mRNA expression which may be examined by Northern blotting, quantitative RT-PCR, in-situ hybridisation or microarray hybridisation, inter alia.
  • the invention provides a method of treating a subject suffering from a disease accompanied by an elevated level of the pro-apoptotic genes of the present invention, the method comprising administering to the subject a compound of the invention in a therapeutically effective dose thereby treating the subject.
  • the invention provides an oligoribonucleotide wherein one strand comprises consecutive nucleotides having, from 5′ to 3′, the sequence set forth in any one of SEQ ID NOS:277-50970 and 50993-68654, shown also in Table B, or a homolog thereof wherein in up to two of the ribonucleotides in each terminal region is altered.
  • nucleic acids according to the present invention comprise at least 14 contiguous nucleotides of any one of the polynucleotides in Table B (SEQ ID NOS:277-50970 and 50993-68654) and more preferably 14 contiguous nucleotide base pairs at any end of the double-stranded structure comprised of the first strand and second strand as described above.
  • siRNA molecules of the present invention may be delivered to the target tissue by direct application of the naked molecules prepared with a carrier or a diluent.
  • naked siRNA refers to siRNA molecules that are free from any delivery vehicle that acts to assist, promote or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like.
  • siRNA in PBS is “naked siRNA”.
  • the siRNA molecules of the invention are delivered in liposome formulations and lipofectin formulations and the like and can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein incorporated by reference.
  • siRNA has recently been successfully used for inhibition of gene expression in primates (see for example, Tolentino et al., Retina 24(4):660).
  • the pharmaceutically acceptable carriers, solvents, diluents, excipients, adjuvants and vehicles as well as implant carriers generally refer to inert, non-toxic solid or liquid fillers, diluents or encapsulating material not reacting with the active ingredients of the invention and they include liposomes and microspheres.
  • delivery systems useful in the present invention include U.S. Pat. Nos. 5,225,182; 5,169,383; 5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224; 4,439,196; and 4,475,196. Many other such implants, delivery systems, and modules are well known to those skilled in the art.
  • topical and transdermal formulations may be selected.
  • the siRNAs or pharmaceutical compositions of the present invention are administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual subject, the disease to be treated, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners.
  • the “therapeutically effective dose” for purposes herein is thus determined by such considerations as are known in the art.
  • the dose must be effective to achieve improvement including but not limited to improved survival rate or more rapid recovery, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art.
  • the active dose of compound for humans is in the range of from 1 ng/kg to about 20-100 mg/kg body weight per day, preferably about 0.01 mg to about 2-10 mg/kg body weight per day, in a regimen of one dose per day or twice or three or more times per day for a period of 1-4 weeks or longer.
  • the compounds of the present invention can be administered by any of the conventional routes of administration. It should be noted that the compound can be administered as the compound or as pharmaceutically acceptable salt and can be administered alone or as an active ingredient in combination with pharmaceutically acceptable carriers, solvents, diluents, excipients, adjuvants and vehicles.
  • the compounds can be administered orally, subcutaneously or parenterally including intravenous, intraarterial, intramuscular, intraperitoneally, and intranasal administration as well as intrathecal and infusion techniques. Implants of the compounds are also useful.
  • Liquid forms may be prepared for injection, the term including subcutaneous, transdermal, intravenous, intramuscular, intrathecal, and other parental routes of administration.
  • the liquid compositions include aqueous solutions, with and without organic co-solvents, aqueous or oil suspensions, emulsions with edible oils, as well as similar pharmaceutical vehicles.
  • the administration comprises intravenous administration.
  • the administration comprises topical or local administration.
  • compositions for use in the novel treatments of the present invention may be formed as aerosols, for example for intranasal administration.
  • oral compositions such as tablets, suspensions, solutions
  • oral compositions may be effective for local delivery to the oral cavity such as oral composition suitable for mouthwash for the treatment of oral mucositis.
  • the present invention relates to a method for the treatment of a subject in need of treatment for a disease or disorder associated with the abnormal expression of the proapoptotic genes of Table A, comprising administering to the subject an amount of an inhibitor which reduces or inhibits expression of these genes.
  • the subject being treated is a warm-blooded animal and, in particular, mammals including human.
  • the methods of the invention comprise administering to the subject one or more inhibitory compounds which down-regulate expression of the proapoptotic genes of Table A; and in particular siRNA in a therapeutically effective dose so as to thereby treat the subject.
  • the inhibitor is selected from the group consisting of siRNA, shRNA, an aptamer, an antisense molecule, miRNA, a ribozyme, and an antibody.
  • the inhibitor is siRNA.
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) pro-apoptotic-related disorder as listed above.
  • Those in need of treatment include those already experiencing the disease or condition, those prone to having the disease or condition, and those in which the disease or condition is to be prevented.
  • the compounds of the invention may be administered before, during or subsequent to the onset of the disease or condition or symptoms associated therewith.
  • the present invention relates to a method for delaying the onset of or averting the development of the disease or disorder.
  • the present invention relates to the use of compounds which down-regulate the expression of the pro-apoptotic genes of the invention particularly to novel small interfering RNAs (siRNAs), in the treatment of the following diseases or conditions in which inhibition of the expression of the pro-apoptotic genes is beneficial: hearing loss, acute renal failure (ARF), glaucoma, acute respiratory distress syndrome (ARDS) and other acute lung and respiratory injuries, ischemia-reperfusion injury following lung transplantation, organ transplantation including lung, liver, heart, bone marrow, pancreas, cornea and kidney transplantation, spinal cord injury, pressure sores, age-related macular degeneration (AMD), dry eye syndrome, oral mucositis and chronic obstructive pulmonary disease (COPD).
  • siRNAs small interfering RNAs
  • oligomer sequences useful in the preparation of siRNA directed to selected pro-apoptotic genes are set forth in SEQ ID NOS:277-50970 and 50993-68654, listed in Table B.
  • the method of the invention includes administering a therapeutically effective amount of one or more compounds which down-regulate expression of the pro-apoptotic genes, particularly the novel siRNAs of the present invention, small molecule inhibitors of the pro-apoptotic genes as described herein or antibodies to the pro-apoptotic proteins.
  • the methods of the invention are applied to various conditions of hearing loss.
  • the hearing loss may be due to apoptotic inner ear hair cell damage or loss, wherein the damage or loss is caused by infection, mechanical injury, loud sound, aging (presbycusis), or chemical-induced ototoxicity.
  • Ototoxins include therapeutic drugs including antineoplastic agents, salicylates, quinines, and aminoglycoside antibiotics, contaminants in foods or medicinals, and environmental or industrial pollutants.
  • treatment is performed to prevent or reduce ototoxicity, especially resulting from or expected to result from administration of therapeutic drugs.
  • a therapeutically effective composition is given immediately after the exposure to prevent or reduce the ototoxic effect. More preferably, treatment is provided prophylactically, either by administration of the composition prior to or concomitantly with the ototoxic pharmaceutical or the exposure to the ototoxin.
  • ototoxin in the context of the present invention is meant a substance that through its chemical action injures, impairs or inhibits the activity of the sound receptors component of the nervous system related to hearing, which in turn impairs hearing (and/or balance).
  • ototoxicity includes a deleterious effect on the inner ear hair cells.
  • Ototoxic agents that cause hearing impairments include, but are not limited to, antineoplastic agents such as vincristine, vinblastine, cisplatin and cisplatin-like compounds, taxol and taxol-like compounds, dideoxy-compounds, e.g., dideoxyinosine; alcohol; metals; industrial toxins involved in occupational or environmental exposure; contaminants of food or medicinals; and over-doses of vitamins or therapeutic drugs, e.g., antibiotics such as penicillin or chloramphenicol, and megadoses of vitamins A, D, or B6, salicylates, quinines, loop diuretics, and phosphodiesterase type 5 (PDE5) inhibitors such as sildenafil citrate (Viagra®).
  • antineoplastic agents such as vincristine, vinblastine, cisplatin and cisplatin-like compounds, taxol and taxol-like compounds, dideoxy-compounds, e.g., dideoxyinosine;
  • Exposure to an ototoxic agent is meant that the ototoxic agent is made available to, or comes into contact with, a mammal. Exposure to an ototoxic agent can occur by direct administration, e.g., by ingestion or administration of a food, medicinal, or therapeutic agent, e.g., a chemotherapeutic agent, by accidental contamination, or by environmental exposure, e.g., aerial or aqueous exposure.
  • Hearing may be due to end-organ lesions involving inner ear hair cells, e.g., acoustic trauma, viral endolymphatic labyrinthitis, Meniere's disease.
  • Hearing impairments include tinnitus, which is a perception of sound in the absence of an acoustic stimulus, and may be intermittent or continuous, wherein there is diagnosed a sensorineural loss.
  • Hearing loss may be due to bacterial or viral infection, such as in herpes zoster oticus, purulent labyrinthitis arising from acute otitis media, purulent meningitis, chronic otitis media, sudden deafness including that of viral origin, e.g., viral endolymphatic labyrinthitis caused by viruses including mumps, measles, influenza, chicken pox, mononucleosis and adenoviruses.
  • viruses including mumps, measles, influenza, chicken pox, mononucleosis and adenoviruses.
  • the hearing loss can be congenital, such as that caused by rubella, anoxia during birth, bleeding into the inner ear due to trauma during delivery, ototoxic drugs administered to the mother, erythroblastosis fetalis, and hereditary conditions including Waardenburg's syndrome and Hurler's syndrome.
  • the hearing loss can be noise-induced, generally due to a noise greater than 85 decibels (db) that damages the inner ear.
  • the hearing loss is caused by an ototoxic drug that effects the auditory portion of the inner ear, particularly inner ear hair cells.
  • Incorporated herein by reference are chapters 196, 197, 198 and 199 of The Merck Manual of Diagnosis and Therapy, 14th Edition, (1982), Merck Sharp & Dome Research Laboratories, N.J. and corresponding chapters in the most recent 16th edition, including Chapters 207 and 210) relating to description and diagnosis of hearing and balance impairments.
  • One embodiment of the invention is a method for treating a hearing disorder or impairment wherein the ototoxicity results from administration of a therapeutically effective amount of an ototoxic pharmaceutical drug.
  • Typical ototoxic drugs are chemotherapeutic agents, e.g. antineoplastic agents, and antibiotics.
  • Other possible candidates include loop-diuretics, quinines or a quinine-like compound, and salicylate or salicylate-like compounds.
  • Ototoxic aminoglycoside antibiotics include but are not limited to neomycin, paromomycin, ribostamycin, lividomycin, kanamycin, amikacin, tobramycin, viomycin, gentamicin, sisomicin, netilmicin, streptomycin, dibekacin, fortimicin, and dihydrostreptomycin, or combinations thereof.
  • Particular antibiotics include neomycin B, kanamycin A, kanamycin B, gentamicin C1, gentamicin C1a, and gentamicin C2.
  • compositions of the present invention are also effective when the ototoxic compound is a antineoplastic agent such as vincristine, vinblastine, cisplatin and cisplatin-like compounds and taxol and taxol-like compounds.
  • a antineoplastic agent such as vincristine, vinblastine, cisplatin and cisplatin-like compounds and taxol and taxol-like compounds.
  • the methods and compositions of the present invention are also effective in the treatment of acoustic trauma or mechanical trauma, preferably acoustic or mechanical trauma that leads to inner ear hair cell loss.
  • Acoustic trauma to be treated in the present invention may be caused by a single exposure to an extremely loud sound, or following long-term exposure to everyday loud sounds above 85 decibels.
  • Mechanical inner ear trauma to be treated in the present invention is for example the inner ear trauma following an operation of electronic device insertion in the inner ear.
  • the compositions of the present invention prevent or minimize the damage to inner ear hair cells associated with the operation.
  • composition of the invention is co-administered with an ototoxin.
  • an improved method for treatment of infection of a mammal by administration of an aminoglycoside antibiotic, the improvement comprising administering a therapeutically effective amount of one or more compounds (particularly novel siRNAs) which down-regulate expression of the pro-apoptotic genes, to the subject in need of such treatment to reduce or prevent ototoxin-induced hearing impairment associated with the antibiotic.
  • the compounds which down-regulate expression of the pro-apoptotic genes particularly novel siRNAs are preferably administered locally within the inner ear.
  • an improved method for treatment of cancer in a mammal by administration of a chemotherapeutic compound comprises administering a therapeutically effective amount of a composition of the invention to the subject in need of such treatment to reduce or prevent ototoxin-induced hearing impairment associated with the chemotherapeutic drug.
  • the compounds which reduce or prevent the ototoxin-induced hearing impairment, eg. the novel siRNAs inter alia are preferably administered locally within the inner ear.
  • the methods of treatment are applied to treatment of hearing loss resulting from the administration of a chemotherapeutic agent in order to treat its ototoxic side-effect.
  • Ototoxic chemotherapeutic agents amenable to the methods of the invention include, but are not limited to an antineoplastic agent, including cisplatin or cisplatin-like compounds, taxol or taxol-like compounds, and other chemotherapeutic agents believed to cause ototoxin-induced hearing impairments, e.g., vincristine, an antineoplastic drug used to treat hematological malignancies and sarcomas.
  • Cisplatin-like compounds include carboplatin (Paraplatin®), tetraplatin, oxaliplatin, aroplatin and transplatin inter alia.
  • the methods of the invention are applied to hearing impairments resulting from the administration of quinine and its synthetic substitutes, typically used in the treatment of malaria, to treat its ototoxic side-effect.
  • the methods of the invention are applied to hearing impairments resulting from administration of a diuretic to treat its ototoxic side-effect.
  • Diuretics particularly “loop” diuretics, i.e. those that act primarily in the Loop of Henle, are candidate ototoxins.
  • Illustrative examples, not limiting to the invention method, include furosemide, ethacrylic acid, and mercurials.
  • Diuretics are typically used to prevent or eliminate edema.
  • Diuretics are also used in nonedematous states for example hypertension, hypercalcemia, idiopathic hypercalciuria, and nephrogenic diabetes insipidus.
  • the compounds of the invention are used for treating acute renal failure, in particular acute renal failure due to ischemia in post surgical patients, and acute renal failure due to chemotherapy treatment such as cisplatin administration or sepsis-associated acute renal failure.
  • a preferred use of the compounds of the invention is for the prevention of acute renal failure in high-risk patients undergoing major cardiac surgery or vascular surgery.
  • the patients at high-risk of developing acute renal failure can be identified using various scoring methods such as the Cleveland Clinic algorithm or that developed by US Academic Hospitals (QMMI) and by Veterans' Administration (CICSS).
  • Other preferred uses of the compounds of the invention are for the prevention of ischemic acute renal failure in kidney transplant patients or for the prevention of toxic acute renal failure in patients receiving chemotherapy.
  • the compounds of the invention are used for treating glaucoma.
  • Main types of glaucoma are primary open angle glaucoma (POAG), angle closure glaucoma, normal tension glaucoma and pediatric glaucoma. These are marked by an increase of intraocular pressure (IOP), or pressure inside the eye. When optic nerve damage has occurred despite a normal IOP, this is called normal tension glaucoma.
  • Secondary glaucoma refers to any case in which another disease causes or contributes to increased eye pressure, resulting in optic nerve damage and vision loss.
  • the compounds of the invention are used for treating or preventing the damage caused by nephrotoxins such as diuretics, ⁇ -blockers, vasodilator agents, ACE inhibitors, cyclosporin, aminoglycoside antibiotics (e.g.
  • gentamicin amphotericin B
  • cisplatin e.g aspirin, ibuprofen, diclofenac
  • radiocontrast media immunoglobulins
  • mannitol e.g aspirin, ibuprofen, diclofenac
  • NSAIDs eg aspirin, ibuprofen, diclofenac
  • cyclophosphamide methotrexate
  • amciclovir polyethylene glycol
  • ⁇ -lactam antibiotics vancomycin, rifampicin, sulphonamides, ciprofloxacin
  • ranitidine cimetidine, furosemide, thiazides
  • phenyloin penicillamine
  • lithium salts fluoride, demeclocycline, foscamet, aristolochic acid.
  • the compounds of the invention are used for treating or preventing the damage caused by spinal-cord injury especially spinal cord trauma caused by motor vehicle accidents, falls, sports injuries, industrial accidents, gunshot wounds, spinal cord trauma caused by spine weakening (such as from rheumatoid arthritis or osteoporosis) or if the spinal canal protecting the spinal cord has become too narrow (spinal stenosis) due to the normal aging process, direct damage that occur when the spinal cord is pulled, pressed sideways, or compressed, damage to the spinal-cord following bleeding, fluid accumulation, and swelling inside the spinal cord or outside the spinal cord (but within the spinal canal).
  • the compounds of the invention are also used for treating or preventing the damage caused by spinal-cord injury due to disease such as polio or spina bifida.
  • the compounds and methods of the invention are useful for treating or preventing the incidence or severity of acute lung injury, in particular conditions which result from ischemic/reperfusion injury or oxidative stress.
  • acute respiratory distress syndrome due to coronavirus infection or endotoxins, severe acute respiratory syndrome (SARS), ischemia reperfusion injury associated with lung transplantation and other acute lung injuries.
  • SARS severe acute respiratory syndrome
  • the compounds and methods of the invention are useful for treating or preventing damage following organ transplantation including lung, liver, heart, bone pancreas, intestine, skin, blood vessels, heart valve, bone and kidney transplantation.
  • organ transplant is meant to encompass transplant of any one or more of the following organs including, inter alia, lung, kidney, heart, skin, vein, bone, cartilage, liver transplantation. Although a xenotransplant can be contemplated in certain situations, an allotransplant is usually preferable.
  • An autograft can be considered for bone marrow, skin, bone, cartilage and or blood vessel transplantation.
  • siRNA compounds of the present invention are particularly useful in treating a subject experiencing the adverse effects of organ transplant, including ameliorating, treating or preventing perfusion injury.
  • the present invention relates to a method of treating an organ donor or an organ recipient comprising the step of administering to the organ donor or organ recipient a therapeutically effective amount of a compound according to the present invention.
  • the invention further relates to a method for preserving an organ comprising contacting the organ with an effective amount of compound of the present invention. Also provided is a method for reducing or preventing injury (in particular reperfusion injury) of an organ during surgery and/or following removal of the organ from a subject comprising placing the organ in an organ preserving solution wherein the solution comprises a compound according to the present invention.
  • the compounds and methods of the invention are useful for treating or preventing the incidence or severity of other diseases and conditions in a patient.
  • diseases and conditions include stroke and stroke-like situations (e.g. cerebral, renal, cardiac failure), neuronal cell death, brain injuries with or without reperfusion issues, chronic degenerative diseases e.g. neurodegenerative disease including Alzheimer's disease, Huntington's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, spinobulbar atrophy, prion disease, and apoptosis resulting from traumatic brain injury (TBI).
  • TBI traumatic brain injury
  • the compounds and methods of the invention are directed to providing neuroprotection, or to provide cerebroprotection, or to prevent and/or treat cytotoxic T cell and natural killer cell-mediated apoptosis associated with autoimmune disease and transplant rejection, to prevent cell death of cardiac cells including heart failure, cardiomyopathy, viral infection or bacterial infection of heart, myocardial ischemia, myocardial infarct, and myocardial ischemia, coronary artery by-pass graft, to prevent and/or treat mitochondrial drug toxicity e.g.
  • hair loss due-to male-pattern baldness, or hair loss due to radiation, chemotherapy or emotional stress or to treat or ameliorate skin damage whereby the skin damage may be due to exposure to high levels of radiation, heat, chemicals, sun, or to burns and autoimmune diseases), or to prevent cell death of bone marrow cells in myelodysplastic syndromes (MDS), to treat pancreatitis, to treat rheumatoid arthritis, psoriasis, glomerulonephritis, atherosclerosis, and graft versus host disease (GVHD), or to treat retinal pericyte apoptosis, retinal damages resulting from ischemia, diabetic retinopathy, or to treat any disease states associated with an increase of apoptotic cell death.
  • MDS myelodysplastic syndromes
  • GVHD graft versus host disease
  • siRNA compounds directed to two pro-apoptotic genes are combined in order to obtain a synergistic therapeutic effect.
  • siRNA compounds directed to RhoA whose mRNA sequence is set forth as SEQ ID NO:46
  • siRNA compounds directed to Casp2 whose mRNA sequence is set forth as SEQ ID NOS: 10-11).
  • the present invention also provides for a process of preparing a pharmaceutical composition, which comprises:
  • the present invention also provides for a process of preparing a pharmaceutical composition, which comprises admixing one or more compounds of the present invention with a pharmaceutically acceptable carrier.
  • the compound used in the preparation of a pharmaceutical composition is admixed with a carrier in a pharmaceutically effective dose.
  • the compound of the present invention is conjugated to a steroid or to a lipid or to another suitable molecule e.g. to cholesterol.
  • Modifications or analogs of nucleotides can be introduced to improve the therapeutic properties of the nucleotides. Improved properties include increased nuclease resistance and/or increased ability to permeate cell membranes.
  • the present invention also includes all analogs of, or modifications to, a oligonucleotide of the invention that does not substantially affect the function of the polynucleotide or oligonucleotide.
  • such modification is related to the base moiety of the nucleotide, to the sugar moiety of the nucleotide and/or to the phosphate moiety of the nucleotide.
  • the modification is a modification of the phosphate moiety, whereby the modified phosphate moiety is selected from the group comprising phosphothioate.
  • the compounds of the present invention can be synthesized by any of the methods that are well-known in the art for synthesis of ribonucleic (or deoxyribonucleic) oligonucleotides. Such synthesis is, among others, described in Beaucage and Iyer, Tetrahedron 1992; 48:2223-2311; Beaucage and Iyer, Tetrahedron 1993; 49: 6123-6194 and Caruthers, et. al., Methods Enzymol. 1987; 154: 287-313; the synthesis of thiolates is, among others, described in Eckstein, Annu. Rev. Biochem.
  • oligonucleotides of the present invention can be synthesized separately and joined together post-synthetically, for example, by ligation (Moore et al., 1992, Science 256, 9923; Draper et al., International Patent Publication No. WO 93/23569; Shabarova et al., 1991, NAR 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204), or by hybridization following synthesis and/or deprotection.
  • oligonucleotides are prepared according to the sequences disclosed herein. Overlapping pairs of chemically synthesized fragments can be ligated using methods well known in the art (e.g., see U.S. Pat. No. 6,121,426). The strands are synthesized separately and then are annealed to each other in the tube. Then, the double-stranded siRNAs are separated from the single-stranded oligonucleotides that were not annealed (e.g. because of the excess of one of them) by HPLC. In relation to the siRNAs or siRNA fragments of the present invention, two or more such sequences can be synthesized and linked together for use in the present invention.
  • the compounds of the invention can also be synthesized via tandem synthesis methodology, as described for example in US Patent Publication No. US 2004/0019001 (McSwiggen), wherein both siRNA strands are synthesized as a single contiguous oligonucleotide fragment or strand separated by a cleavable linker which is subsequently cleaved to provide separate siRNA fragments or strands that hybridize and permit purification of the siRNA duplex.
  • the linker can be a polynucleotide linker or a non-nucleotide linker.
  • the present invention further provides for a pharmaceutical composition
  • a pharmaceutical composition comprising two or more siRNA molecules for the treatment of any of the diseases and conditions mentioned herein, whereby said two molecules may be physically mixed together in the pharmaceutical composition in amounts which generate equal or otherwise beneficial activity, or may be covalently or non-covalently bound, or joined together by a nucleic acid linker of a length ranging from 2-100, preferably 2-50 or 2-30 nucleotides.
  • the siRNA molecules are comprised of a double-stranded nucleic acid structure as described herein, wherein the two siRNA sequences are selected from the nucleic acids set forth in Table B.
  • the siRNA molecules may be covalently or non-covalently bound or joined by a linker to form a tandem siRNA compound.
  • tandem siRNA compounds comprising two siRNA sequences are typically of 38-150 nucleotides in length, more preferably 38 or 40-60 nucleotides in length, and longer accordingly if more than two siRNA sequences are included in the tandem molecule.
  • a longer tandem compound comprised of two or more longer sequences which encode siRNA produced via internal cellular processing, e.g., long dsRNAs, is also envisaged, as is a tandem molecule encoding two or more shRNAs.
  • tandem molecules are also considered to be a part of the present invention.
  • a tandem compound comprising two or more siRNAs sequences of the invention is envisaged.
  • the tandem comprises RhoA siRNA covalently linked to one or more of the other siRNAs of the invention.
  • the tandem compound may comprise a sequence comprising siRNA to RhoA and a sequence comprising siRNA to Casp2.
  • RhoA is a small GTPase that when activated inhibits neurite outgrowth and its inhibition is relevant for spinal cord injury.
  • a tandem compound for this indication can comprise RhoA siRNA sequence and one or more siRNA sequences to anti-apoptotic siRNAs of the invention. The latter will protect, and siRNA to RhoA will promote regeneration, and so a combined or even synergistic effect is produced.
  • siRNA molecules that target the pro-apoptotic genes of the invention may be the main active component in a pharmaceutical composition, or may be one active component of a pharmaceutical composition containing two or more siRNAs (or molecules which encode or endogenously produce two or more siRNAs, be it a mixture of molecules or one or more tandem molecules which encode two or more siRNAs), said pharmaceutical composition further being comprised of one or more additional siRNA molecule which targets one or more additional gene. Simultaneous inhibition of said additional gene(s) will likely have an additive or synergistic effect for treatment of the diseases disclosed herein.
  • the pro-apoptotic siRNA disclosed herein or any nucleic acid molecule comprising or encoding such siRNA can be linked or bound (covalently or non-covalently) to antibodies (including aptamer molecules) against cell surface internalizable molecules expressed on the target cells, in order to achieve enhanced targeting for treatment of the diseases disclosed herein.
  • antibodies including aptamer molecules
  • anti-Fas antibody preferably a neutralizing antibody
  • an aptamer which can act like a ligand/antibody may be combined (covalently or non-covalently) with any pro-apoptotic siRNA.
  • the compounds of the present invention can be delivered either directly or with viral or non-viral vectors.
  • the sequences When delivered directly the sequences are generally rendered nuclease resistant.
  • the sequences can be incorporated into expression cassettes or constructs such that the sequence is expressed in the cell as discussed herein below.
  • the construct contains the proper regulatory sequence or promoter to allow the sequence to be expressed in the targeted cell.
  • Vectors optionally used for delivery of the compounds of the present invention are commercially available, and may be modified for the purpose of delivery of the compounds of the present invention by methods known to one of skill in the art.
  • a long oligonucleotide (typically 25-500 nucleotides in length) comprising one or more stem and loop structures, where stem regions comprise the sequences of the oligonucleotides of the invention, may be delivered in a carrier, preferably a pharmaceutically acceptable carrier, and may be processed intracellularly by endogenous cellular complexes (e.g. by DROSHA and DICER as described above) to produce one or more smaller double stranded oligonucleotides (siRNAs) which are oligonucleotides of the invention.
  • This oligonucleotide can be termed a tandem shRNA construct.
  • this long oligonucleotide is a single stranded oligonucleotide comprising one or more stem and loop structures, wherein each stem region comprises a sense and corresponding antisense siRNA sequence of the pro-apoptotic genes of the invention.
  • this oligonucleotide comprises sense and antisense siRNA sequences as depicted in Table B, set forth in SEQ ID NOS:277-50970 and 50993-68654.
  • nucleotide/oligonucleotide may be employed with the present invention, provided that said analogue or modification does not substantially affect the function of the nucleotide/oligonucleotide.
  • the nucleotides can be selected from naturally occurring or synthetic modified bases. Naturally occurring bases include adenine, guanine, cytosine, thymine and uracil.
  • Modified bases of nucleotides include inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thioalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thioalkyl guanines, 8-hydroxyl guanine and other substituted guanines, other aza and deaza adenines, other aza and deaza guanines, 5-trifluoromethyl uracil and 5-trifluoro cyto
  • analogues of polynucleotides can be prepared wherein the structure of one or more nucleotide is fundamentally altered and better suited as therapeutic or experimental reagents.
  • An example of a nucleotide analogue is a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in DNA (or RNA is replaced with a polyamide backbone which is similar to that found in peptides.
  • PNA analogues have been shown to be resistant to enzymatic degradation and to have extended stability in vivo and in vitro.
  • oligonucleotides include polymer backbones, cyclic backbones, acyclic backbones, thiophosphate-D-ribose backbones, triester backbones, thiolate backbones, 2′-5′ bridged backbone, artificial nucleic acids, morpholino nucleic acids, locked nucleic acid (LNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), arabinoside, and mirror nucleoside (for example, beta-L-deoxynucleoside instead of beta-D-deoxynucleoside).
  • LNA locked nucleic acid
  • GNA glycol nucleic acid
  • TAA threose nucleic acid
  • arabinoside arabinoside
  • mirror nucleoside for example, beta-L-deoxynucleoside instead of beta-D-deoxynucleoside.
  • siRNA compounds comprising LNA nucleotides are disclosed in El
  • the compounds of the present invention can be synthesized using one or more inverted nucleotides, for example inverted thymidine or inverted adenine (see, for example, Takei, et al., 2002, JBC 277(26):23800-06).
  • inverted nucleotides for example inverted thymidine or inverted adenine
  • a “mirror” nucleotide is a nucleotide with reversed chirality to the naturally occurring or commonly employed nucleotide, i.e., a mirror image (L-nucleotide) of the naturally occurring (D-nucleotide).
  • the nucleotide can be a ribonucleotide or a deoxyribonucleotide and my further comprise at least one sugar, base and or backbone modification.
  • U.S. Pat. No. 6,602,858 discloses nucleic acid catalysts comprising at least one L-nucleotide substitution.
  • inhibitors of the present invention are preferably siRNA molecules
  • other inhibitors contemplated to be used in the methods of the invention to inhibit a pro-apoptotic gene and to treat the diseases and conditions described herein are inter alia antibodies, preferably neutralizing antibodies or fragments thereof, including single chain antibodies, antisense oligonucleotides, antisense DNA or RNA molecules, ribozymes, proteins, polypeptides and peptides including peptidomimetics and dominant negatives, and also expression vectors expressing all the above.
  • Additional inhibitors may be small chemical molecules, which generally have a molecular weight of less than 2000 daltons, more preferably less than 1000 daltons, even more preferably less than 500 daltons.
  • inhibitors may act as follows: small molecules may affect expression and/or activity; antibodies may affect activity; all kinds of antisense may affect the pro-apoptotic gene expression; and dominant negative polypeptides and peptidomimetics may affect activity; expression vectors may be used inter alia for delivery of antisense or dominant-negative polypeptides or antibodies.
  • antibody refers to IgG, IgM, IgD, IgA, and IgE antibody, inter alia.
  • the definition includes polyclonal antibodies or monoclonal antibodies. This term refers to whole antibodies or fragments of antibodies comprising an antigen-binding domain, e.g. antibodies without the Fc portion, single chain antibodies, miniantibodies, fragments consisting of essentially only the variable, antigen-binding domain of the antibody, etc.
  • antibody may also refer to antibodies against polynucleotide sequences obtained by cDNA vaccination. The term also encompasses antibody fragments which retain the ability to selectively bind with their antigen or receptor and are exemplified as follows, inter alia:
  • genes of the present invention that are preferably inhibited using specific antibodies for the treatment of a desired disease are reticulon 4 receptor (RTN4R) and annexin A2 (ANXA2).
  • AS antisense
  • AS antisense fragment
  • An AS polynucleotide is a polynucleotide which comprises consecutive nucleotides having a sequence of sufficient length and homology to a sequence present within the sequence of the target gene to permit hybridization of the AS to the gene.
  • AS oligonucleotide sequences may be short sequences of DNA, typically 15-30 mer but may be as small as 7 mer (Wagner et al, 1996 Nat Biotechnol. 14(7):840-4), designed to complement a target mRNA of interest and form an RNA:AS duplex. This duplex formation can prevent processing, splicing, transport or translation of the relevant mRNA. Moreover, certain AS nucleotide sequences can elicit cellular RNase H activity when hybridized with their target mRNA, resulting in mRNA degradation (Calabretta et al, 1996 Semin Oncol. 23(1):78-87).
  • RNase H will cleave the RNA component of the duplex and can potentially release the AS to further hybridize with additional molecules of the target RNA.
  • An additional mode of action results from the interaction of AS with genomic DNA to form a triple helix which can be transcriptionally inactive.
  • the sequence target segment for the antisense oligonucleotide is selected such that the sequence exhibits suitable energy related characteristics important for oligonucleotide duplex formation with their complementary templates, and shows a low potential for self-dimerization or self-complementation (Anazodo et al., 1996 BBRC. 229(1):305-9).
  • the computer program OLIGO Primary Analysis Software, Version 3.4
  • the program allows the determination of a qualitative estimation of these two parameters (potential self-dimer formation and self-complimentary) and provides an indication of “no potential” or “some potential” or “essentially complete potential”.
  • target segments are generally selected that have estimates of no potential in these parameters.
  • segments can be used that have “some potential” in one of the categories.
  • a balance of the parameters is used in the selection as is known in the art.
  • the oligonucleotides are also selected as needed so that analogue substitution do not substantially affect function.
  • Phosphorothioate antisense oligonucleotides do not normally show significant toxicity at concentrations that are effective and exhibit sufficient pharmacodynamic half-lives in animals (Agarwal et al., 1996) and are nuclease resistant. Antisense induced loss-of-function phenotypes related with cellular development were shown for a variety of different genes including integrin (Galileo et al., Neuron. 1992 9(6):1117-31.) and for the N-myc protein (Rosolen et al., 1990 Prog Clin Biol Res. 366:29-36).
  • bFGF basic fibroblast growth factor
  • a “ribozyme” is an RNA molecule that possesses RNA catalytic ability (see Cech Biochem Soc Trans. 2002 November; 30(Pt 6):1162-6, for review) and cleaves a specific site in a target RNA.
  • ribozymes which cleave mRNA may be utilized as inhibitors. This may be necessary in cases where antisense therapy is limited by stoichiometric considerations (Sarver et al., 1990, Gene Regulation and Aids, pp. 305-325). Ribozymes can then be used that will target the a gene associated with a bone marrow disease.
  • RNA molecules that are cleaved by a ribozyme is greater than the number predicted by stochiochemistry (Hampel and Tritz, Biochem. 1989, 28(12):4929-33; Uhlenbeck, Nature. 1987 328(6131):596-600).
  • Ribozymes catalyze the phosphodiester bond cleavage of RNA.
  • ribozyme structural families include Group I introns, RNase P, the hepatitis delta virus ribozyme, hammerhead ribozymes and the hairpin ribozyme originally derived from the negative strand of the tobacco ringspot virus satellite RNA (sTRSV) (Sullivan, 1994; U.S. Pat. No. 5,225,347).
  • the latter two families are derived from viroids and virusoids, in which the ribozyme is believed to separate monomers from oligomers created during rolling circle replication (Symons, 1989 and 1992).
  • ribozyme motifs are most commonly adapted for trans-cleavage of mRNAs for gene therapy (Sullivan, 1994).
  • the ribozyme has a length of from about 30-100 nucleotides. Delivery of ribozymes is similar to that of AS fragments and/or siRNA molecules.
  • Some of the compounds and compositions of the present invention may be used in a screening assay for identifying and isolating compounds that modulate the activity of a pro-apoptotic gene, in particular compounds that modulate a disorder accompanied by an elevated level of the pro-apoptotic genes of the invention.
  • the compounds to be screened comprise inter alia substances such as small chemical molecules and antisense oligonucleotides.
  • the inhibitory activity of the compounds of the present invention on pro-apoptotic genes or binding of the compounds of the present invention to pro-apoptotic genes may be used to determine the interaction of an additional compound with the pro-apoptotic polypeptide, e.g., if the additional compound competes with the oligonucleotides of the present invention for inhibition of a pro-apoptotic gene, or if the additional compound rescues said inhibition.
  • the inhibition or activation can be tested by various means, such as, inter alia, assaying for the product of the activity of the pro-apoptotic polypeptide or displacement of binding compound from the pro-apoptotic polypeptide in radioactive or fluorescent competition assays.
  • PCR Polymerase chain reaction
  • In situ (In cell) PCR in combination with Flow Cytometry can be used for detection of cells containing specific DNA and mRNA sequences (Testoni et al., 1996, Blood 87:3822.) Methods of performing RT-PCR are also well known in the art.
  • test cells HeLa cells or 293T cells for siRNA targeting the human gene and NRK52 cells or NMUMG cells for siRNA targeting the rat/mouse gene
  • test cells were seeded per well in 6 wells plate (70-80% confluent).
  • siRNA oligos As positive control for cells transfection PTEN-Cy3 labeled siRNA oligos were used. As negative control for siRNA activity GFP siRNA oligos were used.
  • siRNAs used in the in vitro experiments described in Example 1 were 19-mers or 23-mers, having alternating ribonucleotides modified in both the antisense and the sense strands of the compound.
  • the modification was such that a 2′-O-methyl (Me) group was present on the first, third, fifth, seventh, ninth, eleventh, thirteenth, fifteenth, seventeenth and nineteenth nucleotide of the antisense strand, whereby the very same modification, i.e. a 2′-O-Me group, was present at the second, fourth, sixth, eighth, tenth, twelfth, fourteenth, sixteenth and eighteenth nucleotide of the sense strand.
  • These particular siRNA compounds were also blunt ended and were non-phosphorylated at the termini; however, comparative experiments have shown that siRNAs phosphorylated at the 3′-termini have similar activity.
  • the percent of inhibition of gene expression using specific siRNAs was determined using qPCR analysis of target gene in cells expressing the endogenous gene.
  • the data in Tables C1, C2 and C3 below demonstrate the percent of residual expression of the target gene in cells following treatment with specific siRNA molecules (Tables C1 and C3: 19-mer siRNA compounds; Table C2: 23-mer siRNA compounds).
  • the siRNAs having specific sequences that were selected for in vitro testing were specific for both human and the rat/rabbit genes. Similar results of reduced expression of specific genes are obtained with other siRNAs, the sequences of which are listed in Table B.
  • ARF is a clinical syndrome characterized by rapid deterioration of renal function that occurs within days.
  • the acute kidney injury may be the result of renal ischemia-reperfusion injury such as renal ischemia-reperfusion injury in patients undergoing major surgery such as major cardiac surgery.
  • the principal feature of ARF is an abrupt decline in glomerular filtration rate (GFR), resulting in the retention of nitrogenous wastes (urea, creatinine).
  • GFR glomerular filtration rate
  • urea nitrogenous wastes
  • Ischemia-reperfusion injury was induced in rats following 45 minutes bilateral kidney arterial clamp and subsequent release of the clamp to allow 24 hours of reperfusion.
  • a dose of 12 mg/kg of the following siRNA compounds was injected into the jugular vein of individual experimental animals 30 minutes prior to and 4 hours following the clamp.
  • ARF progression was monitored by measurement of serum creatinine levels before (baseline) and 24 hrs post surgery.
  • SEQ ID NO:213 Rac1_2: Sense sequence: GAGUCCUGCAUCAUUUGAA,; SEQ ID NO:214 Antisense sequence: UUCAAAUGAUGCAGGACUC,.
  • SEQ ID NO:99 TP53BP2_2 Sense sequence: CACCCAGAGAACAUUUAUU,; SEQ ID NO:100 Antisense sequence: AAUAAAUGUUCUCUGGGUG,.
  • Casp2_4 Sense sequence: GCCAGAAUGUGGAACUCCU,; SEQ ID NO:140 Antisense sequence: AGGAGUUCCACAUUCUGGC,.
  • the rats were perfused via an indwelling femoral line with warm PBS followed by 4% paraformaldehyde.
  • the left kidneys were surgically removed and stored in 4% paraformaldehyde for subsequent histological analysis.
  • Acute renal failure is frequently defined as an acute increase of the serum creatinine level from baseline.
  • An increase of at least 0.5 mg per dL or 44.2 ⁇ mol per L of serum creatinine is considered as an indication for acute renal failure.
  • Serum creatinine was measured at time zero before the surgery and at 24 hours post ARF surgery. Tables D1-D3 below demonstrate the results obtained in the ARF model in rats.
  • RAC1, TP53BP2 and Casp2 siRNA compounds reduced creatinine levels following ischemia-reperfusion induced ARF in an experimental rat model.
  • Values represent creatinine levels [in mg/dL] prior to (Baseline) and 24 hours following ischemia-reperfusion induced ARF in placebo group (PBS), and in RAC1 — 2 siRNA treated rats 30 min prior to the ischemic injury ( ⁇ 30′) and in RAC1 — 2 siRNA treated rats 4 hours post ischemic injury (+4 h).
  • Table D2 Treatment with TP53BP2 — 2 siRNA (SEQ ID NOS:99-100). Values represent creatinine levels 24 hours following ischemia-reperfusion induced ARF in placebo group (PBS), in non-relevant GFP siRNA treated rats 4 hours post ischemic injury (GFP siRNA (+4 h)), in TP53BP2 — 2 siRNA treated rats 30 min prior to the ischemic injury (TP53BP2 — 2 siRNA ( ⁇ 30′)) and in TP53BP2 — 2 siRNA treated rats 4 hours post ischemic injury (TP53BP2 — 2 siRNA (+4 h)).
  • Table D3 Treatment with Casp2 — 4 siRNA (SEQ ID NO:139-140). Values represent creatinine levels prior to (baseline) and 24 hours following ischemia-reperfusion induced ARF in placebo group (PBS), in Casp2 — 4 siRNA treated rats 30 min prior to the ischemic injury ( ⁇ 30′) and in Casp2 — 4 siRNA treated rats 4 hours post ischemic injury (+4 h).
  • siRNAs directed to particular genes TP53BP2, LRDD, CYBA, ATF3, CASP2, HRK, CIQBP, BNIP3, MAPK8, MAPK14, RAC1, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, CX43, TYROBP, CTGF, and SPP1.
  • Pressure sores or pressure ulcers including diabetic ulcers are areas of damaged skin and tissue that develop when sustained pressure (usually from a bed or wheelchair) cuts off circulation to vulnerable parts of the body, especially the skin on the buttocks, hips and heels.
  • the lack of adequate blood flow leads to ischemic necrosis and ulceration of the affected tissue.
  • Pressure sores occur most often in patients with diminished or absent sensation or who are debilitated, emaciated, paralyzed, or long bedridden. Tissues over the sacrum, ischia, greater trochanters, external malleoli, and heels are especially susceptible; other sites may be involved depending on the patient's situation.
  • siRNA according to Table B and specifically compounds directed to genes CIQBP, RAC1, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, CX43, or TYROBP are tested in animal models where it is shown that these siRNA compounds treat and prevent pressure sores and ulcers.
  • COPD Chronic Obstructive Pulmonary Disease
  • COPD chronic obstructive pulmonary disease
  • emphysema a permanent destruction of peripheral air spaces distal to terminal bronchioles.
  • Emphysema is also characterized by accumulation of inflammatory cells such as macrophages and neutrophils in bronchioles and alveolar structures.
  • Emphysema and chronic bronchitis may occur as part of COPD or independently.
  • siRNA according to Table B and in particular to siRNA to genes CIQBP, BNIP3, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, CX43, TYROBP, CTGF, and DUOX1 are tested in these animal models, which show that these siRNA compounds may treat and/or prevent emphysema, chronic bronchitis and COPD.
  • Traumatic injury can be due to automobile accidents, falls, gunshot, diving accidents inter alia, and diseases which can affect the spinal cord include polio, spina bifida, tumors and Friedreich's ataxia.
  • Cy3 labeled siRNA (delivered by injection into the injured cord) in different types of cells was examined following spinal cord contusion in 18 rats and in uninjured rats (9 rats). Sagittal cryosections were produced and immunostaining using four different groups of antibodies was performed in order to determine whether uptake has occurred in neurons, astroglia, oligodendroglia and/or macrophages/microglia.
  • Markers for neurons were NeuN, or GAP43; markers for astroglia and potential neural stem cells were GFAP, nestin or vimentin; markers for oligodendroglia were NG2 or APC; markers for macrophages/microglia were ED1 or Iba-1 (Hasegawa et al., 2005. Exp Neurol 193 394-410).
  • RhoA — 4 siRNA Prior to injury, three point injections of RhoA — 4 siRNA (Sense sequence: GCCACUUAAUGUAUGUUAC, SEQ ID NO:235; Antisense sequence: GUAACAUACAUUAAGUGGC, SEQ ID NO:236) at the concentration of 1 ⁇ g/ ⁇ l were performed at the injury epicenter 2 mm rostral and caudal to the epicenter (total dose of 30 ⁇ g).
  • GFP siRNA was injected in additional five rats as a control. Each injection was conducted slowly during a period of 10 min into dorsal column ( ⁇ 1 mm depth) of T10 using a Hamilton syringe. Following injections, muscles and skin were closed separately. Cefazolin (25 mg/kg) was administered for 7 days after surgery. The behavioral assessment of the recovery following the spinal cord contusion was preformed using an open field locomotor test as described by Basso et al (the BBB locomotor rating scale).
  • Table D4 below demonstrates the results obtained in the open field locomotor test following spinal-cord injury in rats.
  • RhoA siRNA compounds protect against spinal-cord injury in an experimental rat model as revealed by significantly higher BBB locomotor score up to 6 weeks post injury in the RhoA siRNA treated rats.
  • Table D4 Treatment with RhoA — 4 siRNA (SEQ ID NOS:235-236). Values represent mean BBB locomotor score following spinal-cord injury in placebo group (GFP siRNA) and in RhoA — 4 siRNA treated group.
  • siRNA compounds according to Table B and in particular siRNA directed to genes LRDD, CYBA, ATF3, CASP2, HRK, CIQBP, BNIP3, MAPK8, MAPK14, RAC1, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, CX43, TYROBP, CTGF, and RHOA are tested in this animal model, which show that these siRNA compounds promote functional recovery following spinal cord injury and thus may be used to treat spinal cord injury.
  • testing the active inhibitors of the invention for treating or preventing glaucoma is done in the animal model for example as described by Pease et al., J. Glaucoma, 2006, 15(6):512-9 (Manometric calibration and comparison of TonoLab and TonoPen tonometers in rats with experimental glaucoma and in normal mice).
  • siRNA according to Table B in particular to genes TP53BP2, LRDD, CYBA, ATF3, CASP2, HRK, BNIP3, MAPK8, MAPK14, RAC1, and RHOA are tested in this animal model which show that these siRNA compounds treat and/or prevent glaucoma.
  • testing the active inhibitors of the invention for treating or preventing ischemia/reperfusion injury or hypoxic injury following lung transplantation is done in one or more of the experimental animal models, for example as described by Mizobuchi et al., (2004. J. Heart Lung Transplant, 23:889-93); Huang, et al., (1995. J. Heart Lung Transplant. 14: S49); Matsumura, et al., (1995. Transplantation 59: 1509-1517); Wilkes, et al., (1999. Transplantation 67:890-896); Naka, et al., (1996. Circulation Research, 79: 773-783).
  • siRNA according to Table B and in particular to TP53BP2, LRDD, CYBA, CASP2, BNIP3, RAC1, and DUOX1 are tested in these animal models, which show that these siRNA compounds treat and/or prevent ischemia-reperfusion injury following lung transplantation and thus may be used in conjunction with transplant surgery.
  • siRNA active inhibitors of the invention
  • testing the active inhibitors of the invention is done in the animal model as described by Chen, et al (J Biomed Sci. 2003; 10(6 Pt 1):588-92).
  • siRNA compounds according to Table B in particular to genes CYBA, HRK, BNIP3, MAPK8, MAPK14, RAC1, GSK3B, P2RX7, TRPM2, PARG, SPP1, and DUOX1 are tested in this animal model which shows that these siRNAs treat and/or prevent acute respiratory distress syndrome and thus may be used to treat this condition.
  • Cy3-PTEN siRNA (total of 0.3-0.4 ⁇ g) PBS was applied to the round window of chinchillas.
  • the Cy3-labelled cells within the treated cochlea were analyzed 24-48 hours post siRNA round window application after sacrifice of the chinchillas.
  • the pattern of labeling within the cochlea was similar following 24 h and 48 h and includes labeling in the basal turn of cochlea, in the middle turn of cochlea and in the apical turn of cochlea.
  • Application of Cy3-PTEN siRNA onto scala tympani revealed labelling mainly in the basal turn of the cochlea and the middle turn of the cochlea.
  • the Cy3 signal was persistence to up to 15 days after the application of the Cy3-PTEN siRNA. These results indicated for the first time that local application of siRNA molecules within the round window led to significant penetration of the siRNA molecules to the basal, middle and apical turns of the cochlea.
  • the siRNA compounds of the invention are tested in this animal model which shows that there is significant penetration of these siRNA compounds to the basal, middle and apical turns of the cochlea, and that these compounds may be used in the treatment of hearing loss.
  • Chinchilla model of carboplatin-induced or cisplatin-induced cochlea hair cell death Chinchillas are pre-treated by direct administration of specific siRNA in saline to the left ear of each animal. Saline is given to the right ear of each animal as placebo. Two days following the administration of the specific siRNA compounds of the invention, the animals are treated with carboplatin (75 mg/kg ip) or cisplatin (intraperitoneal infusion of 13 mg/kg over 30 minutes).
  • the % of dead cells of inner hair cells (IHC) and outer hair cells (OHC) is calculated in the left ear (siRNA treated) and in the right ear (saline treated). It is calculated that the % of dead cells of inner hair cells (IHC) and outer hair cells (OHC) is lower in the left ear (siRNA treated) than in the right ear (saline treated).
  • the activity of specific siRNA in an acoustic trauma model is studied in chinchilla.
  • the animals are exposed to an octave band of noise centered at 4 kHz for 2.5 h at 105 dB.
  • the left ear of the noise-exposed chinchillas is pre-treated (48 h before the acoustic trauma) with 30 ⁇ g of siRNA in ⁇ 10 ⁇ L of saline; the right ear is pre-treated with vehicle (saline).
  • CAP compound action potential
  • the CAP is recorded by placing an electrode near the base of the cochlea in order to detect the local field potential that is generated when a sound stimulus, such as click or tone burst, is abruptly turned on.
  • the functional status of each ear is assessed 2.5 weeks after the acoustic trauma.
  • the mean threshold of the compound action potential recorded from the round window is determined 2.5 weeks after the acoustic trauma in order to determine if the thresholds in the siRNA-treated ear are lower (better) than the untreated (saline) ear.
  • the amount of inner and outer hair cell loss is determined in the siRNA-treated and the control ear.
  • siRNA molecules according to Table B in particular to genes TP53BP2, LRDD, CYBA, ATF3, CASP2, NOX3, HRK, CIQBP, BNIP3, MAPK8, MAPK14, RAC1, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, CX43, TYROBP, and CTGF are tested in this animal model which shows that the thresholds in the siRNA-treated ear are lower (better) than in the untreated (saline) ear. In addition, the amount of inner and outer hair cell loss is lower in the siRNA-treated ear than in the control ear.
  • Collagen induced arthritis CIA in mice is described in Trentham et al. (1977. J. Exp. Med. 146: 857-868).
  • Adjuvant-induced arthritis AA is described in Kong et al., (1999. Nature, 402:304-308).
  • a menisectomy model is described in Han et al., (1999. Nagoya J Med Sci 62(3-4):115-26).
  • siRNA inhibitors such as siRNA to SSP1
  • siRNA compounds according to Table B in particular to SSP1 are tested in these animal models which show that these siRNAs treat and/or prevent OA and thus may be used to treat this condition.
  • siRNAs used in the in vivo experiments described herein were all 19-mers, having alternating ribonucleotides modified in both the antisense and the sense strands of the compound.
  • the modification was such that a 2′-O-methyl (Me) group was present on the first, third, fifth, seventh, ninth, eleventh, thirteenth, fifteenth, seventeenth and nineteenth nucleotide of the antisense strand, whereby the very same modification, i.e. a 2′-O-Me group, was present at the second, fourth, sixth, eighth, tenth, twelfth, fourteenth, sixteenth and eighteenth nucleotide of the sense strand.
  • These particular siRNA compounds were also blunt ended and were non-phosphorylated at the termini; however, comparative experiments have shown that siRNAs phosphorylated at the 3′-termini have similar activity.
  • the sequences of many potential siRNAs were generated.
  • some of the 23-mer oligomer sequences were generated by 5′ and/or 3′ extension of the 19-mer sequences.
  • the sequences that have been generated using this method are fully complementary to the corresponding mRNA sequence.
  • Table B shows siRNAs for the following pro-apoptotic genes: TP53BP2, LRDD, CYBA, ATF3, CASP2, NOX3, HRK, CIQBP, BNIP3, MAPK8, MAPK14, RAC1, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, CX43, TYROBP, CTGF, SPP1, RHOA, and DUOX1
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Publication number Priority date Publication date Assignee Title
US20090156524A1 (en) * 2006-10-25 2009-06-18 Elena Feinstein Novel siRNAS and methods of use thereof
US20100130595A1 (en) * 2008-08-25 2010-05-27 Dean Nicholas M Antisense oligonucleotides directed against connective tissue growth factor and uses thereof
US20100266574A1 (en) * 2005-06-10 2010-10-21 Orna Mor Oligoribonucleotides and Methods of Use Thereof for Treatment of Fibrotic Conditions and Other Diseases
US20100292301A1 (en) * 2007-02-28 2010-11-18 Elena Feinstein Novel sirna structures
US20110021420A1 (en) * 2003-01-10 2011-01-27 Imperial Innovations Limited Modification of feeding behaviour
US20110028531A1 (en) * 2008-03-20 2011-02-03 Elena Feinstein Novel sirna compounds for inhibiting rtp801
US20110034534A1 (en) * 2008-01-15 2011-02-10 Elena Feinstein siRNA compounds and methods of use thereof
US20110092575A1 (en) * 2008-06-06 2011-04-21 Assignment for Published Patent Application Gene Techno Science Co., Ltd. Sirna of human osteopontin
US20110105584A1 (en) * 2007-12-12 2011-05-05 Elena Feinstein Rtp80il sirna compounds and methods of use thereof
US20110112168A1 (en) * 2007-10-03 2011-05-12 Elena Feinstein Novel sirna structures
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US20110142917A1 (en) * 2008-06-06 2011-06-16 Egvenia Alpert Compositions and methods for treatment of ear disorders
US20110190380A1 (en) * 2008-10-23 2011-08-04 Elena Feinstein Methods for delivery of sirna to bone marrow cells and uses thereof
US20110230543A1 (en) * 2007-03-21 2011-09-22 Quark Pharmaceuticals, Inc. Oligoribonucleotide inhibitors of nrf2 and methods of use thereof for treatment of cancer
WO2012118911A1 (en) 2011-03-03 2012-09-07 Quark Pharmaceuticals, Inc. Oligonucleotide modulators of the toll-like receptor pathway
WO2012110457A3 (en) * 2011-02-14 2013-03-28 Santaris Pharma A/S Compounds for the modulation of osteopontin expression
WO2013067076A2 (en) 2011-11-03 2013-05-10 Quark Pharmaceuticals, Inc. Methods and compositions for neuroprotection
WO2013070821A1 (en) 2011-11-08 2013-05-16 Quark Pharmaceuticals, Inc. Methods and compositions for treating diseases, disorders or injury of the nervous system
US20130131142A1 (en) * 2010-03-24 2013-05-23 Lyn Libertine Rna interference in ocular indications
US8614311B2 (en) 2007-12-12 2013-12-24 Quark Pharmaceuticals, Inc. RTP801L siRNA compounds and methods of use thereof
US8796239B2 (en) 2009-11-26 2014-08-05 Quark Pharmaceuticals, Inc. Sirna compounds comprising terminal substitutions
US9173894B2 (en) 2011-02-02 2015-11-03 Excaliard Pharamaceuticals, Inc. Method of treating keloids or hypertrophic scars using antisense compounds targeting connective tissue growth factor (CTGF)
US20160145617A1 (en) * 2013-07-19 2016-05-26 Ionis Pharmaceuticals, Inc. Compositions for modulating tau expression
US20160304879A1 (en) * 2012-01-04 2016-10-20 Quark Pharmaceuticals, Inc. Double-Stranded RNA Compounds to CASP2 and Uses Thereof
US9796979B2 (en) 2011-03-03 2017-10-24 Quark Pharmaceuticals Inc. Oligonucleotide modulators of the toll-like receptor pathway
RU2656154C2 (ru) * 2013-07-05 2018-05-31 Байонир Корпорейшн СВЯЗАННАЯ С РЕСПИРАТОРНЫМ ЗАБОЛЕВАНИЕМ ГЕН-СПЕЦИФИЧЕСКАЯ миРНК, ДВУСПИРАЛЬНАЯ КОНСТРУКЦИЯ ОЛИГО-РНК, СОДЕРЖАЩАЯ миРНК, И СОДЕРЖАЩАЯ ЕЕ КОМПОЗИЦИЯ ДЛЯ ПРЕДУПРЕЖДЕНИЯ ИЛИ ЛЕЧЕНИЯ РЕСПИРАТОРНОГО ЗАБОЛЕВАНИЯ
US10407680B2 (en) 2016-09-29 2019-09-10 Ionis Pharmaceuticals, Inc. Compounds and methods for reducing Tau expression
US10563265B2 (en) * 2013-09-06 2020-02-18 Chugai Seiyaku Kabushiki Kaisha Use of RHOA in cancer diagnosis and inhibitor screening
US11155815B2 (en) 2013-03-14 2021-10-26 Ionis Pharmaceuticals, Inc. Compositions and methods for modulating Tau expression
US11781135B2 (en) 2012-03-30 2023-10-10 Washington University Methods for treating Alzheimer's disease

Families Citing this family (48)

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Publication number Priority date Publication date Assignee Title
WO2006006948A2 (en) * 2002-11-14 2006-01-19 Dharmacon, Inc. METHODS AND COMPOSITIONS FOR SELECTING siRNA OF IMPROVED FUNCTIONALITY
EP1602926A1 (en) * 2004-06-04 2005-12-07 University of Geneva Novel means and methods for the treatment of hearing loss and phantom hearing
US7825099B2 (en) * 2006-01-20 2010-11-02 Quark Pharmaceuticals, Inc. Treatment or prevention of oto-pathologies by inhibition of pro-apoptotic genes
US20110224281A1 (en) * 2008-06-19 2011-09-15 Anja Molhart Hog Rna antagonists targeting hsp70-2
US8946172B2 (en) 2008-08-25 2015-02-03 Excaliard Pharmaceuticals, Inc. Method for reducing scarring during wound healing using antisense compounds directed to CTGF
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EP2440214A4 (en) 2009-06-08 2013-07-31 Quark Pharmaceuticals Inc METHODS OF TREATING CHRONIC RENAL DISEASE
GB0910723D0 (en) 2009-06-22 2009-08-05 Sylentis Sau Novel drugs for inhibition of gene expression
EP2862929B1 (en) 2009-12-09 2017-09-06 Quark Pharmaceuticals, Inc. Compositions and methods for treating diseases, disorders or injury of the CNS
WO2011084193A1 (en) 2010-01-07 2011-07-14 Quark Pharmaceuticals, Inc. Oligonucleotide compounds comprising non-nucleotide overhangs
EP2366432A1 (en) * 2010-03-16 2011-09-21 Helmholtz Zentrum München Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Inhibitors of glycogen synthase kinase 3 for use in therapeutic methods and screening method relating thereto
EP2550002B1 (en) 2010-03-24 2019-05-08 Phio Pharmaceuticals Corp. Rna interference in dermal and fibrotic indications
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EP2390327A1 (en) 2010-05-27 2011-11-30 Sylentis S.A. siRNA and their use in methods and compositions for the treatment and/or prevention of eye conditions
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SG190412A1 (en) 2010-12-06 2013-06-28 Quark Pharmaceuticals Inc Double stranded oligonucleotide compounds comprising threose modifications
JP6000287B2 (ja) * 2011-03-03 2016-09-28 クォーク ファーマシューティカルズ インコーポレーティッドＱｕａｒｋ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ，Ｉｎｃ． 肺疾患および損傷を治療するための組成物および方法
US8802839B2 (en) * 2011-07-15 2014-08-12 Fibrogen, Inc. Connective tissue growth factor antisense oligonucleotides
WO2014043291A1 (en) 2012-09-12 2014-03-20 Quark Pharmaceuticals, Inc. Double-stranded nucleic acid compounds
WO2014170786A1 (en) 2013-04-17 2014-10-23 Pfizer Inc. N-piperidin-3-ylbenzamide derivatives for treating cardiovascular diseases
BR112016000160B1 (pt) 2013-07-05 2021-06-08 Bioneer Corporation estrutura de oligonucleotídeo tipo nanopartícula melhorada tendo alta eficiência e método para a preparação da mesma
US9695421B2 (en) 2013-07-05 2017-07-04 Bioneer Corporation Dengue virus-specific siRNA, double helix oligo-RNA structure comprising siRNA, and composition for suppressing proliferation of dengue virus comprising RNA structure
WO2015015498A1 (en) 2013-07-31 2015-02-05 Qbi Enterprises Ltd. Methods of use of sphingolipid polyalkylamine oligonucleotide compounds
US9889200B2 (en) 2013-07-31 2018-02-13 Qbi Enterprises Ltd. Sphingolipid-polyalkylamine-oligonucleotide compounds
EP2865756A1 (en) 2013-10-22 2015-04-29 Sylentis, S.A.U. siRNA and their use in methods and compositions for inhibiting the expression of the FLAP gene.
EP2865757A1 (en) 2013-10-22 2015-04-29 Sylentis, S.A.U. siRNA and their use in methods and compositions for inhibiting the expression of the PDK1 gene.
EP2865758A1 (en) 2013-10-22 2015-04-29 Sylentis, S.A.U. siRNA and their use in methods and compositions for inhibiting the expression of the ORAI1 gene
US10208309B2 (en) 2014-04-04 2019-02-19 Bioneer Corporation Double-stranded oligo RNA targeted to amphiregulin and pharmaceutical composition comprising same for preventing or treating fibrosis or respiratory diseases
KR101652957B1 (ko) * 2014-05-08 2016-09-02 대한민국 ATF3 유전자 발현을 억제하는 신규 siRNA 및 이의 용도
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MA44908A (fr) 2015-09-08 2018-07-18 Sylentis Sau Molécules d'arnsi et leur utilisation dans des procédés et des compositions pour inhiber l'expression du gène nrarp
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CN108245528A (zh) * 2018-02-01 2018-07-06 河南省立眼科医院（河南省眼科研究所） 一种视神经保护的药物
EA202092748A1 (ru) 2018-05-22 2021-04-19 Айонис Фармасьютикалз, Инк. Модуляторы экспрессии apol1
TW202023573A (zh) 2018-09-19 2020-07-01 美商Ionis製藥公司 Pnpla3表現之調節劑
EA202192755A1 (ru) * 2019-04-24 2022-03-23 Стоук Терапьютикс, Инк. Способы и композиции для модуляции сплайсинга и трансляции
KR20210063137A (ko) * 2019-11-22 2021-06-01 (주)바이오니아 Ctgf 유전자 특이적 이중가닥 올리고뉴클레오티드 및 이를 포함하는 섬유증 관련 질환 및 호흡기 관련 질환 예방 및 치료용 조성물
US20240093188A1 (en) * 2020-05-26 2024-03-21 Olix Pharmaceuticals, Inc. RNAi AGENT TARGETING MYD88 AND USE THEREOF
WO2021255262A1 (en) 2020-06-19 2021-12-23 Sylentis Sau siRNA AND COMPOSITIONS FOR PROPHYLACTIC AND THERAPEUTIC TREATMENT OF VIRUS DISEASES
EP4015634A1 (en) 2020-12-15 2022-06-22 Sylentis, S.A.U. Sirna and compositions for prophylactic and therapeutic treatment of virus diseases

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5898031A (en) * 1996-06-06 1999-04-27 Isis Pharmaceuticals, Inc. Oligoribonucleotides for cleaving RNA
US6372249B1 (en) * 1991-12-16 2002-04-16 Baylor College Of Medicine Senscent cell-derived inhibitors of DNA synthesis
US6506559B1 (en) * 1997-12-23 2003-01-14 Carnegie Institute Of Washington Genetic inhibition by double-stranded RNA
US20050080246A1 (en) * 2002-11-05 2005-04-14 Charles Allerson Compositions comprising alternating 2'-modified nucleosides for use in gene modulation
US20060069056A1 (en) * 2004-09-28 2006-03-30 Elena Feinstein Oligoribonucleotides and methods of use thereof for treatment of alopecia, acute renal failure and other diseases
US20060217329A1 (en) * 2004-08-16 2006-09-28 Elena Feinstein Therapeutic uses of inhibitors of RTP801

Family Cites Families (74)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4475196A (en) 1981-03-06 1984-10-02 Zor Clair G Instrument for locating faults in aircraft passenger reading light and attendant call control system
US4447233A (en) 1981-04-10 1984-05-08 Parker-Hannifin Corporation Medication infusion pump
US4439196A (en) 1982-03-18 1984-03-27 Merck & Co., Inc. Osmotic drug delivery system
US4447224A (en) 1982-09-20 1984-05-08 Infusaid Corporation Variable flow implantable infusion apparatus
US4487603A (en) 1982-11-26 1984-12-11 Cordis Corporation Implantable microinfusion pump system
US4486194A (en) 1983-06-08 1984-12-04 James Ferrara Therapeutic device for administering medicaments through the skin
US4666828A (en) 1984-08-15 1987-05-19 The General Hospital Corporation Test for Huntington's disease
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4801531A (en) 1985-04-17 1989-01-31 Biotechnology Research Partners, Ltd. Apo AI/CIII genomic polymorphisms predictive of atherosclerosis
US4959217A (en) 1986-05-22 1990-09-25 Syntex (U.S.A.) Inc. Delayed/sustained release of macromolecules
US4925678A (en) 1987-04-01 1990-05-15 Ranney David F Endothelial envelopment drug carriers
US5080646A (en) 1988-10-03 1992-01-14 Alza Corporation Membrane for electrotransport transdermal drug delivery
US5272057A (en) 1988-10-14 1993-12-21 Georgetown University Method of detecting a predisposition to cancer by the use of restriction fragment length polymorphism of the gene for human poly (ADP-ribose) polymerase
US5270030A (en) 1988-12-29 1993-12-14 Bio-Technology General Corp. Fibrin binding domain polypeptide and method of producing
US5703055A (en) 1989-03-21 1997-12-30 Wisconsin Alumni Research Foundation Generation of antibodies through lipid mediated DNA delivery
US5192659A (en) 1989-08-25 1993-03-09 Genetype Ag Intron sequence analysis method for detection of adjacent and remote locus alleles as haplotypes
US5225347A (en) 1989-09-25 1993-07-06 Innovir Laboratories, Inc. Therapeutic ribozyme compositions and expression vectors
US5167616A (en) 1989-12-14 1992-12-01 Alza Corporation Iontophoretic delivery method
US5378825A (en) 1990-07-27 1995-01-03 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogs
US5225182A (en) 1991-10-31 1993-07-06 Sharma Yash P Vectored drug delivery system using a cephaloplastin linking agent and a methed of using the system
CA2126691C (en) 1991-12-24 2003-05-06 Phillip Dan Cook Gapped 2' modified oligonucleotides
CA2135646A1 (en) 1992-05-11 1993-11-25 Kenneth G. Draper Method and reagent for inhibiting viral replication
US5593972A (en) 1993-01-26 1997-01-14 The Wistar Institute Genetic immunization
WO1995000160A1 (en) 1993-06-24 1995-01-05 The General Hospital Corporation Programmed cell death genes and proteins
US5858678A (en) 1994-08-02 1999-01-12 St. Louis University Apoptosis-regulating proteins
US5929042A (en) 1997-03-03 1999-07-27 The Trustees Of Columbia University In The City Of New York Antisense compounds which prevent cell death and uses thereof
US7223856B2 (en) 1997-03-03 2007-05-29 The Trustees Of Columbia University In The City Of New York Antisense compounds which prevent cell death and uses thereof
US6251666B1 (en) 1997-03-31 2001-06-26 Ribozyme Pharmaceuticals, Inc. Nucleic acid catalysts comprising L-nucleotide analogs
AU9016898A (en) 1997-08-07 1999-03-01 United States Of America, Represented By The Secretary, Department Of Health And Human Services, The Methods and compositions for treatment of restenosis
US5877309A (en) 1997-08-13 1999-03-02 Isis Pharmaceuticals, Inc. Antisense oligonucleotides against JNK
DE69829857T2 (de) 1997-08-21 2006-02-02 Quark Biotech, Inc., Pleasanton Hypoxie-regulierte gene
US6180597B1 (en) 1998-03-19 2001-01-30 Brigham And Women's Hospital, Inc. Upregulation of Type III endothelial cell nitric oxide synthase by rho GTPase function inhibitors
US20030125277A1 (en) 2001-11-08 2003-07-03 Isis Pharmaceuticals Inc. Antisense modulation of activating transcription factor 3 expression
US6821724B1 (en) * 1998-09-17 2004-11-23 Affymetrix, Inc. Methods of genetic analysis using nucleic acid arrays
US6410323B1 (en) 1999-08-31 2002-06-25 Isis Pharmaceuticals, Inc. Antisense modulation of human Rho family gene expression
US7169783B2 (en) * 1998-11-02 2007-01-30 Universite De Montreal (+)-Trans-4-(1-aminoethyl)-1-(4-pyridycarbamoyl)-cyclohexane and method for promoting neural growth in the central nervous system and in a patient at a site of neuronal lesion
EP1146908B1 (en) 1999-01-27 2005-08-03 Becker, David, Dr. Formulations comprising antisense nucleotides to connexins
DE19956568A1 (de) 1999-01-30 2000-08-17 Roland Kreutzer Verfahren und Medikament zur Hemmung der Expression eines vorgegebenen Gens
US20040171566A1 (en) 1999-04-06 2004-09-02 Monia Brett P. Antisense modulation of p38 mitogen activated protein kinase expression
US6140124A (en) 1999-04-06 2000-10-31 Isis Pharmaceuticals Inc. Antisense modulation of P38 mitogen activated protein kinase expression
WO2001018037A2 (en) 1999-09-07 2001-03-15 University Health Network A p53-induced protein with a death domain that can promote apoptosis
GB9927444D0 (en) 1999-11-19 2000-01-19 Cancer Res Campaign Tech Inhibiting gene expression
DE10160151A1 (de) 2001-01-09 2003-06-26 Ribopharma Ag Verfahren zur Hemmung der Expression eines vorgegebenen Zielgens
US6323029B1 (en) 2000-01-19 2001-11-27 Isis Pharmaceuticals, Inc. Antisense modulation of glycogen synthase kinase 3 beta expression
US8202979B2 (en) * 2002-02-20 2012-06-19 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid
WO2003070918A2 (en) 2002-02-20 2003-08-28 Ribozyme Pharmaceuticals, Incorporated Rna interference by modified short interfering nucleic acid
ES2336887T5 (es) 2000-03-30 2019-03-06 Whitehead Inst Biomedical Res Mediadores de interferencia por ARN específicos de secuencias de ARN
WO2002024720A1 (en) 2000-09-20 2002-03-28 Isis Pharmaceuticals, Inc. Antisense modulation of caspase 2 expression
FR2815541B1 (fr) 2000-10-24 2008-02-29 Lipha Utilisation d'inhibiteurs de l'apoptose des pericytes pour le traitement et/ou la prevention de la retinopathie diabetique
DK2813582T3 (da) * 2000-12-01 2017-07-31 Max-Planck-Gesellschaft Zur Förderung Der Wss E V Små RNA-molekyler, som formidler RNA-interferens
US20040019001A1 (en) 2002-02-20 2004-01-29 Mcswiggen James A. RNA interference mediated inhibition of protein typrosine phosphatase-1B (PTP-1B) gene expression using short interfering RNA
GB0116955D0 (en) 2001-07-11 2001-09-05 Syngenta Ltd Weed control process
WO2003030836A2 (en) * 2001-10-12 2003-04-17 Case Western Reserve University Neuronal regeneration
AU2003207708A1 (en) 2002-02-20 2003-09-09 Sirna Therapeutics, Inc. Rna interference mediated inhibition of map kinase genes
WO2003087368A2 (en) 2002-04-18 2003-10-23 Lynkeus Bio Tech Gmbh Means and methods for the specific modulation of target genes in the cns and the eye and methods for their identification
SE523714C2 (sv) * 2002-07-05 2004-05-11 Packetfront Sweden Ab Ett filter i ett gränssnitt inom ett öppet system av typ skikt2 för trafikseparation i minst en router för åtkomstomkoppling inom ett nät, och ett förfarande för detta
WO2004009780A2 (en) 2002-07-22 2004-01-29 University Of Miami Preventing ischemia-induced cell damage
WO2004009797A2 (en) 2002-07-23 2004-01-29 Incyte Corporation Protein modification and maintenance molecules
BRPI0313202A8 (pt) * 2002-08-05 2016-08-16 Atugen Ag Formas adicionais para interferir com as moléculas de rna
US20040142865A1 (en) 2002-10-02 2004-07-22 Weber Georg F. Osteopontin-based cancer therapies
JP2006517916A (ja) 2002-10-30 2006-08-03 ザ シービーアール インスティテュート フォー バイオメディカル リサーチ インコーポレーティッド Ｒｎａ干渉物質を用いてアポトーシス関連疾患を治療および予防する方法
AU2002952550A0 (en) 2002-11-08 2002-11-21 The University Of Queensland Modulating TNFalpha Secretion
EP2305812A3 (en) * 2002-11-14 2012-06-06 Dharmacon, Inc. Fuctional and hyperfunctional sirna
AU2003300815A1 (en) 2002-12-05 2004-06-30 Incyte Corporation Protein modification and maintenance molecules
DE602004030315D1 (de) 2003-01-17 2011-01-13 Max Planck Gesellschaft Induzierbare sirna expressionskonstrukte zur gezielten genabschaltung
CA2526493A1 (en) 2003-05-22 2004-12-02 Theraptosis S.A. Means for preventing and treating cellular death and their biological applications
US20080249038A1 (en) 2003-10-07 2008-10-09 Quark Biotech, Inc. Bone Morphogenetic Protein (Bmp) 2A and Uses Thereof
EP2330111A3 (en) * 2004-01-30 2011-08-17 Quark Pharmaceuticals, Inc. Oligoribonucleotides and methods of use thereof for treatment of fibrotic conditions and other diseases
WO2005103297A1 (en) 2004-03-31 2005-11-03 Georgetown University METHODS AND COMPOSITIONS RELATED TO MODULATION OF p22phox
US8173600B2 (en) 2004-04-30 2012-05-08 Chiesi Farmaceutici S.P.A. Caspase-2 inhibitors and their biological applications
EP1602926A1 (en) 2004-06-04 2005-12-07 University of Geneva Novel means and methods for the treatment of hearing loss and phantom hearing
WO2006130472A2 (en) * 2005-05-27 2006-12-07 The Regents Of The University Of California Identification of rac1b as a marker and mediator of matrix metalloproteinase-induced malignancy
GB0606269D0 (en) 2006-03-29 2006-05-10 Bioinvent Int Ab Biological materials and uses thereof
JP2010507387A (ja) * 2006-10-25 2010-03-11 クアーク・ファーマスーティカルス、インコーポレイテッド 新規のｓｉＲＮＡおよびその使用方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6372249B1 (en) * 1991-12-16 2002-04-16 Baylor College Of Medicine Senscent cell-derived inhibitors of DNA synthesis
US5898031A (en) * 1996-06-06 1999-04-27 Isis Pharmaceuticals, Inc. Oligoribonucleotides for cleaving RNA
US6107094A (en) * 1996-06-06 2000-08-22 Isis Pharmaceuticals, Inc. Oligoribonucleotides and ribonucleases for cleaving RNA
US6506559B1 (en) * 1997-12-23 2003-01-14 Carnegie Institute Of Washington Genetic inhibition by double-stranded RNA
US20050080246A1 (en) * 2002-11-05 2005-04-14 Charles Allerson Compositions comprising alternating 2'-modified nucleosides for use in gene modulation
US20060217329A1 (en) * 2004-08-16 2006-09-28 Elena Feinstein Therapeutic uses of inhibitors of RTP801
US20060069056A1 (en) * 2004-09-28 2006-03-30 Elena Feinstein Oligoribonucleotides and methods of use thereof for treatment of alopecia, acute renal failure and other diseases

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Far et al., The activity of siRNA in mammalian cells is related to structural target accessibility: a comparison with antisense oligonucleotides, 2003, Nucleic Acids Research, volume 31, pages 4417-4424. *
Miyagishi et al., Comparison of the suppressive effects of antisense oligonucleotides and siRNAs directed against the same targets in mammalian cells, 2003, Antisense and Nucleic Acid Drug Development, volume 13, pages 1-7. *

Cited By (53)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110021420A1 (en) * 2003-01-10 2011-01-27 Imperial Innovations Limited Modification of feeding behaviour
US20100266574A1 (en) * 2005-06-10 2010-10-21 Orna Mor Oligoribonucleotides and Methods of Use Thereof for Treatment of Fibrotic Conditions and Other Diseases
US20090156524A1 (en) * 2006-10-25 2009-06-18 Elena Feinstein Novel siRNAS and methods of use thereof
US20100292301A1 (en) * 2007-02-28 2010-11-18 Elena Feinstein Novel sirna structures
US8410069B2 (en) 2007-03-21 2013-04-02 Quark Pharmaceuticals, Inc. Oligoribonucleotide inhibitors of Nrf2 and methods of use thereof for treatment of cancer
US20110230543A1 (en) * 2007-03-21 2011-09-22 Quark Pharmaceuticals, Inc. Oligoribonucleotide inhibitors of nrf2 and methods of use thereof for treatment of cancer
US8614309B2 (en) 2007-10-03 2013-12-24 Quark Pharmaceuticals, Inc. Double-stranded RNA directed to CASP2 and methods of use thereof
US20110112168A1 (en) * 2007-10-03 2011-05-12 Elena Feinstein Novel sirna structures
US8614311B2 (en) 2007-12-12 2013-12-24 Quark Pharmaceuticals, Inc. RTP801L siRNA compounds and methods of use thereof
US20110105584A1 (en) * 2007-12-12 2011-05-05 Elena Feinstein Rtp80il sirna compounds and methods of use thereof
US20110034534A1 (en) * 2008-01-15 2011-02-10 Elena Feinstein siRNA compounds and methods of use thereof
US20110028531A1 (en) * 2008-03-20 2011-02-03 Elena Feinstein Novel sirna compounds for inhibiting rtp801
US20110142917A1 (en) * 2008-06-06 2011-06-16 Egvenia Alpert Compositions and methods for treatment of ear disorders
US9089591B2 (en) 2008-06-06 2015-07-28 Quark Pharmaceuticals, Inc. Compositions and methods for treatment of ear disorders
US20110092575A1 (en) * 2008-06-06 2011-04-21 Assignment for Published Patent Application Gene Techno Science Co., Ltd. Sirna of human osteopontin
US8431692B2 (en) 2008-06-06 2013-04-30 Quark Pharmaceuticals, Inc. Compositions and methods for treatment of ear disorders
US20190323013A1 (en) * 2008-08-25 2019-10-24 Excaliard Pharmaceuticals, Inc. Antisense oligonucleotides directed against connective tissue growth factor and uses thereof
US9096851B2 (en) 2008-08-25 2015-08-04 Excaliard Pharmaceuticals, Inc. Antisense oligonucleotides directed against connective tissue growth factor and uses thereof
US9688987B2 (en) 2008-08-25 2017-06-27 Excaliard Pharmaceuticals, Inc. Antisense oligonucleotides directed against connective tissue growth factor and uses thereof
US8252762B2 (en) * 2008-08-25 2012-08-28 Excaliard Pharmaceuticals, Inc. Antisense oligonucleotides directed against connective tissue growth factor and uses thereof
US8772260B2 (en) 2008-08-25 2014-07-08 Isis Pharmaceuticals, Inc Methods for inhibiting expression of connective tissue growth factor
US20100130595A1 (en) * 2008-08-25 2010-05-27 Dean Nicholas M Antisense oligonucleotides directed against connective tissue growth factor and uses thereof
US20110190380A1 (en) * 2008-10-23 2011-08-04 Elena Feinstein Methods for delivery of sirna to bone marrow cells and uses thereof
US8901097B2 (en) 2009-11-08 2014-12-02 Quark Pharmaceuticals, Inc. Methods for delivery of siRNA to the spinal cord and therapies arising therefrom
CN102666856A (zh) * 2009-11-08 2012-09-12 夸克医药公司 用于向脊髓递送siRNA的方法和由此产生的疗法
WO2011057171A1 (en) * 2009-11-08 2011-05-12 Quark Pharmaceuticals, Inc. METHODS FOR DELIVERY OF siRNA TO THE SPINAL CORD AND THERAPIES ARISING THEREFROM
US9701960B2 (en) 2009-11-26 2017-07-11 Quark Pharmaceuticals, Inc. siRNA compounds comprising terminal substitutions
US8796239B2 (en) 2009-11-26 2014-08-05 Quark Pharmaceuticals, Inc. Sirna compounds comprising terminal substitutions
US10494631B2 (en) 2009-11-26 2019-12-03 Quark Pharmaceuticals, Inc. siRNA compounds comprising terminal substitutions
US11584933B2 (en) 2010-03-24 2023-02-21 Phio Pharmaceuticals Corp. RNA interference in ocular indications
US10662430B2 (en) 2010-03-24 2020-05-26 Phio Pharmaceuticals Corp. RNA interference in ocular indications
US9095504B2 (en) * 2010-03-24 2015-08-04 Rxi Pharmaceuticals Corporation RNA interference in ocular indications
US10184124B2 (en) 2010-03-24 2019-01-22 Phio Pharmaceuticals Corp. RNA interference in ocular indications
US20130131142A1 (en) * 2010-03-24 2013-05-23 Lyn Libertine Rna interference in ocular indications
US9173894B2 (en) 2011-02-02 2015-11-03 Excaliard Pharamaceuticals, Inc. Method of treating keloids or hypertrophic scars using antisense compounds targeting connective tissue growth factor (CTGF)
WO2012110457A3 (en) * 2011-02-14 2013-03-28 Santaris Pharma A/S Compounds for the modulation of osteopontin expression
US9796979B2 (en) 2011-03-03 2017-10-24 Quark Pharmaceuticals Inc. Oligonucleotide modulators of the toll-like receptor pathway
US9487778B2 (en) 2011-03-03 2016-11-08 Quark Pharmaceuticals, Inc. Oligonucleotide modulators of the toll-like receptor pathway
WO2012118911A1 (en) 2011-03-03 2012-09-07 Quark Pharmaceuticals, Inc. Oligonucleotide modulators of the toll-like receptor pathway
WO2013067076A2 (en) 2011-11-03 2013-05-10 Quark Pharmaceuticals, Inc. Methods and compositions for neuroprotection
WO2013070821A1 (en) 2011-11-08 2013-05-16 Quark Pharmaceuticals, Inc. Methods and compositions for treating diseases, disorders or injury of the nervous system
US10570394B2 (en) * 2012-01-04 2020-02-25 Quark Pharmaceuticals, Inc. Double-stranded RNA compounds to CASP2 and uses thereof
US20160304879A1 (en) * 2012-01-04 2016-10-20 Quark Pharmaceuticals, Inc. Double-Stranded RNA Compounds to CASP2 and Uses Thereof
US11781135B2 (en) 2012-03-30 2023-10-10 Washington University Methods for treating Alzheimer's disease
US11155815B2 (en) 2013-03-14 2021-10-26 Ionis Pharmaceuticals, Inc. Compositions and methods for modulating Tau expression
RU2656154C2 (ru) * 2013-07-05 2018-05-31 Байонир Корпорейшн СВЯЗАННАЯ С РЕСПИРАТОРНЫМ ЗАБОЛЕВАНИЕМ ГЕН-СПЕЦИФИЧЕСКАЯ миРНК, ДВУСПИРАЛЬНАЯ КОНСТРУКЦИЯ ОЛИГО-РНК, СОДЕРЖАЩАЯ миРНК, И СОДЕРЖАЩАЯ ЕЕ КОМПОЗИЦИЯ ДЛЯ ПРЕДУПРЕЖДЕНИЯ ИЛИ ЛЕЧЕНИЯ РЕСПИРАТОРНОГО ЗАБОЛЕВАНИЯ
US20160145617A1 (en) * 2013-07-19 2016-05-26 Ionis Pharmaceuticals, Inc. Compositions for modulating tau expression
US10793856B2 (en) 2013-07-19 2020-10-06 Biogen Ma Inc. Compositions for modulating Tau expression
US11591595B2 (en) 2013-07-19 2023-02-28 Biogen Ma Inc. Compositions for modulating Tau expression
US9683235B2 (en) * 2013-07-19 2017-06-20 Ionis Pharmaceuticals, Inc. Compositions for modulating Tau expression
US10563265B2 (en) * 2013-09-06 2020-02-18 Chugai Seiyaku Kabushiki Kaisha Use of RHOA in cancer diagnosis and inhibitor screening
US11053498B2 (en) 2016-09-29 2021-07-06 Biogen Ma Inc. Compounds and methods for reducing Tau expression
US10407680B2 (en) 2016-09-29 2019-09-10 Ionis Pharmaceuticals, Inc. Compounds and methods for reducing Tau expression

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