Source: https://patents.google.com/patent/US7939652?oq=6%2C460%2C050
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US7939652B2 - Oligoribonucleotides and methods of use thereof for treatment of fibrotic conditions and other diseases - Google Patents
Oligoribonucleotides and methods of use thereof for treatment of fibrotic conditions and other diseases Download PDF
US7939652B2
US7939652B2 US12072941 US7294108A US7939652B2 US 7939652 B2 US7939652 B2 US 7939652B2 US 12072941 US12072941 US 12072941 US 7294108 A US7294108 A US 7294108A US 7939652 B2 US7939652 B2 US 7939652B2
US12072941
US20080287386A1 (en )
The invention relates to a double-stranded compound, preferably an oligoribonucleotide (siRNA), which down-regulates the expression of a human TGaseII gene at the post-transcriptional level. The invention also relates to a pharmaceutical composition comprising the compound, or a vector capable of expressing the oligoribonucleotide compound, and a pharmaceutically acceptable carrier. The present invention also contemplates a method of treating a patient suffering from a fibrotic disease such as pulmonary, kidney and liver fibrosis or ocular, scarring comprising administering to the patient the pharmaceutical composition in a therapeutically effective dose so as to thereby treat the patient. The invention also relates to treatment of fibrotic and other diseases by use of antibodies to TGaseII polypeptide.
This application claims priority of U.S. Ser. No. 11/045,780, filed Jan. 27, 2005 now abandoned, which claims benefit of U.S. Provisional patent applications No. 60/540,687, filed Jan. 30, 2004 and No. 60/641,522 filed Jan. 4, 2005, the contents of all of which are hereby incorporated by reference into this application.
Throughout this application various patent and scientific publications are cited. The disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
RNA interference (RNAi) is a phenomenon involving double-stranded (ds) RNA-dependent gene specific posttranscriptional silencing. Originally, attempts to study this phenomenon and to manipulate mammalian cells experimentally were frustrated by an active, non-specific antiviral defense mechanism which was activated in response to long dsRNA molecules; see Gil et al. 2000, Apoptosis, 5:107-114. Later it was discovered that synthetic duplexes of 21 nucleotide RNAs could mediate gene specific RNAi in mammalian cells, without the stimulation of the generic antiviral defence mechanisms see Elbashir et al. Nature 2001, 411:494-498 and Caplen et al. Proc Natl Acad Sci 2001, 98:9742-9747. As a result, small interfering RNAs (siRNAs), which are short double-stranded RNAs, have become powerful tools in attempting to understand gene function.
Thus, RNA interference (RNAi) refers to the process of sequence-specific post-transcriptional gene silencing in mammals mediated by small interfering RNAs (siRNAs) (Fire et al, 1998, Nature 391, 806) or microRNAs (miRNAs) (Ambros V. Nature 431:7006, 350-355 (2004); and Bartel D P. Cell. 2004 Jan. 23; 116(2): 281-97 MicroRNAs: genomics, biogenesis, mechanism, and function). The corresponding process in plants is commonly referred to as specific post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. An siRNA is a double-stranded RNA molecule which down-regulates or silences (prevents) the expression of a gene/mRNA of its endogenous (cellular) counterpart. RNA interference is based on the ability of dsRNA species to enter a specific protein complex, where it is then targeted to the complementary cellular RNA and specifically degrades it. Thus, 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. 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 2001, Genes Dev., 15, 188). In more detail, longer dsRNAs are digested into short (17-29 bp) dsRNA fragments (also referred to as short inhibitory RNAs—“siRNAs”) by type III RNAses (DICER, DROSHA, etc., Bernstein et al., Nature, 2001, v. 409, p. 363-6; Lee et al., Nature, 2003, 425, p. 415-9). The RISC protein complex recognizes these fragments and complementary mRNA. The whole process is culminated by endonuclease cleavage of target mRNA (McManus & Sharp, Nature Rev Genet, 2002, v. 3, p. 737-47; Paddison & Hannon, Curr Opin Mol. Ther. 2003 June; 5(3): 217-24). For information on these terms and proposed mechanisms, see Bernstein E., Denli A M. Hannon G J: 2001The rest is silence. RNA. I; 7(11): 1509-21; Nishikura K.: 2001 A short primer on RNAi: RNA-directed RNA polymerase acts as a key catalyst. Cell. 116; 107(4): 415-8 and PCT publication WO 01/36646 (Glover et al).
The selection and synthesis of siRNA corresponding to known genes has been widely reported; see for example Chalk A M, Wahlestedt C, Sonnhammer E L. 2004 Improved and automated prediction of effective siRNA Biochem. Biophys. Res. Commun. Jun. 18; 319(1): 264-74; Sioud M, Leirdal M., 2004, Potential design rules and enzymatic synthesis of siRNAs, Methods Mol. Biol.; 252:457-69; Levenkova N, Gu Q, Rux J J. 2004, Gene specific siRNA selector Bioinformatics. I 12; 20(3): 430-2. and Ui-Tei K, Naito Y, Takahashi F, Haraguchi T, Ohki-Hamazaki H, Juni A, Ueda R, Saigo K., Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference Nucleic Acids Res. 2004 I 9; 32(3):936-48. See also Liu Y, Braasch D A, Nulf C J, Corey D R. Efficient and isoform-selective inhibition of cellular gene expression by peptide nucleic acids, Biochemistry, 2004 I 24; 43(7):1921-7. See also PCT publications WO 2004/015107 (Atugen) and WO 02/44321 (Tuschl et al), and also Chiu Y L, Rana T M. siRNA function in RNAi: a chemical modification analysis, RNA 2003 September; 9(9):1034-48 and I U.S. Pat. Nos. 5,898,031 and 6,107,094 (Crooke) for production of modified/more stable siRNAs.
Transglutaminase (TGase) Family
Transglutaminases (EC 2.3.2.13) are a family of enzymes that catalyze the crosslinking of proteins by epsilon-gamma glutamyl lysine isopeptide bonds. The family comprises 9 different enzymes among which are the factor XIIIa (plasma transglutaminase), keratinocyte transglutaminase (TGaseI), epidermal transglutaminase (TGaseIII), prostate transglutaminase (TGaseIV), and tissue-type transglutaminase (TGaseII). Although the overall primary structure of these enzymes is different, they all share a common amino acid sequence at the active site (Y-G-Q-C-W) and a strict calcium dependence for their activity (Lesort M, Tucholski J, Miller M L, Johnson G V, Tissue transglutaminase: a possible role in neurodegenerative diseases. Prog Neurobiol. 2000 August; 61(5):439-63).
Transglutaminase II. Transglutaminase II (TGaseII) also known as Protein-glutamine gamma-glutamyltransferase, TGase C, TGC, TG(C), and tissue-type transglutaminase, is a unique multifunctional enzyme with dual enzymatic activity:
1) The enzyme acts as a TGase protein (Ca2+ activated, GTP-inactivated) with crosslinking activities (i.e. it catalyzes reactions resulting in protein cross-links and/or covalent incorporation of biogenic amines). TGase further catalyzes the formation of a covalent glutamyl-lysyl bond, a unique isopeptide bond that is highly resistant to proteolysis and denaturants and that cannot be disrupted by any known vertebrate endopeptidase.
2) The enzyme also acts as a GTP-binding protein that transduces the activating signal from alpha1B and alpha 1D adrenergic receptors, from TP alpha thromboxane A2 receptor and from oxytocin receptor to phospholipase C delta 1 (i.e. it activates inositol phosphate production, Ca2+ mobilization etc.). It has been shown that both alpha 1D adrenergic receptor and PLC act as guanine nucleotide exchanging factor for transglutaminase II (Baek K J, Kang S, Damron D, Im M, Phospholipase C-delta1 is a guanine nucleotide exchanging factor for transglutaminase II (G alpha h) and promotes alpha IB-adrenoreceptor-mediated GTP binding and intracellular calcium release. J Biol. Chem. 2001 Feb. 23; 276(8):5591-7).
The dual function of this enzyme is separate and the active sites are located at different positions (Im M J, Russell M A, Feng J F, Transglutaminase II: a new class of GTP-binding protein with new biological junctions. Cell Signal. 1997 November; 9(7):477-82), and inhibition of crosslinking activity of TGaseII may be achieved without the interference with its G-protein function.
TGaseII: Subcellular Localization and Fibrosis Related Intracellular Signaling Activity
TGaseII is ubiquitously expressed and can be found in association with ECM and intracellularly, both as membrane bound and as cytosolic protein. The GTP-binding activity is higher in the membrane fraction of TGase and the cross-linking activity is higher in the cytosolic and extracellular fraction. On the cell surface, TGase binds to fibronectin via its 42 kDa gelatin-binding domain. The cross-linking of fibronectin with collagen contributes to structural stabilization of the ECM rendering the matrix resistant to proteolysis by matrix mettaloproteinases. Thus TGaseII, by favoring deposition of extracellular proteins and inhibiting breakdown of said proteins, contributes to ECM accumulation, a phenomenon that is central to the formation of tissue fibrosis.
In addition, TGase mediates the binding of latent TGF-beta to the ECM, a required step for a proper subsequent processing of this pro-fibrotic factor, which finally results in the release of active TGF-beta (Le et al., Connect Tissue Res. 2001; 42(4):245-53. Rosenthal et al., Arthritis Rheum. 2000 August; 43(8):1729-33).
In the cytoplasm, TGaseII may activate RhoA, a small G protein of the ras family known to have an important role in cytoskeletal rearrangement, regulation of cell morphology and differentiation. Activation of TGaseII results in an increased transamidation of RhoA, which then functions as a constitutively active G-protein showing increased binding to and activation of its downstream target ROCK-2 (Rho-associated kinase) (Singh et al. EMBO J. 2001 May 15; 20(10):2413-23). The specific ROCK kinase inhibitor, Y-27632, showed anti-fibrotic effect in UUO model of renal fibrosis as well as in the models of liver or lung fibrosis (Nagatoya K, Moriyama T, Kawada N, Takeji M, Oseto S, Murozono T, Ando A, Imai E, Hori M. Y-27632 was found to prevent tubulointerstitial fibrosis in mouse kidneys with unilateral urethral obstruction. Kidney Int. 2002 May; 61(5):1684-95; Murata T, Arii S, Nakamura T, Mori A, Kaido T, Furuyama H, Furumoto K, Nakao T, Isobe N, Imamura M, Inhibitory effect of Y-27632, a ROCK inhibitor, on progression of rat liver fibrosis in association with inactivation of hepatic stellate cells. J Hepatol. 2001 October; 35(4):474-81; Shimizu Y, Dobashi K, Iizuka K, Horie T, Suzuki K, Tukagoshi H, Nakazawa T, Nakazato Y, Mori M, Contribution of small GTPase Rho and its target protein ROCK in a murine model of lung fibrosis. Am J Respir Crit. Care Med. 2001 January; 163(1):210-7). Recently, a unique function of TGaseII contributing to activation of NFκB, a transcription factor with a well-documented pro-fibrotic activity was described (Baud et al., J Soc Biol. 2002; 196(4):269-73, Lee at al. J Biol. Chem. 2004 Dec. 17; 279(51): δ3725-35).
Fibrotic diseases are all characterized by the excess deposition of a fibrous material within the extracellular matrix, which contributes to abnormal changes in tissue architecture and interferes with normal organ function. Unfortunately, although fibrosis is widely prevalent, debilitating and often life threatening, there is no effective treatment currently available.
All tissues damaged by trauma respond by the initiation of a wound-healing program. Fibrosis, a type of disorder characterized by excessive scarring, occurs when the normal self-limiting process of wound healing response is disturbed, and causes excessive production and deposition of collagen. As a result, normal organ tissue is replaced with scar tissue, which eventually leads to the functional failure of the organ.
Fibrosis may be initiated by diverse causes and in various organs. Liver cirrhosis, pulmonary fibrosis, sarcoidosis, keloids and kidney fibrosis are all chronic conditions associated with progressive fibrosis, thereby causing a continuous loss of normal tissue function.
Acute fibrosis (usually with a sudden and severe onset and of short duration) occurs as a common response to various forms of trauma including accidental injuries (particularly injuries to the spine and central nervous system), infections, surgery, ischemic illness (e.g. cardiac scarring following heart attack), burns, environmental pollutants, alcohol and other types of toxins, acute respiratory distress syndrome, radiation and chemotherapy treatments).
For further information on different types of fibrosis see: Molina V, Blank M, Shoenfeld Y. (2002), “Fibrotic diseases”, Harefuah, 141(11): 973-8, 1009; Yu L, Noble N A, Border W A (2002), “Therapeutic strategies to halt renal fibrosis”, Curr Opin Pharmacol. 2(2):177-81; Keane W F, Lyle P A. (2003), “Recent advances in management of type 2 diabetes and nephropathy: lessons from the RENAAL study”, Am J Kidney Dis. 41(3 Suppl 2): S22-5; Bohle A, Kressel G, Muller C A, Muller G A. (1989), “The pathogenesis of chronic renal failure”, Pathol Res Pract. 185(4):421-40; Kikkawa R, Togawa M, Isono M, Isshiki K, Haneda M. (1997), “Mechanism of the progression of diabetic nephropathy to renal failure”, Kidney Int Suppl. 62:S39-40; Bataller R, Brenner D A. (2001), “Hepatic stellate cells as a target for the treatment of liver fibrosis”, Semin Liver Dis. 21(3):437-51; Gross T J, Hunninghake G W, (2001) “Idiopathic pulmonary fibrosis”, N Engl J. Med. 345(7):517-25; Frohlich E D. (2001) “Fibrosis and ischemia: the real risks in hypertensive heart disease”, Am J Hypertens; 14(6 Pt 2): 194S-199S.
Liver fibrosis (LF) is a generally irreversible consequence of hepatic damage of several etiologies. In the Western world, the main etiologic categories are: alcoholic liver disease (30-50%), viral hepatitis (30%), biliary disease (5-10%), primary hemochromatosis (5%), and drug-related and cryptogenic cirrhosis of unknown etiology (10-15%). Wilson's disease, α1-antitrypsin deficiency and other rare diseases also have liver fibrosis as one of the symptoms Scheinberg I H, Sternlieb I., Wilson disease and idiopathic copper toxicosis. Am J Clin Nutr 1996 May; 63(5):842S-5S; Parfrey H, Mahadeva R, Lomas D A., Alpha(1)-antitrypsin deficiency, liver disease and emphysema. Int J Biochem Cell Biol. 2003 July; 35(7):1009-14.
Liver cirrhosis, the end stage of liver fibrosis, frequently requires liver transplantation and is among the top ten causes of death in the Western world.
Anti-inflammatory agents, which cause inhibition of activation of hepatic stellate cells, stimulation of growth of hepatocytes and inhibition of post translational modification of collagen have all been used to treat liver fibrosis. However, due to the lack of selective targeting, these treatments suffer from, inter alia, the drawbacks of severe side effects.
For more information see Friedman S L. (2003), “Liver fibrosis—from bench to bedside”, J Hepatol. 38 Suppl 1:S38-53; Albanis E, Safadi R, Friedman S L. (2003), “Treatment of hepatic fibrosis: almost there”, Curr Gastroenterol Rep. 5(1):48-56. See also Grenard P, Bresson-Hadni S, El Alaoui S, Chevallier M, Vuitton D A, Ricard-Blum S., Transglutaminase-mediated cross-linking is involved in the stabilization of extracellular matrix in human liver fibrosis. J Hepatol. 2001 September; 35(3):367-75; Mirza A, Liu S L, Frizell E, Zhu J, Maddukuri S, Martinez J, Davies P, Schwarting R, Norton P, Zern M A., A role for tissue transglutaminase in hepatic injury and fibrogenesis, and its regulation by NF-kappaB. Am J. Physiol. 1997 February; 272(2 Pt 1):G281-8).
Kidney Fibrosis and Related Conditions
Chronic renal failure is a gradual and progressive loss of the ability of the kidneys to excrete wastes, concentrate urine, and conserve electrolytes. CRF is slowly progressive. It most often results from any disease that causes gradual loss of kidney function, and fibrosis is the main pathology that produces CRF.
Diabetic nephropathy, hallmarks of which are glomerulosclerosis and tubulointerstitial fibrosis, is the single most prevalent cause of end-stage renal disease in the modern world, and diabetic patients constitute the largest population on dialysis. Such therapy is costly and far from optimal. Transplantation offers a better outcome but suffers from a severe shortage of donors. More targeted therapies against diabetic nephropathy (as well as against other types of kidney pathologies) are not developed, since molecular mechanisms underlying these pathologies are largely unknown. Identification of an essential functional target gene that is modulated in the disease and affects the severity of the outcome of diabetes nephropathy has a high diagnostic as well as therapeutic value.
Origins of Kidney Pathology
Many pathological processes in the kidney (e.g., glomerular nephritis, physical obstructions, toxic injuries, metabolic and immunological diseases) eventually culminate in similar or identical morphological changes, namely glomerulosclerosis and tubulointerstitial fibrosis. Thus, different types of insults converge on the same single genetic program resulting in two hallmarks of fibrosis: the proliferation of fibroblasts and overproduction by them of various protein components of connective tissue. In addition, thickening of the basal membrane in the glomeruli accompanies interstitial fibrosis and culminates in glomerulosclerosis. See also Johnson T S, Skill N J, El Nahas A M, Oldroyd S D, Thomas G L, Douthwaite J A, Haylor J L, Griffin M, Transglutaminase transcription and antigen translocation in experimental renal scarring. J Am Soc Nephrol. 1999 October; 10(10):2146-57; Johnson T S, Griffin M, Thomas G L, Skill J, Cox A, Yang B, Nicholas B, Birckbichler P J, Muchaneta-Kubara C, Meguid El Nahas A, The role of transglutaminase in the rat subtotal nephrectomy model of renal fibrosis. J Clin Invest. 1997 Jun. 15; 99(12):2950-60).
Interstitial pulmonary fibrosis (IPF) is scarring of the lung caused by a variety of inhaled agents including mineral particles, organic dusts, and oxidant gases, or by unknown reasons (idiopathic lung fibrosis). The disease afflicts millions of individuals worldwide, and there are no effective therapeutic approaches. A major reason for the lack of useful treatments is that few of the molecular mechanisms of disease have been defined sufficiently to design appropriate targets for therapy (Lasky J A., Brody A R. (2000), “Interstitial fibrosis and growth factors”, Environ Health Perspect.; 108 Suppl 4:751-62).
The cellular substrate of pathogenesis of pulmonary fibrosis includes endothelial and epithelial cell injury, production of inflammatory cells and their mediators, and fibroblast activation., Fibrosis is believed to be related to a dysregulation in cross-talk between inflammatory and structural cells, mediated by various cytokines, chemokines and growth factors, which are responsible for the maintenance of tissue homeostasis and which coordinate the response to injury (Kelly M, Kolb M, Bonniaud P, Gauldie J. (2003), “Re-evaluation of fibrogenic cytokines in lung fibrosis”, Curr Pharm Des. 9(1):39-49).
Conventional therapy consisting of glucocorticoids or cytotoxic drugs is usually ineffective in preventing progression of the disease. It is believed that further understanding of the molecular mechanisms of endothelial and epithelial cell injury, inflammatory reaction, fibroblast proliferation, collagen deposition and lung repair, is necessary for the development of effective treatments against pulmonary fibrosis (Kuwano K, Hagimoto N, Hara N. (2001), “Molecular mechanisms of pulmonary fibrosis and current treatment”, Curr Mol. Med. 1(5):551-73). See also Griffin M, Smith L L, Wynne J., Changes in transglutaminase activity in an experimental model of pulmonary fibrosis induced by Paraquat. Br. J. Exp. Pathol. 1979 December; 60(6):653-61).
Heart failure is unique among the major cardiovascular disorders in that it alone is increasing in prevalence while there has been a striking decrease in other conditions. Some of this can be attributed to the aging of the populations of the United States and Europe. The ability to salvage patients with myocardial damage is also a major factor, as these patients may develop progression of left ventricular dysfunction due to deleterious remodelling of the heart.
The normal myocardium is composed of a variety of cells, cardiac myocytes and noncardiomyocytes, which include endothelial and vascular smooth muscle cells and fibroblasts. (Weber K T. (2000), “Fibrosis and hypertensive heart disease”, Curr Opin Cardiol. 15(4):264-72).
Structural remodeling of the ventricular wall is a key determinant of clinical outcome in heart disease. Such remodeling involves the production and destruction of extracellular matrix proteins, cell proliferation and migration, and apoptotic and necrotic cell death. Cardiac fibroblasts are crucially involved in these processes, producing growth factors and cytokines that act as autocrine and paracrine factors, as well as extracellular matrix proteins and proteinases. Recent studies have shown that the interactions between cardiac fibroblasts and cardiomyocytes are essential for the progression of cardiac remodeling of which the net effect is deterioration in cardiac function and the onset of heart failure (Manabe I, Shindo T, Nagai R. (2002), “Gene expression in fibroblasts and fibrosis: involvement in cardiac hypertrophy”, Circ Res. 13; 91(12):1103-13).
The use of agents to block the renin-angiotensin-aldosterone and sympathetic nervous systems has been shown to inhibit (and sometimes even reverse) cardiac remodelling and to improve the clinical course of patients with cardiac dysfunction. However, drugs aiming at direct inhibition or reduction of fibrosis are not yet available. See also Greenberg B. (2001), “Treatment of heart failure: state of the art and prospectives”, J Cardiovasc Pharmacol. 38 Suppl 2:S59-63, Zhang Z, Vezza R, Plappert T, McNamara P, Lawson J A, Austin S, Pratico D, Sutton M S, FitzGerald G A, COX-2-dependent cardiac failure in Gh/tTG transgenic mice. Circ Res. 2003 May 30; 92(10):1153-61. Epub 2003 Apr. 17; Hwang K C, Gray C D, Sweet W E, Moravec C S, Im M J., Alpha 1-adrenergic receptor coupling with Gh in the failing human heart. Circulation. 1996 Aug. 15; 94(4):718-26.
Polyglutamine diseases are a group of neurological diseases that are caused by expansion of CAG trinucleotide repeats coding for polyglutamine insert. Polyglutamine diseases include Huntington's disease (HD), spinobulbar muscular atrophy, dentatorubral-pallidoluysian atrophy and spinocerebellar ataxias (SCAs) 1, 2, 3, 6, 7 and 17. All these diseases are characterized by the presence of expansion of polyglutamine stretches (exceeding 35-40 glutamines), thus forming intranuclear aggregates, which leads to neuronal death. Alzheimer's disease (AD) is the most common cause of cognitive impairment in older patients and is expected to increase greatly in prevalence. Neurofibrillary degeneration, associated with the formation of paired helical filaments (PHF), is one of the critical neuropathological hallmarks of Alzheimer's disease (AD). Parkinson disease is a neurodegenerative disorder of aging characterized by a selective and progressive loss of dopaminergic neurons within the substantia nigra. See also Mastroberardino P G, Iannicola C, Nardacci R, Bernassola F, De Laurenzi V, Melino G, Moreno S, Pavone F, Oliverio S, Fesus L, Piacentini M. Tissue transglutaminase ablation reduces neuronal death and prolongs survival in a mouse model of Huntington's disease. Cell Death Differ. 2002 September; 9(9):873-80; Karpuj M V, Becher M W, Springer J E, Chabas D, Youssef S, Pedotti R, Mitchell D, Steinman L., Prolonged survival and decreased abnormal movements in transgenic model of Huntington disease, with administration of the transglutaminase inhibitor cystamine. Nat. Med. 2002 February; 8(2): 143-9; Citron B A, Suo Z, SantaCruz K, Davies P J, Qin F, Festoff B W., Protein crosslinking, tissue transglutaminase, alternative splicing and neurodegeneration. Neurochem Int. 2002 January; 40(1):69-78; Chen J S, Mehta K., Tissue transglutaminase: an enzyme with a split personality. Int J Biochem Cell Biol. 1999 August; 31(8):817-36.
Among the main characteristics of osteoarthritis are the degradation of articular cartilage and the formation of new bone at the joint edges, so-called osteophytes. See Van den Berg W B., Growth factors in experimental osteoarthritis: transforming growth factor beta pathogenic? J Rheumatol Suppl. 1995 February; 43:143-5; Scharstuhl A, Glansbeek H L, Van Beuningen H M, Vitters E L, Van der Kraan P M, Van den Berg W B., Inhibition of endogenous TGF-beta during experimental osteoarthritis prevents osteophyte formation and impairs cartilage repair. J. Immunol. 2002 Jul. 1; 169(1):507-14; Karpouzas G A, Terkeltaub R A., New developments in the pathogenesis of articular cartilage calcification. Curr Rheumatol Rep. 1999 December; 1(2):121-7.
Age-related cataracts: Cataracts are characterized by aggregation and covalent cross-linking of the crystallins, the major structural proteins of the eye lens, resulting in increase light scattering, opacification and cataract. Disturbance of calcium homeostasis in the tissue is one of the factors implicated in cataractogenesis, see Shridas et al., FEBS Lett. 2001 Jun. 22; 499(3):245-50; Shin et al, J Biol. Chem. 2004 Apr. 9; 279(15):15032-9; Wan et al., Br J Opthalmol. 2002 November; 86(11):1293-8. and Takeuchi N, Kamei A. Biol Pharm Bull. 2000 March; 23(3):283-90.
Proliferative vitreoretinopathy Proliferative vitreoretinopathy (PVR) is the most common complication following retinal detachment and associated with a retinal hole or break. PVR refers to the growth of cellular membranes within the vitreous cavity and on the front and back surfaces of the retina containing retinal pigment epithelial (RPE) cells. These membranes, which are essentially scar tissues, exert traction on the retina and may result in recurrences of retinal detachment, even after an initially successful retinal detachment procedure.
Migration and adhesion of dislocated retinal pigment epithelial (RPE) cells to a fibronectin-rich extracellular matrix is an initial step in proliferative vitreoretinopathy (PVR). See also Casaroli-Marano R P et al Invest Opthalmol V is Sci. 1999 August; 40(9):2062-72), and Priglinger S et al, Invest Opthalmol V is Sci. 2004 March; 45(3):955-63; Priglinger et al., Invest Opthalmol V is Sci. 2003 January; 44(1):355-64.
Fibrosis following glaucoma filtering operation. The goal of the glaucoma filtration procedure is to create a new passageway by which aqueous fluid inside the eye can escape, thereby lowering the pressure. The filter, therefore, allows the drainage of fluid from inside the anterior chamber of the eye to a “pocket” created between the conjuctiva, which is the outermost covering of the eye, and the sclera, which is the underlying white anatomical structure of the eye. The fluid is eventually absorbed by blood vessels. Unfortunately, due to postoperational scarring, the generated drainage system may be blocked requiring additional surgical intervention(s).
Current anti-scarring regimens (Mitomycin C or 5FU) are limited due to the complications involved (e.g. blindness) e.g. see Cordeiro M Y, Gay J A, Khaw P T., Human anti-transforming growth factor-beta2 antibody: a new glaucoma anti-scarring agent Invest Opthalmol V is Sci. 1999 September; 40(10):2225-34.
In conclusion, there are no effective modes of therapy for the diseases described above, and there is a need, therefore, to develop novel effective compounds and methods of treatment for these purposes.
The invention provides novel double stranded oligoribonucleotides. These oligoribonucleotides inhibit human TGaseII via the mechanism of RNA interference. The invention also provides a pharmaceutical composition comprising such an oligoribonucleotide, and a vector capable of expressing the oligoribonucleotide. The present invention also provides a method of treating a patient suffering from a fibrosis-related pathology comprising administering to the patient the oligoribonucleotide typically as a pharmaceutical composition, in a therapeutically effective dose so as to thereby treat the patient. The present invention also contemplates treating other diseases and conditions. The invention also relates to treatment of fibrotic and other diseases by use of an antibody to TGaseII polypeptide.
FIG. 1. This figure sets forth the nucleotide sequence of the human TGaseII cDNA-(gi|13653650|ref|XM—009482.3|—ORF_)—SEQ ID NO:1.
FIG. 2. This figure sets forth the amino acid sequence of the human TGaseII corresponding polypeptide—SEQ ID NO:2.
FIG. 3. Western Blot results demonstrating the effect of various TGaseII siRNAs on human TGaseII polypeptide expression.
The figure demonstrates the activity of various siRNAs in reducing expression of human TGase polypeptide in HeLa cells, 72 hr following siRNA transfection. GAPDH expression serves as control for protein loading. The numbers 15 and 30 represent concentration of the oligonucleotide in nM. “Cells” (first lane in each panel) stands for nontransfected control cells. The last lane in each panel contains protein extracts from HeLa cells transfected with non-relevant (NR) siRNA, to ensure that the decrease in TGaseII expression is specific to the anti TGase siRNAs activity.
The present invention provides a method for treatment of various pathologies, as recited below, in a subject in need of such treatment which comprises: administering to the subject an amount of an inhibitor of TGaseII polypeptide sufficient to effect a substantial inhibition of the TGaseII activity so as to thereby treat the subject. The TGaseII inhibitor may be L683685, an antibody to TGaseII polypeptide or an siRNA to TGaseII RNA or any of the TGaseII inhibitors known in the art (for further information on such inhibitors see for example: U.S. Pat. Nos. 5,021,440; 4,968,713 and 5098707). In particular the present invention relates to specific siRNAs targeting TGaseII RNA and the use thereof. The use of neutralizing antibodies against TGaseII is also disclosed.
The present invention relates generally to compounds which down-regulate expression of the human TGaseII gene particularly to novel small interfering RNAs (siRNAs), and to the use of these novel siRNAs in the treatment of various diseases and medical conditions in particular fibrotic diseases, diseases related to fibrotic scarring and other diseases related to aberrant expression of Transglutaminase II.
The present invention provides methods and compositions for inhibiting expression of the target TGaseII gene in vivo. In general, the method includes administering oligoribonucleotides, such as small interfering RNAs (i.e., siRNAs) that are targeted to a particular mRNA and hybridize to, or interact with, it under biological conditions (within the cell), or a nucleic acid material that can produce siRNA in a cell, in an amount sufficient to down-regulate expression of a target gene by an RNA interference mechanism. In particular, the subject method can be used to inhibit expression of the TGaseII gene for treatment of disease.
Thus, the inhibitor of TGaseII expression (transcription or translation) or polypeptide activity may be inter alia siRNA, antibodies, preferably neutralizing antibodies or fragments thereof, including single chain antibodies, antisense oligonucleotides, antisense DNA or RNA molecules, proteins, polypeptides and peptides including peptido-mimetics 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. These inhibitors may act as follows: small molecules may affect expression and/or activity; antibodies may affect activity; all kinds of antisense may affect TGaseII 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.
In accordance with the present invention, the siRNA molecules or inhibitors of Transglutaminase II, such as antibodies, may be used as drugs to treat various pathologies including fibrosis related pathologies (as defined below) and also to treat ocular diseases including cataract, cardiovascular diseases, neurological diseases, polyglutamine diseases (including Huntington's disease (HD), spinobulbar muscular atrophy, dentatorubral-pallidoluysian atrophy and spinocerebellar ataxias (SCAs) 1, 2, 3, 6, 7 and 17), Alzheimer's and Parkinson's disease and osteoarthritis.
As used herein, the term “Transglutaminase II gene”, or “TGaseII gene”, or “TGase gene” is defined as any homolog of TGaseII gene having preferably 90% homology, more preferably 95% homology, and even more preferably 98% homology to the amino acid encoding region of SEQ ID NO:1, or nucleic acid sequences which bind to the TGaseII gene under conditions of highly stringent hybridization, which are well-known in the art (for example, see Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1988), updated in 1995 and 1998).
As used herein, the term “Transglutaminase II polypeptide”, or “TGaseII polypeptide”, or “TGase” is defined as any homolog of TGaseII polypeptide having preferably 90% homology, more preferably 95% homology, and even more preferably 98% homology to SEQ ID NO:2, as either to full-length or fragments or a domain thereof, as a mutant of the polypeptide encoded by a spliced variant nucleic acid sequence, as a chimera with other polypeptides, provided that any of the above has the same or substantially the same biological function as the TGaseII polypeptide. TGaseII polypeptide, or a TGaseII polypeptide homolog, may be present in different forms, including but not limited to soluble protein, membrane-bound (either in purified membrane preparations or on a cell surface), bead-bound, or any other form presenting TGaseII protein or fragments and polypeptides derived thereof.
As used herein, an “interactor” is a molecule with which TGaseII binds or interacts or activates in nature; for example, a molecule on the surface of a TGaseII polypeptide expressing cell, a molecule on the surface of a second cell or a cytoplasmic molecule. An interactor may be a ligand that is activated by TGaseII alone or by TGaseII as part of a complex with other components. An interactor may be a component of a signal transduction pathway that facilitates transduction of an extracellular signal from TGaseII through the cell membrane and into the cell. An interactor, for example, can be a second intercellular protein that mediates downstream signaling from TGaseII. The interactor is a molecule with which TGase binds in competition with a known TGase substrate (e.g. fibronectin).
As used herein, the term “lysyl donor” or “K donor” is defined as any polypeptide having the ability to donate a lysyl side chain to allow the formation of gamma-glutamyl-lysine bonds during transglutamination process.
As used herein, the term “glutamyl donor” or “Q donor” is defined as any polypeptide having the ability to donate glutamine side chain to allow the formation of gamma-glutamyl-lysine bonds during transglutamination process.
The present invention provides double-stranded oligoribonucleotides (siRNAs), which down-regulate the expression of TGaseII. The downregulation of the expression of transglutminase II can be measured by e.g., measuring the amount of the lysyl-glutamyl crosslinked material produced in the presence of the siRNAs or by direct assessment of the amounts of TGaseII mRNA or polypeptide. The amount of TGaseII mRNA may be measured by e.g., by Northern blotting, RNase protection, RT-PCR or real-time PCR. The amount of TGaseII polypeptide may be measured by immunoblotting or by immunoprecipitation or by ELISA with TGaseII-specific antibodies.
An siRNA of the invention is a duplex oligoribonucleotide in which the sense strand is derived from the mRNA sequence of gene TGaseII, 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 Nucleic Acids Research 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.
There are at least two variant TGaseII polypeptides, for which the GeneBank references are variant 2 NM—198951.1 GI:39777598 and variant 1NM—004613.2 GI:39777596. The sequence given in FIG. 1 is the nucleotide sequence of variant 2 (GI:39777598). Both variants and any other similar minor variants are included in the definition of TGaseII polypeptide and in the definition of the TGaseII genes encoding them.
As used herein, the term “TGaseII gene” is defined as the naturally-occurring human gene including any allelic variant thereof as well as any homolog of the TGaseII gene having preferably 90% homology, more preferably 95% homology, and even more preferably 98% homology to the amino acid encoding region of SEQ ID NO:1 or nucleic acid sequences which bind to the TGaseII gene under conditions of highly stringent hybridization, which are well-known in the art (for example, see Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1988), updated in 1995 and 1998.
As used herein, the term “TGaseII”, or “TGaseII polypeptide” is defined as the naturally-occurring polypeptide product of the gene including any allelic variant thereof as well as any homolog of the TGaseII polypeptide having preferably 90% homology, more preferably 95% homology, and even more preferably 98% homology to SEQ ID NO:2, as either full-length or a fragment or a domain thereof, as a mutant or the polypeptide encoded by a spliced variant nucleic acid sequence, as a chimera with other polypeptides, provided that any of the above has the same or substantially the same biological function as the TGaseII polypeptide.
More particularly, the invention provides a compound having the structure:
5′(N)x—Z3′ (antisense strand)
and wherein the sequence of (N)x comprises any one of the antisense sequences present in Tables A, B and C
It will be readily understood by those skilled in the art that the compounds of the present invention consist of a plurality of nucleotides which are linked through covalent linkages. Each such covalent linkage may be a phosphodiester linkage, a phosphothioate linkage, or a combination of both, along the length of the nucleotide sequence of the individual strand. Other possible backbone modifications are described inter alia in U.S. Pat. Nos. 5,587,361; 6,242,589; 6,277,967; 6,326,358; 5,399,676; 5,489,677; and 5,596,086.
In some embodiments of the compound of the invention, at least one ribonucleotide is modified in its sugar residue, preferably a modification at the 2′ position. The modification at the 2′ position results in the presence of a moiety which is preferably selected from the group comprising amino, fluoro, methoxy, alkoxy and alkyl groups. In a presently most preferred embodiment the moiety at the 2′ position is methoxy (2′-0-methyl).
In some embodiments of the invention, alternating ribonucleotides are modified in both the antisense and the sense strands of the compound.
In particularly preferred embodiments of the invention, the antisense strand is phosphorylated at the 5′terminus, and may or may not be phosphorylated at the 3′terminus; and the sense strand may or may not be phosphorylated at the 5′terminus and at the 3′terminus.
The invention also provides a composition comprising one or more of the compounds of the invention in a carrier, preferably a pharmaceutically acceptable carrier.
The invention also provides a composition comprising a carrier and one or more of the compounds of the invention in an amount effective to down-regulate expression in a cell of a human TGaseII gene, which compound comprises a sequence substantially complementary to the sequence of (N)x.
The invention also provides a method of down-regulating the expression of a human TGaseII gene by at least 50% as compared to a control comprising contacting an mRNA transcript of the gene with one or more of the compounds of the invention.
In one embodiment the compound is down-regulating TGaseII, whereby the down-regulation of TGaseII is selected from the group comprising down-regulation of TGaseII function (which may be examined by an enzymatic assay or a binding assay with a known interactor of the native gene/polypeptide, inter alia), down-regulation of TGaseII protein (which may be examined by Western blotting, ELISA or immuno-precipitation, inter alia) and down-regulation of TGaseII mRNA expression (which may be examined by Northern blotting, quantitative RT-PCR, in-situ hybridisation or microarray hybridisation, inter alia).
The invention also provides a method of treating a patient suffering from fibrosis or a fibrosis-related pathology, comprising administering to the patient a composition of the invention in a therapeutically effective dose so as to thereby treat the patient.
The invention also provides a method of treating a patient suffering from a pathology related to aberrant cross-linking of cellular proteins via Transglutaminase II comprising administering to the patient a composition of the invention in a therapeutically effective dose so as to thereby treat the patient.
The invention also provides a use of a therapeutically effective dose of one or more compounds of the invention for the preparation of a composition for promoting recovery in a patient suffering from fibrosis or a fibrosis-related pathology or a pathology related to aberrant crosslinking of cellular proteins via Transglutaminase II.
Fibrotic diseases or diseases in which fibrosis is evident (fibrosis-related pathology) include both acute and chronic forms of fibrosis of organs, including all etiological variants of the following: pulmonary fibrosis, including interstitial lung disease and fibrotic lung disease, liver fibrosis, cardiac fibrosis including myocardial fibrosis, kidney fibrosis including chronic renal failure, skin fibrosis including scleroderma, keloids and hypertrophic scars; myelofibrosis (bone marrow fibrosis); all types of ocular scarring including proliferative vitreoretinopathy (PVR) and scarring resulting from surgery to treat cataract or glaucoma; inflammatory bowel disease of variable etiology, macular degeneration, Grave's ophthalmopathy, drug induced ergotism, psoriasis, glioblastoma in Li-Fraumeni syndrome, sporadic glioblastoma, myleoid leukemia, acute myelogenous leukemia, myelodysplastic syndrome, myeloproferative syndrome, gynecological cancer, Kaposi's sarcoma, Hansen's disease, and collagenous colitis.
The compounds of the invention may be used to treat many other diseases and conditions apart from fibrotic diseases. Other indications may be ocular diseases including cataract, cardiovascular diseases especially cardiac hypertrophy, atherosclerosis/restenosis, neurological diseases, including polyglutamine diseases (such as Huntington's disease), spinobulbar muscular atrophy, dentatorubral-pallidoluysian atrophy and spinocerebellar ataxias (SCAs) 1, 2, 3, 6, 7 and 17, Alzheimer's disease and Parkinson's disease.
The compound may have homologs wherein up to two of the ribonucleotides in each terminal region a base is altered; the terminal region refers to the four terminal ribonucleotides e.g. refers to bases 1-4 and/or 16-19 in a 19-mer sequence and to bases 1-4 and/or 18-21 in a 21-mer sequence.
The preferred oligonucleotides of the invention are the oligonucleotides listed in Tables A, B and C, preferably the oligonucleotides targeting human cDNA. The most preferred oligonucleotides of the invention are the oligonucleotides having inhibitory activity as demonstrated in Table D, preferably oligonucleotides targeting human TGaseII cDNA.
The presently most preferred compound of the invention is a blunt-ended 19-mer oligonucleotide, i.e. x=y=19 and Z and Z′ are both absent; the oligonucleotide is phosphorylated at the 5′position of the antisense strand and at the 3′ position of the sense strand wherein alternating ribonucleotides are modified at the 2′ position in both the antisense and the sense strands, wherein the moiety at the 2′ position is methoxy (2′-0-methyl) and wherein 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 presently most preferred such compound is TG_HMRG1. The antisense strand of TG_HFMRG1 has SEQ ID NO: 22 and the sense strand has SEQ ID NO: 4. Other preferred compounds are TG_HMG1 and TG_HM1, which have the antisense strand represented by SEQ ID NOS: 23 and 25 respectively and the sense strand represented by SEQ ID NO: 5 and 7 respectively.
the first stretch is either complementary or identical to a nucleic acid sequence coding for TGaseII and whereby the second stretch is either identical or complementary to a nucleic acid sequence coding for TGaseII.
In an embodiment the first stretch and/or the second stretch comprises from about 14 to 40 nucleotides, preferably about 18 to 30 nucleotides, more preferably from about 19 to 27 nucleotides and most preferably from about 19 to 23 nucleotides, in particular from about 19 to 21 nucleotides. In such an aspect the oligonucleotide may be from 17-40 nucleotides in length. Additionally, further nucleic acids according to the present invention comprise at least 14 contiguous nucleotides of any one of the SEQ. ID. NO.3 to SEQ ID NO: 416 and more preferably 14 contiguous nucleotide base pairs at any end of the double-stranded structure comprised of the first stretch and second stretch as described above.
The term “treatment” as used herein refers to administration of a therapeutic substance effective to ameliorate symptoms associated with a disease or condition, to lessen the severity or cure the disease or condition, or to prevent the disease or condition from occurring.
In a particular embodiment, the administration comprises intravenous administration. In another particular embodiment the administration comprises topical or local administration.
Additionally, the present invention provides a method of regulating a pathology or disease (as recited above) in a patient in need of such treatment by administering to a patient a therapeutically effective dose of at least one antisense (AS) oligonucleotide or at least one siRNA against the nucleic acid sequences or a dominant negative peptide directed against the TGaseII sequences or TGaseII proteins or an antibody directed against the TGaseII polypeptide.
Delivery: Delivery systems aimed specifically at the enhanced and improved delivery of siRNA into mammalian cells have been developed, see, for example, Shen et al (FEBS letters 539: 111-114 (2003)), Xia et al., Nature Biotechnology 20: 1006-1010 (2002), Reich et al., Molecular Vision 9: 210-216 (2003), Sorensen et al. (J. Mol. Biol. 327: 761-766 (2003), Lewis et al., Nature Genetics 32: 107-108 (2002) and Simeoni et al., Nucleic Acids Research 31, 11: 2717-2724 (2003). siRNA has recently been successfully used for inhibition in primates; for further details see Tolentino et al., Retina 24(1) February 2004 I 132-138 Respiratory formulations for siRNA are described in U.S. patent application No. 2004/0063654 of Davis et al. Cholesterol-conjugated siRNAs (and other steroid and lipid conjugated siRNAs) can been used for delivery (see Soutschek et al Nature 432: 173-177 (2004) Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs; and Lorenz et al. Bioorg. Med. Chemistry. Lett. 14:4975-4977 (2004) Steroid and lipid conjugates of siRNAs to enhance cellular uptake and gene silencing in liver cells.
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 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 cosolvents, aqueous or oil suspensions, emulsions with edible oils, as well as similar pharmaceutical vehicles. In addition, under certain circumstances the compositions for use in the novel treatments of the present invention may be formed as aerosols, for intranasal and like administration. The patient being treated is a warm-blooded animal and, in particular, mammals including man. 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. Examples of 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. In one specific embodiment of this invention topical and transdermal formulations are particularly preferred.
In general, 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. Treatment for many years or even lifetime treatment is also envisaged for some of the indications disclosed herein.
obtaining at least one double stranded siRNA compound of the invention; and
The present invention also provides for a process of preparing a pharmaceutical composition, which comprises admixing a compound of the present invention with a pharmaceutically acceptable carrier.
In embodiments of the invention, the nucleotides can be selected from naturally occurring or synthetically modified bases. Naturally occurring bases include adenine, guanine, cytosine, thymine and uracil. Modified bases of the oligonucleotides 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-thiuracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl 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 cytosine.
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 S. L. and Iyer R. P., Tetrahedron 1992; 48: 2223-2311, Beaucage S. L. and Iyer R. P., Tetrahedron 1993; 49: 6123-6194 and Caruthers M. H. et. al., Methods Enzymol. 1987; 154: 287-313, the synthesis of thioates is, among others, described in Eckstein F., Annu. Rev. Biochem. 1985; 54: 367-402, the synthesis of RNA molecules is described in Sproat B., in Humana Press 2005 Edited by Herdewijn P.; Kap. 2: 17-31 and respective downstream processes are, among others, described in Pingoud A. et. al., in IRL Press 1989 Edited by Oliver R. W. A.; Kap. 7: 183-208 and Sproat B., in Humana Press 2005 Edited by Herdewijn P.; Kap. 2: 17-31 (supra).
It is also envisaged that a long double stranded oligonucleotide (typically 25-500 nucleotides in length) comprising one or more of the sequences of the oligonucleotides of the invention may be delivered and may be processed intracellularly by endogenous cellular complexes (e.g. by DICER as described above) to produce smaller double stranded oligonucleotides which are oligonucleotides of the invention.
Animal model systems: Testing the active siRNAs of the invention may be done in predictive animal models. Several models for kidney fibrosis are described in Example 3.
Two models of liver fibrosis in rats are the Bile Duct Ligation (BDL) with sham operation as controls, and CCl4 poisoning, with olive oil fed animals as controls, as described in the following references: Lotersztajn S, Julien B, Teixeira-Clerc F, Grenard P, Mallat A, Hepatic Fibrosis: Molecular Mechanisms and Drug Targets. Annu Rev Pharmacol Toxicol. 2004 Oct. 7; Uchio K, Graham M, Dean N M, Rosenbaum J, Desmouliere A., Down-regulation of connective tissue growth factor and type I collagen mRNA expression by connective tissue growth factor antisense oligonucleotide during experimental liver fibrosis. Wound Repair Regen. 2004 January-February; 12(1):60-6; and. Xu X Q, Leow C K, Lu X, Zhang X, Liu J S, Wong W H, Asperger A, Deininger S, Eastwood Leung H C., Molecular classification of liver cirrhosis in a rat model by proteomics and bioinformatics Proteomics. 2004 October; 4(10):3235-45.
Models for ocular scarring are well known in the art e.g. Sherwood M B et al., J Glaucoma. 2004 October; 13(5):407-12. A new model of glaucoma filtering surgery in the rat; Miller M H et al., Ophthalmic Surg. 1989 May; 20(5):350-7. Wound healing in an animal model of glaucoma fistulizing surgery in the rabbit; vanBockxmeer F M et al., Retina. 1985 Fall-Winter; 5(4): 239-52. Models for assessing scar tissue inhibitors; Wiedemann P et al., J Pharmacol Methods. 1984 August; 12(1): 69-78. Proliferative vitreoretinopathy: the rabbit cell injection model for screening of antiproliferative drugs.
Models of cataract are described in the following publications: The role of Src family kinases in cortical cataract formation. Zhou J, Menko A S. Invest Opthalmol V is Sci. 2002 July; 43(7):2293-300; Bioavailability and anticataract effects of a topical ocular drug delivery system containing disulfuram and hydroxypropyl-beta-cyclodextrin on selenite-treated rats. Wang S, Li D, Ito Y, Nabekura T, Wang S, Zhang J, Wu C. Curr Eye Res. 2004 July; 29(1):51-8; and Long-term organ culture system to study the effects of UV-Airradiation on lens transglutaminase. Weinreb O, Dovrat A.; Curr Eye Res. 2004 July; 29(1):51-8.
By the term “antibody” as used in the present invention is meant both poly- and mono-clonal complete antibodies as well as fragments thereof, such as Fab, F(ab′)2, and Fv, which are capable of binding the epitopic determinant. These antibody fragments retain the ability to selectively bind with its antigen or receptor and are exemplified as follows, inter alia:
(1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule can be produced by digestion of whole antibody with the enzyme papain to yield a light chain and a portion of the heavy chain;
Such fragments having antibody functional activity can be prepared by methods known to those skilled in the art (e.g. Bird et al. (1988) Science 242:423-426)
Conveniently, antibodies may be prepared against the immunogen or portion thereof, for example, a synthetic peptide based on the sequence, or prepared recombinantly by cloning techniques or the natural gene product and/or portions thereof may be isolated and used as the immunogen. Immunogens can be used to produce antibodies by standard antibody production technology well known to those skilled in the art, as described generally in Harlow and Lane (1988), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., and Borrebaeck (1992), Antibody Engineering—A Practical Guide, W.H. Freeman and Co., NY.
For producing polyclonal antibodies a host, such as a rabbit or goat, is immunized with the immunogen or immunogen fragment, generally with an adjuvant and, if necessary, coupled to a carrier; antibodies to the immunogen are collected from the sera. Further, the polyclonal antibody can be absorbed such that it is monospecific; that is, the sera can be absorbed against related immunogens so that no cross-reactive antibodies remain in the sera, rendering it monospecific.
For producing recombinant antibody see generally Huston et al. (1991) “Protein engineering of single-chain Fv analogs and fusion proteins” in Methods in Enzymology (J J Langone, ed., Academic Press, New York, N.Y.) 203:46-88; Johnson and Bird (1991) “Construction of single-chain Fvb derivatives of monoclonal antibodies and their production in Escherichia coli in Methods in Enzymology (J J Langone, ed.; Academic Press, New York, N.Y.) 203:88-99; Mernaugh and Mernaugh (1995) “An overview of phage-displayed recombinant antibodies” in Molecular Methods In Plant Pathology (RP Singh and US Singh, eds.; CRC Press Inc., Boca Raton, Fla.: 359-365). In particular scFv antibodies are described in WO 2004/007553 (Tedesco and Marzari). Additionally, messenger RNAs from antibody-producing B-lymphocytes of animals, or hybridoma can be reverse-transcribed to obtain complementary DNAs (cDNAs). Antibody cDNA, which can be full or partial length, is amplified and cloned into a phage or a plasmid. The cDNA can be a partial length of heavy and light chain cDNA, separated or connected by a linker. The antibody, or antibody fragment, is expressed using a suitable expression system to obtain recombinant antibody. Antibody cDNA can also be obtained by screening pertinent expression libraries.
The antibody can be bound to a solid support substrate or conjugated with a detectable moiety or be both bound and conjugated as is well known in the art. (For a general discussion of conjugation of fluorescent or enzymatic moieties see Johnstone & Thorpe (1982), Immunochemistry in Practice, Blackwell Scientific Publications, Oxford). The binding of antibodies to a solid support substrate is also well known in the art (for a general discussion, see Harlow & Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Publications, New York; and Borrebaeck (1992), Antibody Engineering—A Practical Guide, W.H. Freeman and Co.). The detectable moieties contemplated with the present invention can include, but are not limited to, fluorescent, metallic, enzymatic and radioactive markers such as biotin, gold, ferritin, alkaline phosphatase, β-galactosidase, peroxidase, urease, fluorescein, rhodamine, tritium, 14C and iodination.
Additional compounds which are also considered to be useful in the treatment of the diseases and disorders discussed herein may be antisense DNA molecules (which can be generated using the sequence in FIG. 1 by methods known in the art), catalytic RNAs such as ribozymes, polypeptides such as dominant negative peptides (which can be generated using the sequence in FIG. 2 by methods known in the art) or other polypeptide inhibitors. Antisense DNA molecules which comprise the siRNA sequences disclosed herein (with the appropriate nucleic acid modifications stemming from the differences between DNA and RNA) are particularly desirable and may be used in the same capacity as their corresponding siRNAs for all uses and methods disclosed herein.
Screening of TGase Inactivating Compounds
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 TGaseII, in particular compounds that modulate fibrotic disease or fibrosis-related pathology. The compounds to be screened comprise inter alia substances such as small chemical molecules, antibodies especially neutralizing antibodies, inhibitory polypeptides and dominant negative peptides.
The inhibitory activity of the compounds of the present invention on TGaseII polypeptide enzymatic activity or binding of the compounds of the present invention to TGaseII may be used to determine the interaction of an additional compound with the TGaseII polypeptide, e.g., if the additional compound competes with the antibodies or dominant-negative peptides of the present invention for TGaseII inhibition, 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 TGaseII polypeptide or displacement of binding compound from the TGaseII polypeptide in radioactive or fluorescent competition assays.
The present invention additionally provides for a process of obtaining a compound capable of inhibiting the enzymatic activity of a TGaseII polypeptide, preferably a human TGaseII polypeptide, that comprises the steps of:
(i) contacting the TGaseII polypeptide or cells expressing the TGaseII polypeptide with the compound, a lysyl donor and with a glutamyl donor which is a biological molecule;
(ii) removing excess lysyl donor from the mixture of step (i);
(iii) measuring the amount of the lysyl-glutamyl crosslinked material produced in the presence of the compound;
(iv) comparing the amount of lysyl-glutamyl crosslinked material measured in step (iii) to that measured in the absence of the compound under controlled conditions, wherein a decrease in the amount of lysyl-glutamyl crosslinked material is indicative of inhibition of the TGaseII polypeptide activity by the compound; and
(v) identifying the compound as an inhibitor of TGaseII polypeptide activity.
In one embodiment of all the processes described herein, the TGaseII polypeptide used in such process comprises consecutive amino acids, the sequence of which is set forth in SEQ ID NO: 2.
In another embodiment, the lysyl donor of step (i) is tagged and the measurement in step (iii) is of the tag related read-out, and wherein, a decrease in the read-out level is indicative of inhibition of the TGaseII polypeptide activity by the compound.
The glutamyl donor which is a biological molecule may be e.g. fibronectin. or collagen.
In another embodiment, the lysyl donor used in such process is cadaverin tagged with a dansyl fluorescent tag.
In another embodiment the lysyl donor used in such process is tagged with biotin, and in a specific embodiment, the lysyl donor is biotin cadaverine. Biotin is detected by streptavidin (or a modified streptavidin such as nutravidin) conjugated to readouts known in the art (e.g. conjugated to horseradish peroxidase, wherein the amount of horseradish peroxidase is quantified using tetramethylbenzidine as a substrate).
In yet another embodiment of the invention, the glutamyl donor in the provided process may be an extra-cellular matrix protein or a cellular protein. The extra cellular matrix protein is selected from the group consisting of: fibronectin, collagen, osteonectin, ECM-gel (e.g. Sigma E-1270), and the intracellular protein is selected inter alia from the group consisting of: RhoA, fialmin, spectrin, vimentin, HSP90, HSP60, (see Orru S, Caputo I, D'Amato A, Ruoppolo M, Esposito C Proteomics identification of acyl-acceptor and acyl-donor substrates for transglutaminase in a human intestinal epithelial cell line. Implications for celiac disease. J Biol. Chem. 2003 Aug. 22; 278(34):31766-73).
In a different embodiment, the compound is contacted with cells expressing the TGaseII polypeptide. The cells that may be used in such process may either be separated or present in a tissue. The cells may express the TGaseII polypeptide naturally or as a result of having been transfected with TGaseII gene, either transiently or stably or may over-express the TGaseII gene and the activity of TGaseII will be compared between over-expressors to normal TGaseII expressing cells. In a further embodiment, the glutamyl donor is either a natural product of the cells or it is added from outside.
In a different embodiment, the TGaseII polypeptide is contacted with the compound. The TGaseII polypeptide can be either immobilized or free in a solution. In an embodiment of the provided screening process the glutamyl donor is immobilized.
In a further embodiment, the processes provided for obtaining a compound capable of inhibiting the activity of human TGaseII may comprise a further step in which prior to step (i) TGaseII is contacted with a second compound known to bind TGaseII.
In one embodiment of the invention, the tag read-out in the provided processes described herein is the interaction of biotin with neutravidin conjugated to horseradish peroxidase, wherein the amount of cell-bound horseradish peroxidase is quantified using TMB (tetramethylbenzidine) as a substrate.
In yet a further embodiment, the compound obtained by the provided process inhibits the activity of the TGaseII polypeptide at least 2-fold more effectively than it inhibits the activity of at least one other member of the TGase family, and more preferably the inhibition is at least 10-fold, 50-fold and even 100-fold more effective. In such process, either the TGaseII polypeptide or the second compound may be immobilized.
In one another embodiment, the compound obtained by any of the provided processes is an antibody.
The invention further provides a process of obtaining a compound which modulates the activity of a TGaseII polypeptide, preferably a human TGaseII polypeptide, which comprises the steps of:
(i) contacting the TGaseII polypeptide with an interactor with which the TGaseII polypeptide interacts specifically in vivo;
(ii) contacting the TGaseII polypeptide or the interactor with said compound; and
(iii) measuring the effect of the compound on the interaction between TGaseII polypeptide and the interactor by measuring a parameter related to fibrosis; and
(iv) comparing the effect measured in step (iii) with the effect measured in the absence of the compound, a change in the effect measured indicating that the compound modulates the activity of the human TGaseII polypeptide.
In one embodiment of the invention the compound obtained by the provided process inhibits the activity of a human TGaseII polypeptide.
In another embodiment of the invention the TGaseII polypeptide in such process comprises consecutive amino acids, the sequence of which is set forth in SEQ ID NO: 2. In a further embodiment either the TGaseII polypeptide or the interactor are immobilized.
In a further embodiment of the invention, the parameter measured in any of the provided processes is related to a pathology characterized by TGaseII up-regulation in general and to fibrosis related pathologies as defined above, and also to cataract, cardiovascular diseases, neurological disorders, polyglutamine diseases including Huntington's disease (HD), spinobulbar muscular atrophy, dentatorubral-pallidoluysian atrophy and spinocerebellar ataxias (SCAs) 1, 2, 3, 6, 7 and 17), Alzheimer's and Parkinson's disease, coeliac disease and osteoarthritis. It is another object of the present invention to provide a process of obtaining a compound capable of inhibiting the activity of a human TGaseII polypeptide that comprises the steps of:
(i) contacting the TGaseII polypeptide or cells expressing the TGaseII polypeptide with a plurality of compounds, a tagged lysyl donor and with a glutamyl donor which is a biological molecule;
(ii) washing excess tagged lysyl donor from the mixture of step (i); and
(iii) measuring the tag related read-out in the presence of the plurality of compounds;
(iv) comparing the tag read-out measured in step (iii) to that measured in the absence of the plurality of compounds under controlled conditions, wherein a decrease in the read-out level is indicative of inhibition of the TGaseII polypeptide activity by the plurality of compounds;
(v) separately determining which compound or compounds present in the plurality inhibit the activity of a human TGaseII.
It is another aspect of the present invention to provide a cell-based process of obtaining a compound capable of inhibiting the activity of a TGaseII polypeptide, preferably human TGaseII polypeptide, that comprises the steps of:
(i) contacting cells expressing active TGaseII polypeptide with the compound and with a lysyl donor;
(iii) measuring the amount of lysyl-glutamyl crosslinked material produced in the presence of the compound;
(v) identifying the compound as an inhibitor of TGaseII polypeptide
It is another aspect of the present invention to provide a cell-based process of obtaining a compound capable of inhibiting the activity of a human TGaseII polypeptide that comprises the steps of:
a. contacting cells expressing the TGaseII polypeptide with a plurality of compounds and with a lysyl donor;
b. removing excess lysyl donor from the mixture of step (i); and
c. measuring the amount of the lysyl-glutamyl crosslinked material produced in the presence of the plurality of compounds;
d. comparing the amount of lysyl-glutamyl crosslinked material measured in step (iii) to that measured in the absence of the plurality of compounds under controlled conditions, wherein a decrease in the amount of lysyl-glutamyl crosslinked material is indicative of inhibition of the TGaseII polypeptide activity by the plurality of compounds; and
(e) separately determining which compound or compounds present in the plurality inhibit the activity of a human TGaseII.
Standard molecular biology techniques known in the art and not specifically described were generally followed as in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1989), and as in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989) and as in Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, New York (1988), and as in Watson et al., Recombinant DNA, Scientific American Books, New York and in Birren et al (eds) Genome Analysis: A Laboratory Manual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press, New York (1998) and methodology as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057 and incorporated herein by reference. Polymerase chain reaction (PCR) was carried out generally as in PCR Protocols: A Guide To Methods And Applications, Academic Press, San Diego, Calif. (1990). In situ (in cell) PCR in combination with Flow Cytometry can be used for detection of cells containing specific DNA and mRNA sequences (e.g. Testoni et al., 1996, Blood 87:3822.) Methods of performing RT-PCR are also well known in the art.
EXAMPLES Example 1 Design of Active siRNA Compounds
Using proprietary algorithms and the known sequence of cDNA of TGaseII (SEQ ID NO:1), the sequences of many potential siRNAs were generated. These are shown in the Tables below. Note that the abbreviations for species are: H: human; M: mouse; R: rat; G: guinea pig; C: cow.
Table A shows 18 siRNAs that have so far been chemically synthesized and tested for activity (see Example 2). All these siRNAs are 19-mers. In Table A the sense strands of siRNAs 1-18 have SEQ ID NOS: 3-20 respectively, and the antisense strands of siRNAs 1-18 have SEQ ID NOS: 21-38 respectively.
Table B below shows 153 additional 19-mer siRNAs, which have been generated by the proprietary algorithms but not yet tested for activity. In Table B, the sense strands of siRNAs 1-153 have SEQ ID NOS: 39-191, respectively, and the antisense strands of siRNAs 1-153 have SEQ ID NOS: 192-344 respectively.
Table C below shows 36 additional 21-mer siRNAs that have been generated by the proprietary algorithms. In Table C, the sense strands of siRNAs 1-36 have SEQ ID NOS: 345-380 respectively, and the antisense strands of siRNAs 1-36 have SEQ ID NOS: 381-416, respectively.
No. name Sense strand Antisense strand
1 TG_HMR1 ACAAGAGCGAGAUGAUCUG CAGAUCAUCUCGCUCUUGU
2 TG_HMRG1 AGAGCGAGAUGAUCUGGAA UUCCAGAUCAUCUCGCUCU
3 TG_HMG1 ACCCCAAGUUCCUGAAGAA UUCUUCAGGAACUUGGGGU
4 TG_HMG2 GCGAGAUGAUCUGGAACUU AAGUUCCAGAUCAUCUCGC
5 TG_HM1 CAAAUCCAUCAACCGUUCC GGAACGGUUGAUGGAUUUG
6 TG_M1 GAACAUCCAUGAGAAACUU AAGUUUCUCAUGGAUGUUC
7 TG_M2 CGACCUAUGCCAAGAGAAA UUUCUCUUGGCAUAGGUCG
8 TG_M3 GGUGUGAUUUGGAGAUUCA UGAAUCUCCAAAUCACACC
9 TG_M4 CCAACCACCUGAACAAACU AGUUUGUUCAGGUGGUUGG
10 TG_M5 GAACAAACUGGCAGAGAAA UUUCUCUGCCAGUUUGUUC
11 TG_M6 GCUCUGUCAAGUUCAUCAA UUGAUGAACUUGACAGAGC
12 TG_M7 GAUCCCUACUCUGAGAACA UGUUCUCAGAGUAGGGAUC
13 TG_M8 CCAGAGUGGUGACCAACUA UAGUUGGUCACCACUCUGG
14 TG_M9 GCAACAAGAGCGAGAUGAU AUCAUCUCGCUCUUGUUGC
15 TG_M10 GCAACCUGCUCAUCGAGUA UACUCGAUGAGCAGGUUGC
16 TG_H1 GUGACCUAACCACUUAGCA UGCUAAGUGGUUAGGUCAC
17 TG_H2 GUAGUGACCUAACCACUUA UAAGUGGUUAGGUCACUAC
18 TG_HG1 GACGCUGGGACAACAACUA UAGUUGUUGUCCCAGCGUC
NM_198951 NM_009373 NM_198851 M19646.1
No. Species (human) (mouse) (RAT) (GP)
5 hum_mus_rat [1077-1095] [1069-1087] [1033-1051]
2 hum_mus_rat_GP [1080-1098] [1072-1090] [1036-1054] [1048-1066]
3 hum_mus_GP [699-717] [691-709] [667-685]
4 hum_mus_GP [1083-1101] [1075-1093] [1051-1069]
5 hum_mus [1339-1357] [1331-1349]
6 mus 2599-2617
7 mus 164-182
8 mus 118-136
9 mus_rat [1468-1486] [1432-1450]
10 mus_rat [1478-1496] [1442-1460]
11 mus_rat 601-619
12 mus_rat 1692-1637
13 mus_rat [976-994] [940-958]
14 mus_rat [1066-1084] [1030-1048]
15 mus_rat [1018-1036] [982-1000]
16 hum 1773-1791
17 hum 1770-1788
18 hum_GP [816-834] [784-802]
No. Method Sense siRNA AntiSense siRNA
1 Cross Sp UUCCACUGCUGGGUGGAGU ACUCCACCCAGCAGUGGAA
2 Cross Sp AAGAGCGAGAUGAUCUGGA UCCAGAUCAUCUCGCUCUU
3 Cross Sp CAAGAGCGAGAUGAUCUGG CCAGAUCAUCUCGCUCUUG
4 Cross Sp AACCACCUGAACAAACUGG CCAGUUUGUUCAGGUGGUU
5 Cross Sp ACCACCUGAACAAACUGGC GCCAGUUUGUUCAGGUGGU
6 Cross Sp CUGGAACUUCCACUGCUGG CCAGCAGUGGAAGUUCCAG
7 Cross Sp AACUUCCACUGCUGGGUGG CCACCCAGCAGUGGAAGUU
8 Cross Sp AGGAGAAGAGCGAAGGGAC GUCCCUUCGCUCUUCUCCU
9 Cross Sp UGAUCUGGAACUUCCACUG CAGUGGAAGUUCCAGAUCA
10 Cross Sp GAGCGAGAUGAUCUGGAAC GUUCCAGAUCAUCUCGCUC
11 Cross Sp UCUGGAACUUCCACUGCUG CAGCAGUGGAAGUUCCAGA
12 Cross Sp GAUCUGGAACUUCCACUGC GCAGUGGAAGUUCCAGAUC
13 Cross Sp GAGAUGAUCUGGAACUUCC GGAAGUUCCAGAUCAUCUC
14 Cross Sp AGCGAGAUGAUCUGGAACU AGUUCCAGAUCAUCUCGCU
15 Cross Sp AGAUGAUCUGGAACUUCCA UGGAAGUUCCAGAUCAUCU
16 Cross Sp CCCCAAGUUCCUGAAGAAC GUUCUUCAGGAACUUGGGG
17 Cross Sp AUCUGGAACUUCCACUGCU AGCAGUGGAAGUUCCAGAU
18 Cross Sp AGCUUUGUGCUGGGCCACU AGUGGCCCAGCACAAAGCU
19 Cross Sp AACCCCAAGUUCCUGAAGA UCUUCAGGAACUUGGGGUU
20 Cross Sp CAGGAGAAGAGCGAAGGGA UCCCUUCGCUCUUCUCCUG
21 Cross Sp UUGUGCUGGGCCACUUCAU AUGAAGUGGCCCAGCACAA
22 Cross Sp GAACUUCCACUGCUGGGUG CACCCAGCAGUGGAAGUUC
23 Cross Sp GGAACUUCCACUGCUGGGU ACCCAGCAGUGGAAGUUCC
24 Cross Sp ACUUCCACUGCUGGGUGGA UCCACCCAGCAGUGGAAGU
25 Cross Sp CGAGAUGAUCUGGAACUUC GAAGUUCCAGAUCAUCUCG
26 Cross Sp AUGAUCUGGAACUUCCACU AGUGGAAGUUCCAGAUCAU
27 Cross Sp UUUGUGCUGGGCCACUUCA UGAAGUGGCCCAGCACAAA
28 Cross Sp GAUGAUCUGGAACUUCCAC GUGGAAGUUCCAGAUCAUC
29 Cross Sp UGGAACUUCCACUGCUGGG CCCAGCAGUGGAAGUUCCA
30 Cross Sp CUUUGUGCUGGGCCACUUC GAAGUGGCCCAGCACAAAG
31 Cross Sp GCUUUGUGCUGGGCCACUU AAGUGGCCCAGCACAAAGC
32 Cross Sp UCAGCACUAAGAGCGUGGG CCCACGCUCUUAGUGCUGA
33 Cross Sp AGAUCAGCACUAAGAGCGU ACGCUCUUAGUGCUGAUCU
34 Cross Sp GAAGAUCAGCACUAAGAGC GCUCUUAGUGCUGAUCUUC
35 Cross Sp GAUCAGCACUAAGAGCGUG CACGCUCUUAGUGCUGAUC
36 Cross Sp AGUAUGGCCAGUGCUGGGU ACCCAGCACUGGCCAUACU
37 Cross Sp AAGUAUGGCCAGUGCUGGG CCCAGCACUGGCCAUACUU
38 Cross Sp AUCAGCACUAAGAGCGUGG CCACGCUCUUAGUGCUGAU
39 Cross Sp AAGAUCAGCACUAAGAGCG CGCUCUUAGUGCUGAUCUU
40 Cross Sp GGAUCCUAGACAUCUGCCU AGGCAGAUGUCUAGGAUCC
41 Cross Sp CGCUGGGACAACAACUACG CGUAGUUGUUGUCCCAGCG
42 Cross Sp CUUGGAAUUUUGGGCAGUU AACUGCCCAAAAUUCCAAG
43 Cross Sp GUCAACUGCAACGAUGACC GGUCAUCGUUGCAGUUGAC
44 Cross Sp CAAGAACAUACCUUGGAAU AUUCCAAGGUAUGUUCUUG
45 Cross Sp CUCAUCGAGUACUUCCGCA UGCGGAAGUACUCGAUGAG
46 Cross Sp GCAUGGUCAACUGCAACGA UCGUUGCAGUUGACCAUGC
47 Cross Sp CGGAUGCUGUGUACCUGGA UCCAGGUACACAGCAUCCG
48 Cross Sp CCAUGACCAGAACAGCAAC GUUGCUGUUCUGGUCAUGG
49 Cross Sp ACAUACCUUGGAAUUUUGG CCAAAAUUCCAAGGUAUGU
50 Cross Sp UCGAGUACUUCCGCAAUGA UCAUUGCGGAAGUACUCGA
51 Cross Sp AUGGUCAACUGCAACGAUG CAUCGUUGCAGUUGACCAU
52 Cross Sp ACCUUGGAAUUUUGGGCAG CUGCCCAAAAUUCCAAGGU
53 Cross Sp CAAGUUCAUCAAGAACAUA UAUGUUCUUGAUGAACUUG
54 Cross Sp UUCAUCAAGAACAUACCUU AAGGUAUGUUCUUGAUGAA
55 Cross Sp AUACCUUGGAAUUUUGGGC GCCCAAAAUUCCAAGGUAU
56 Cross Sp AGUACUUCCGCAAUGAGUU AACUCAUUGCGGAAGUACU
57 Cross Sp UGGGUGGAGUCGUGGAUGA UCAUCCACGACUCCACCCA
58 Cross Sp CCCAUGACCAGAACAGCAA UUGCUGUUCUGGUCAUGGG
59 Cross Sp CCAAGUUCAUCAAGAACAU AUGUUCUUGAUGAACUUGG
60 Cross Sp GUCAACCCCAAGUUCCUGA UCAGGAACUUGGGGUUGAC
61 Cross Sp AUCAAGAACAUACCUUGGA UCCAAGGUAUGUUCUUGAU
62 Cross Sp UGGUCAACUGCAACGAUGA UCAUCGUUGCAGUUGACCA
63 Cross Sp CAUGGUCAACUGCAACGAU AUCGUUGCAGUUGACCAUG
64 Cross Sp UGGAAUUUUGGGCAGUUUG CAAACUGCCCAAAAUUCCA
65 Cross Sp ACUGCAACGAUGACCAGGG CCCUGGUCAUCGUUGCAGU
66 Cross Sp AUCCUAGACAUCUGCCUGA UCAGGCAGAUGUCUAGGAU
67 Cross Sp CAACUGCAACGAUGACCAG CUGGUCAUCGUUGCAGUUG
68 Cross Sp GGAAUUUUGGGCAGUUUGA UCAAACUGCCCAAAAUUCC
69 Cross Sp AGCGGAUGCUGUGUACCUG CAGGUACACAGCAUCCGCU
70 Cross Sp UUUGCCCACAUCACCAACA UGUUGGUGAUGUGGGCAAA
71 Cross Sp AAGAACAUACCUUGGAAUU AAUUCCAAGGUAUGUUCUU
72 Cross Sp UCCUAGACAUCUGCCUGAU AUCAGGCAGAUGUCUAGGA
73 Cross Sp CGAGUACUUCCGCAAUGAG CUCAUUGCGGAAGUACUCG
74 Cross Sp UGUCAACCCCAAGUUCCUG CAGGAACUUGGGGUUGACA
75 Cross Sp GACGUCUUUGCCCACAUCA UGAUGUGGGCAAAGACGUC
76 Cross Sp GAUGCUGUGUACCUGGACU AGUCCAGGUACACAGCAUC
77 Cross Sp AGAACAUACCUUGGAAUUU AAAUUCCAAGGUAUGUUCU
78 Cross Sp ACGUCUUUGCCCACAUCAC GUGAUGUGGGCAAAGACGU
79 Cross Sp AUCGAGUACUUCCGCAAUG CAUUGCGGAAGUACUCGAU
80 Cross Sp AAGUUCAUCAAGAACAUAC GUAUGUUCUUGAUGAACUU
81 Cross Sp UGGAGUCGUGGAUGACCAG CUGGUCAUCCACGACUCCA
82 Cross Sp GUGACAAGAGCGAGAUGAU AUCAUCUCGCUCUUGUCAC
83 Cross Sp UCAUCGAGUACUUCCGCAA UUGCGGAAGUACUCGAUGA
84 Cross Sp GUUCGUGCCAUCAAGGAGG CCUCCUUGAUGGCACGAAC
85 Cross Sp CAUCAAGAACAUACCUUGG CCAAGGUAUGUUCUUGAUG
86 Cross Sp UACCUUGGAAUUUUGGGCA UGCCCAAAAUUCCAAGGUA
87 Cross Sp GACAAGAGCGAGAUGAUCU AGAUCAUCUCGCUCUUGUC
88 Cross Sp CGUCUUUGCCCACAUCACC GGUGAUGUGGGCAAAGACG
89 Cross Sp AUGCUGUGUACCUGGACUC GAGUCCAGGUACACAGCAU
90 Cross Sp UUGGAAUUUUGGGCAGUUU AAACUGCCCAAAAUUCCAA
91 Cross Sp GCCAAGUUCAUCAAGAACA UGUUCUUGAUGAACUUGGC
92 Cross Sp AGUUCAUCAAGAACAUACC GGUAUGUUCUUGAUGAACU
93 Cross Sp GUCUUUGCCCACAUCACCA UGGUGAUGUGGGCAAAGAC
94 Cross Sp GGGAUCCUAGACAUCUGCC GGCAGAUGUCUAGGAUCCC
95 Cross Sp GAUCCUAGACAUCUGCCUG CAGGCAGAUGUCUAGGAUC
96 Cross Sp AUGUCAACCCCAAGUUCCU AGGAACUUGGGGUUGACAU
97 Cross Sp UCAUCAAGAACAUACCUUG CAAGGUAUGUUCUUGAUGA
98 Cross Sp UCAAGAACAUACCUUGGAA UUCCAAGGUAUGUUCUUGA
99 Cross Sp CAUCGAGUACUUCCGCAAU AUUGCGGAAGUACUCGAUG
100 Cross Sp GGAUGCUGUGUACCUGGAC GUCCAGGUACACAGCAUCC
101 Cross Sp GAUGUCAACCCCAAGUUCC GGAACUUGGGGUUGACAUC
102 Cross Sp CAUGACCAGAACAGCAACC GGUUGCUGUUCUGGUCAUG
103 Cross Sp GCCCAUGACCAGAACAGCA UGCUGUUCUGGUCAUGGGC
104 Cross Sp GAACAUACCUUGGAAUUUU AAAAUUCCAAGGUAUGUUC
105 Cross Sp GUGGAGUCGUGGAUGACCA UGGUCAUCCACGACUCCAC
106 Cross Sp UCAACUGCAACGAUGACCA UGGUCAUCGUUGCAGUUGA
107 Cross Sp AUUUUGGGCAGUUUGAAGA UCUUCAAACUGCCCAAAAU
108 Cross Sp AACAUACCUUGGAAUUUUG CAAAAUUCCAAGGUAUGUU
109 Cross Sp UGACAAGAGCGAGAUGAUC GAUCAUCUCGCUCUUGUCA
110 Cross Sp UUCGUGCCAUCAAGGAGGG CCCUCCUUGAUGGCACGAA
111 Cross Sp UCAGCCCCAUGUCCUGGAU AUCCAGGACAUGGGGCUGA
112 Cross Sp GGUCAACUGCAACGAUGAC GUCAUCGUUGCAGUUGACC
113 Cross Sp AAUUUUGGGCAGUUUGAAG CUUCAAACUGCCCAAAAUU
114 Cross Sp CAGCGGAUGCUGUGUACCU AGGUACACAGCAUCCGCUG
115 Cross Sp AACUGCAACGAUGACCAGG CCUGGUCAUCGUUGCAGUU
116 Cross Sp AUGACCAGAACAGCAACCU AGGUUGCUGUUCUGGUCAU
117 Cross Sp CAUACCUUGGAAUUUUGGG CCCAAAAUUCCAAGGUAUG
118 Cross Sp CUUUGCCCACAUCACCAAC GUUGGUGAUGUGGGCAAAG
119 Cross Sp GAGUACUUCCGCAAUGAGU ACUCAUUGCGGAAGUACUC
120 Cross Sp GGCAUGGUCAACUGCAACG CGUUGCAGUUGACCAUGCC
121 Cross Sp UCUUUGCCCACAUCACCAA UUGGUGAUGUGGGCAAAGA
122 Cross Sp GAAUUUUGGGCAGUUUGAA UUCAAACUGCCCAAAAUUC
123 Cross Sp CCUUGGAAUUUUGGGCAGU ACUGCCCAAAAUUCCAAGG
124 Cross Sp GUUCAUCAAGAACAUACCU AGGUAUGUUCUUGAUGAAC
125 Cross Sp UGUAUCGCCUCAGCCUGGA UCCAGGCUGAGGCGAUACA
126 Cross Sp GCUCGGCCAAGUUCAUCAA UUGAUGAACUUGGCCGAGC
127 Cross Sp GGCUCGGCCAAGUUCAUCA UGAUGAACUUGGCCGAGCC
128 Cross Sp GGACGCUGGGACAACAACU AGUUGUUGUCCCAGCGUCC
129 Cross Sp GGCAGUUUGAAGAUGGGAU AUCCCAUCUUCAAACUGCC
130 Cross Sp AGUUUGAAGAUGGGAUCCU AGGAUCCCAUCUUCAAACU
131 Cross Sp CAGUUUGAAGAUGGGAUCC GGAUCCCAUCUUCAAACUG
132 Cross Sp AGGGCUCGGCCAAGUUCAU AUGAACUUGGCCGAGCCCU
133 Cross Sp GCAGUUUGAAGAUGGGAUC GAUCCCAUCUUCAAACUGC
134 Cross Sp CAACCCCAAGUUCCUGAAG CUUCAGGAACUUGGGGUUG
135 Cross Sp GGGCAGUUUGAAGAUGGGA UCCCAUCUUCAAACUGCCC
136 Cross Sp ACGCUGGGACAACAACUAC GUAGUUGUUGUCCCAGCGU
137 Cross Sp UACUGCUGUGGCCCAGUUC GAACUGGGCCACAGCAGUA
138 Single Sp UUGCCCACAUCACCAACAA UUGUUGGUGAUGUGGGCAA
139 Single Sp CCAAGUACCUGCUCAACCU AGGUUGAGCAGGUACUUGG
140 Single Sp CGGCCAAGUUCAUCAAGAA UUCUUGAUGAACUUGGCCG
141 Single Sp CGAUGGGUCUGUGCACAAA UUUGUGCACAGACCCAUCG
142 Single Sp CAGUGACUUUGACGUCUUU AAAGACGUCAAAGUCACUG
143 Single Sp GCAUGAACAUGGGCAGUGA UCACUGCCCAUGUUCAUGC
144 Single Sp CAACUACGAGGCCAGUGUA UACACUGGCCUCGUAGUUG
145 Single Sp CAUGGGCAGUGACUUUGAC GUCAAAGUCACUGCCCAUG
146 Single Sp GCCCACAUCACCAACAACA UGUUGUUGGUGAUGUGGGC
147 Single Sp CGAACCACCUGAACAAACU AGUUUGUUCAGGUGGUUCG
148 Single Sp GGCCCGUUUUCCACUAAGA UCUUAGUGGAAAACGGGCC
149 Single Sp UGACGUCUUUGCCCACAUC GAUGUGGGCAAAGACGUCA
150 Single Sp GAACAUGGGCAGUGACUUU AAAGUCACUGCCCAUGUUC
151 Single Sp GCAGUGACUUUGACGUCUU AAGACGUCAAAGUCACUGC
152 Single Sp CUGUGUUCCUGGAGCAUUU AAAUGCUCCAGGAACACAG
153 Single Sp CAACCUGGAGCCUUUCUCU AGAGAAAGGCUCCAGGUUG
human GI Mouse GI Rat GI Guinea-pig Cow GI
No. 39777598 31543859 42476286 GI 387604 31343554 species
1 1100-1118 1092-1110 1056-1074 1068-1086 1046-1064 hmrgc
2 1079-1097 1071-1089 1035-1053 1047-1065 1025-1043 hmrgc
3 1078-1096 1070-1088 1034-1052 1046-1064 1024-1042 hmrgc
4 1478-1496 1470-1488 1434-1452 — 1424-1442 hmr
5 1479-1497 1471-1489 1435-1453 — 1425-1442 hmr
6 1093-1111 1085-1103 1049-1067 1061-1079 1039-1057 hm
7 1097-1115 1089-1107 1056-1071 1065-1083 1043-1061 hm
8 1185-1203 1177-1195 1141-1156 — 1131-1149 hm
9 1089-1107 1081-1099 — 1057-1075 1035-1053 hm
10 1081-1099 1073-1091 1037-1054 1049-1067 1027-1045 hm
11 1092-1110 1084-1102 1048-1066 1060-1078 1038-1056 hm
12 1090-1108 1082-1100 — 1058-1076 1036-1054 hm
13 1085-1103 1077-1095 1041-1059 1053-1071 1031-1049 hm
14 1082-1100 1074-1092 1038-1054 1050-1068 1028-1046 hm
15 1086-1104 1078-1096 1042-1060 1054-1072 1032-1050 hm
16 700-718 692-710 656-671 668-685 646-659 hm
17 1091-1109 1083-1101 — 1059-1077 1037-1055 hm
18 485-503 477-495 — — — hm
19 698-716 690-708 654-671 666-684 644-659 hm
20 1184-1202 1176-1194 1140-1156 1152-1170 1130-1148 hm
21 489-507 481-499 — 462-475 — hm
22 1096-1114 1088-1106 1056-1070 1064-1082 1042-1060 hm
23 1095-1113 1087-1105 1051-1069 1063-1081 1041-1059 hm
24 1098-1116 1090-1108 1056-1072 1066-1084 1044-1062 hm
25 1084-1102 1076-1094 1040-1054 1052-1070 1030-1048 hm
26 1088-1106 1080-1098 1044-1062 1056-1074 1034-1052 hm
27 488-506 480-498 — 462-474 — hm
28 1087-1105 1079-1097 1043-1061 1055-1073 1033-1051 hm
29 1094-1112 1086-1104 1050-1068 1062-1080 1040-1058 hm
30 487-505 479-497 — 462-473 — hm
31 486-504 478-496 — 462-472 — hm
32 1377-1395 1369-1387 1333-1351 — — hr
33 1374-1392 1366-1381 1330-1348 — — hr
34 1372-1390 1364-1381 1328-1346 — 1318-1329 hr
35 1375-1393 1367-1381 1331-1349 — — hr
36 918-936 918-928 874-892 886-904 864-882 hr
37 917-935 — 873-891 885-903 863-881 hr
38 1376-1394 1368-1386 1332-1350 — — hr
39 1373-1391 1365-1381 1329-1347 — 1319-1329 hr
40 663-681 — — — 609-627 hc
41 818-836 810-826 774-787 786-803 764-782 hc
42 636-654 628-646 592-610 — 582-600 hc
43 782-800 774-792 738-756 750-760 728-746 hc
44 625-643 — — 599-610 571-589 hc
45 1034-1052 1026-1042 990-1006 1002-1018 980-998 hc
46 777-795 770-784 733-748 746-760 723-741 hc
47 534-552 — — — 480-498 hc
48 1012-1030 1008-1022 972-986 984-998 958-976 hc
49 630-648 — — 599-610 576-594 hc
50 1038-1056 1030-1048 994-1006 1006-1018 984-1002 hc
51 779-797 771-789 735-753 747-760 725-743 hc
52 634-652 — — — 580-598 hc
53 616-634 608-621 572-585 584-595 562-580 hc
54 620-638 — — — 566-584 hc
55 632-650 — — 600-610 578-596 hc
56 1041-1059 — — — 987-1005 hc
57 1109-1127 1101-1119 1065-1083 1077-1095 1055-1073 hc
58 1011-1029 1003-1021 967-985 979-997 957-975 hc
59 615-633 608-621 572-585 583-595 561-579 hc
60 695-713 690-705 654-669 665-681 641-659 hc
61 623-641 — — 599-609 569-587 hc
62 780-798 772-790 736-754 748-760 726-744 hc
63 778-796 770-784 734-748 746-760 724-742 hc
64 638-656 630-646 594-612 — 584-602 hc
65 786-804 778-796 742-760 — 732-750 hc
66 665-683 — — — 611-629 hc
67 784-802 — — — 730-748 hc
68 639-657 631-646 595-613 615-625 585-603 hc
69 532-550 — — 500-511 478-496 hc
70 1574-1592 1566-1577 1530-1541 — 1520-1538 hc
71 626-644 — — 599-610 572-590 hc
72 666-684 — — — 612-630 hc
73 1039-1057 1031-1049 995-1006 1007-1018 985-1003 hc
74 694-712 690-704 654-668 665-680 640-658 hc
75 1568-1586 1560-1577 1524-1541 — 1514-1532 hc
76 536-554 — — — 482-500 hc
77 627-645 — — 599-610 573-591 hc
78 1569-1587 1561-1577 1525-1541 — 1515-1533 hc
79 1037-1055 1029-1047 993-1006 1005-1018 983-1001 hc
80 617-635 609-621 573-585 585-595 563-581 hc
81 1113-1131 1105-1123 1069-1087 1081-1099 1059-1077 hc
82 1074-1092 1069-1084 1033-1048 1045-1060 1020-1038 hc
83 1035-1053 1027-1042 991-1006 1003-1018 981-999 hc
84 1226-1244 1224-1236 1188-1200 1194-1212 1172-1190 hc
85 622-640 — — — 568-586 hc
86 633-651 — — — 579-597 hc
87 1076-1094 1069-1086 1033-1050 1045-1062 1022-1040 hc
88 1570-1588 1566-1577 1530-1541 — 1516-1534 hc
89 537-555 — — — 483-501 hc
90 637-655 629-646 593-611 — 583-601 hc
91 614-632 608-621 572-585 582-595 560-578 hc
92 618-636 610-621 574-585 — 564-582 hc
93 1571-1589 1566-1577 1530-1541 — 1517-1535 hc
94 662-680 — — — 608-626 hc
95 664-682 — — — 610-628 hc
96 693-711 690-703 649-667 665-679 639-657 hc
97 621-639 — — — 567-585 hc
98 624-642 — — 599-610 570-588 hc
99 1036-1054 1028-1042 992-1006 1004-1018 982-1000 hc
100 535-553 — — — 481-499 hc
101 692-710 690-702 648-666 665-678 638-656 hc
102 1013-1031 1008-1023 972-987 984-999 959-977 hc
103 1010-1028 1002-1020 966-984 978-996 956-974 hc
104 628-646 — — 599-610 574-592 hc
105 1112-1130 — — 1080-1098 1058-1076 hc
106 783-801 — — — 729-747 hc
107 642-660 636-646 600-613 615-628 588-606 hc
108 629-647 — — 599-610 575-593 hc
109 1075-1093 1069-1085 1033-1049 1045-1061 1021-1039 hc
110 1227-1245 1224-1237 1188-1201 1195-1213 1173-1191 hc
111 846-864 — — 814-832 792-810 hc
112 781-799 773-791 737-755 749-760 727-745 hc
113 641-659 636-646 600-613 615-627 587-605 hc
114 531-549 — — 499-511 477-495 hc
115 785-803 — — — 731-749 hc
116 1014-1032 1008-1024 972-988 984-1000 960-978 hc
117 631-649 — — 599-610 577-595 hc
118 1573-1591 1566-1577 1530-1541 — 1519-1537 hc
119 1040-1058 1032-1050 996-1006 1008-1018 986-1004 hc
120 776-794 770-784 732-748 746-760 722-740 hc
121 1572-1590 1566-1577 1530-1541 — 1518-1536 hc
122 640-658 636-646 600-613 615-626 586-604 hc
123 635-653 — — — 581-599 hc
124 619-637 611-621 575-585 — 565-583 hc
125 441-459 — 402-415 409-427 3571-3582 hg
126 609-627 608-619 572-583 577-595 555-573 hg
127 608-626 608-618 572-582 576-594 554-572 hg
128 815-833 810-825 774-787 783-801 764-779 hg
129 648-666 — — 616-634 594-612 hg
130 651-669 — 607-625 619-637 — hg
131 650-668 — — 618-636 596-614 hg
132 606-624 — — 574-592 552-570 hg
133 649-667 — — 617-635 595-613 hg
134 697-715 690-707 654-671 665-683 643-659 hg
135 647-665 — 603-621 615-633 593-611 hg
136 817-835 810-826 774-787 785-803 764-781 hg
137 1205-1223 1197-1213 1161-1177 1173-1191 1151-1169 hg
138 1575-1593 1567-1577 1531-1541 — 1521-1538 h
139 1677-1695 — — 1660-1672 — h
140 612-630 608-621 572-585 580-595 560-576 h
141 1324-1342 1317-1334 1281-1298 — — h
142 1558-1576 — 1517-1528 1535-1553 — h
143 1545-1563 — — 1530-1540 — h
144 244-262 239-250 — — — h
145 1552-1570 — — 1530-1547 — h
146 1577-1595 — — — 1523-1538 h
147 1476-1494 1470-1486 1434-1450 — 1424-1440 h
148 322-340 — — — — h
149 1567-1585 1560-1577 1523-1541 — 1514-1531 h
150 1549-1567 — — 1530-1544 — h
151 1557-1575 — 1517-1528 1534-1552 — h
152 1724-1742 — — — — h
153 1696-1714 — — 1105-1115 — h
1 Single Sp CUAGACAUCUGCCUGAUCCUU AAGGAUCAGGCAGAUGUCUAG
2 Single Sp CAACUGACAACAUGCUAGGUA UACCUAGCAUGUUGUCAGUUG
3 Single Sp CCAAGUUCAUCAAGAACAUAC GUAUGUUCUUGAUGAACUUGG
4 Single Sp CCUGAUCCUUCUAGAUGUCAA UUGACAUCUAGAAGGAUCAGG
5 Cross Sp ACAAGAGCGAGAUGAUCUGGA UCCAGAUCAUCUCGCUCUUGU
6 Single Sp CAAGAACAUACCUUGGAAUUU AAAUUCCAAGGUAUGUUCUUG
7 Single Sp CGGCCAAGUUCAUCAAGAACA UGUUCUUGAUGAACUUGGCCG
8 Single Sp CUGUGUUCCUGGAGCAUUUGU ACAAAUGCUCCAGGAACACAG
9 Cross Sp CAAGAGCGAGAUGAUCUGGAA UUCCAGAUCAUCUCGCUCUUG
10 Single Sp CAACUACGAGGCCAGUGUAGA UCUACACUGGCCUCGUAGUUG
11 Single Sp GGCCCGUUUUCCACUAAGAGA UCUCUUAGUGGAAAACGGGCC
12 Single Sp GCAACCUUCUCAUCGAGUACU AGUACUCGAUGAGAAGGUUGC
13 Cross Sp CGAGAUGAUCUGGAACUUCCA UGGAAGUUCCAGAUCAUCUCG
14 Cross Sp ACUUCCACUGCUGGGUGGAGU ACUCCACCCAGCAGUGGAAGU
15 Cross Sp AACCCCAAGUUCCUGAAGAAC GUUCUUCAGGAACUUGGGGUU
16 Cross Sp AUGAUCUGGAACUUCCACUGC GCAGUGGAAGUUCCAGAUCAU
17 Cross Sp GAACUUCCACUGCUGGGUGGA UCCACCCAGCAGUGGAAGUUC
18 Cross Sp UGGAACUUCCACUGCUGGGUG CACCCAGCAGUGGAAGUUCCA
19 Cross Sp AGCGAGAUGAUCUGGAACUUC GAAGUUCCAGAUCAUCUCGCU
20 Cross Sp GAUGAUCUGGAACUUCCACUG CAGUGGAAGUUCCAGAUCAUC
21 Cross Sp AGAGCGAGAUGAUCUGGAACU AGUUCCAGAUCAUCUCGCUCU
22 Cross Sp CUGGAACUUCCACUGCUGGGU ACCCAGCAGUGGAAGUUCCAG
23 Cross Sp UGAUCUGGAACUUCCACUGCU AGCAGUGGAAGUUCCAGAUCA
24 Cross Sp AAGAGCGAGAUGAUCUGGAAC GUUCCAGAUCAUCUCGCUCUU
25 Cross Sp CAGCUUUGUGCUGGGCCACUU AAGUGGCCCAGCACAAAGCUG
26 Cross Sp GCUUUGUGCUGGGCCACUUCA UGAAGUGGCCCAGCACAAAGC
27 Cross Sp AGAUGAUCUGGAACUUCCACU AGUGGAAGUUCCAGAUCAUCU
28 Cross Sp CUUUGUGCUGGGCCACUUCAU AUGAAGUGGCCCAGCACAAAG
29 Cross Sp GCGAGAUGAUCUGGAACUUCC GGAAGUUCCAGAUCAUCUCGC
30 Cross Sp AUCUGGAACUUCCACUGCUGG CCAGCAGUGGAAGUUCCAGAU
31 Cross Sp UCUGGAACUUCCACUGCUGGG CCCAGCAGUGGAAGUUCCAGA
32 Cross Sp GAUCUGGAACUUCCACUGCUG CAGCAGUGGAAGUUCCAGAUC
33 Cross Sp GAGAUGAUCUGGAACUUCCAC GUGGAAGUUCCAGAUCAUCUC
34 Cross Sp AGCUUUGUGCUGGGCCACUUC GAAGUGGCCCAGCACAAAGCU
35 Cross Sp GAGCGAGAUGAUCUGGAACUU AAGUUCCAGAUCAUCUCGCUC
36 Cross Sp AACUUCCACUGCUGGGUGGAG CUCCACCCAGCAGUGGAAGUU
human mouse rat pig Cow
No. 39777598 31543859 42476286 387604 31343554 file
1 668-688 — — — 614-630 hmrgc
2 1752-1772 — — — — hmrgc
3 615-635 608-621 572-585 583-595 561-581 hmrgc
4 679-699 — — — — hmrgc
5 1077-1097 1069-1089 1033-1053 1045-1065 1023-1043 hmrgc
6 625-645 — — 599-610 571-591 hmrgc
7 612-632 608-621 572-585 580-595 560-578 hmrgc
8 1724-1744 — — — — hmrgc
9 hmrgc
10 244-264 239-250 — — — hmrgc
11 322-342 — — — — hmrgc
12 1026-1046 1018-1038 982-1002 994-1014 972-992 hmrgc
13 1084-1104 1076-1096 1040-1060 1052-1072 1030-1050 hm
14 1098-1118 1090-1110 1056-1074 1066-1086 1044-1064 hm
15 698-718 690-710 654-671 666-685 644-659 hm
16 1088-1108 1080-1100 1044-1064 1056-1076 1034-1054 hm
17 1096-1116 1088-1108 1056-1072 1064-1084 1042-1062 hm
18 1094-1114 1086-1106 1050-1070 1062-1082 1040-1060 hm
19 1082-1102 1074-1094 1038-1054 1050-1070 1028-1048 hm
20 1087-1107 1079-1099 1043-1063 1055-1075 1033-1053 hm
21 1080-1100 1072-1092 1036-1054 1048-1068 1026-1046 hm
22 1093-1113 1085-1105 1049-1069 1061-1081 1039-1059 hm
23 1089-1109 1081-1101 — 1057-1077 1035-1055 hm
24 1079-1099 1071-1091 1035-1054 1047-1067 1025-1045 hm
25 484-504 476-496 — 452-472 — hm
26 486-506 478-498 — 462-474 — hm
27 1086-1106 1078-1098 1042-1062 1054-1074 1032-1052 hm
28 487-507 479-499 — 462-475 — hm
29 1083-1103 1075-1095 1039-1059 1051-1071 1029-1049 hm
30 1091-1111 1083-1103 1047-1067 1059-1079 1037-1057 hm
31 1092-1112 1084-1104 1048-1068 1060-1080 1038-1058 hm
32 1090-1110 1082-1102 1046-1066 1058-1078 1036-1056 hm
33 1085-1105 1077-1097 1041-1061 1053-1073 1031-1051 hm
34 485-505 477-497 — 453-473 — hm
35 1081-1101 1073-1093 1037-1054 1049-1069 1027-1047 hm
36 1097-1117 1089-1109 1056-1073 1065-1085 1043-1063 hm
Example 2 Testing the siRNA Compounds for Anti-TGaseII Activity
I. Preparation of Working Solutions of siRNAs (Double-Stranded Oligonucleotides)
Lyophilized oligonucleotides were dissolved in RNAse-free double-distilled water to produce a final concentration of 100 uM. The diluted oligonucleotides were kept at room temperature for 15 min and immediately frozen in liquid nitrogen. The oligonucleotides were stored at −80° C. and diluted before use with PBS.
II. Establishment of NRK49 Cell Line Stably Overexpressing rat TGaseII
The full length cDNA of rat TGaseII cloned in PLXSN vector (CLONTECH) was used for transfection into a Rat1 fibroblast cell line. Lipofectamine reagent (Invitrogen) was used as the transfection reagent using the protocol described below. 48 hr following transfection, selection of stable clones was performed using G418 antibiotic (neomycin). The stable overexpression of TGaseII in this polyclonal population was verified by Western blotting and TGaseII activity assay. The cells were designated as NRK49-TGaseII (TGASE-OE®).
III. Activity Assay for TGaseII in NRK49
4×103 NRK49-rat TGASE-OE cells were seeded per well in a 96 well plate. TGaseII activity was measured using a cell-based assay for TGaseII activity. Briefly, the growth medium was replaced with cell labeling medium (DMEM in 0.1% BSA, 25 uM BC, 10 uM Calcimycin, 16.2 mM CaCl2) for 45 min at 37° C. 45 min later, the cells were fixed with 4% formaldehyde, treated with 0.6% H2O2 and stained with Netravidin-HRP Ab in order to test the cross-linking of biotin-labeled cadaverin. The absorbance was read at λ=450 nm. For normalization, the cells were stained with Hoechst dye solution. The activity of TGaseII is 75 fold higher in NRK49-TGaseII cells compared to NRK49 control cells.
IV. Transfection by siRNA Oligonucleotides Using Lipofectamine-2000 Reagent
2×105 cells were seeded per well in 6 well-plates. After 24 hrs, the cells were transfected with TGaseII specific siRNA oligonucleotides using Lipofectamine-2000 reagent (Invitrogen) according to the following procedure:
1. Before transfection, the cell medium was replaced with 1500 ul of fresh medium without antibiotics.
2. In a sterile plastic tube, Lipofectamine2000 reagent (the amount is calculated according to 5 ul per well) was added to 250 ul of serum-free medium, and incubated for 5 min at room temperature.
3. In another tube, the siRNA oligonucleotides (varying amounts to fit the desired final concentration per well) were added to 250 ul of serum-free medium.
4. Lipofectamine2000 complex was combined with the siRNA solution and incubated for 20 min at room temperature.
5. The resulting mixture was added dropwise to the cells, and the cells were incubated at 37° C. until analysis of siRNA activity.
V. Testing the Activity of Anti TGaseII siRNAs in Transfected Human, Mouse and Rat Cells
The activity of TGaseII siRNAs listed in Table A was tested in 3 different cell lines originating from different species:
1. Mouse-specific TGaseII siRNAs were tested in mouse cells, which express mouse TGaseII—NMUMG cells.
2. Human-specific TGaseII siRNA were tested in human HeLa cells which express human TgaseII; see FIG. 3.
3. Rat-specific TGaseII siRNAs were tested in NRK49 overexpressing rat TGaseII (TGASE-OE® cells).
48 h to 6 days after transfection of siRNAs, the cells were harvested and expression of TGaseII protein was examined by Western blot analysis with specific antibodies. The decrease in TGaseII polypeptide expression in the cells transfected with siRNA oligonucleotides specific to TGaseII as compared to controls indicated that siRNAs elicited inhibitory effect on TGaseII protein expression, which can be quantified.
Table D below summarizes the information about TGaseII inhibitory activity of each siRNA oligonucleotide. The names of oligonucleotides in Table D correspond to the names used in Table A. Note that the comparison is to the activity in each cell line transfected by different siRNAs, and not between cell lines. The presently most preferred human siRNA is TG_HMRG1, which elicited high TGaseII inhibitory effect and also inhibited mouse, rat and guinea pig to TGaseII. Other preferred human siRNAs are TG_HMG1 and TG_HM1.
name Species TGII TGII TGII
TG_HMR1 hum_mus_rat + − +
TG_HMRG1 hum_mus_rat_GP ++++ +++ ++++
TG_HMG1 hum_mus_GP ++++ + ND
TG_HMG2 hum_mus_GP ++++ ++++ ND
(toxic) (toxic)
TG_HM1 hum_mus ++++ + ND
TG_M1 Mus + +++ ND
TG_M2 Mus − +++ ND
TG_M3 Mus +++ ++++ ND
TG_M4 mus_rat ND ++ ++
TG_M5 mus_rat +++ +++ +++
TG_M6 mus_rat ND ++ +++
TG_M7 mus_rat ND ++ ++++
TG_M8 mus_rat ND ++ ++
TG_M9 mus_rat +++ +++ +++
TG_M10 mus_rat ND + +++
TG_H1 Hum − ND ND
TG_H2 Hum − ND ND
TG_HG1 hum_GP ++ ND ND
Example 3 Animal Models of Kidney Fibrosis
Testing of the in vivo therapeutic activity of anti-TGaseII siRNAs, or of neutralizing antibodies against TGaseII or of other TGaseII inhibitors may be done in the following animal models of kidney fibrosis in which disease-associated overexpression of TGaseII was found (as detected by in situ hybridization; see below).
A. Rat diabetic and Aging Models of Kidney Fibrosis
A1. ZDF Rats
Kidney samples of 9-month-old ZDF rats (Zucker diabetic fatty rats) presented hydronephrotic changes with dilated calyces. Microscopically these samples displayed the features of glomerulosclerosis and tubulointerstitial fibrosis. In accordance with these morphological changes, the expression of fibrosis marker genes (osteopontin (OPN), transforming growth factor β1 (TGF-β1) and procollagen α1(I) (Col1)), as measured by in situ hybridization, was significantly increased compared to normal kidneys. Strong OPN expression was detectable in all tubular structures in both kidney cortex and medulla. TGF-β1 expression was widespread throughout interstitial cells. Some epithelial cells also showed TGF-β1 expression. Col1 expression was detectable by in situ hybridization in most interstitial cells within the medulla, while cortical expression was “focal”.
A2. Aged fa/fa (Obese Zucker) Rats
Fa/fa rats are genetically deficient for leptin receptor; and, as a result, develop insulin resistant diabetes (type II diabetes) with progressive diabetic nephropathy,
Kidney samples of 12-month-old fa/fa rats presented with high degree glomerulosclerosis and diffuse tubulointerstitial fibrosis throughout both the cortex and the medulla. The pattern of fibrosis marker gene expression corresponded to the morphological changes. OPN was expressed in tubular structures in the cortex and in the medulla. Multiple interstitial cells expressed TGF-β1. Significantly, multiple foci and single interstitial cells showed strong Col1 expression in both cortex and medulla so that the number of Col1-expressing cells appeared to be higher in fa/fa kidney samples than in ZDF samples.
A3. Aged Sprague-Dawley (SD) (Normal) Rats
Kidney samples of aged SD rats also showed increased accumulation of collagen in glomeruli and interstitial space and increased expression of fibrosis marker genes. Significantly, the intensity of fibrotic changes varied among samples. Thus, one of the four samples studied displayed very few changes compared with young animals; in the second sample, fibrotic changes were confined only to “polar” regions, and the remaining two samples displayed uniform accumulation of collagen and elevated expression of marker genes throughout the sections
A4. Goto Kakizaki (GK)48-Week-Old Rats
GK rats are an inbred strain derived from Wistar rats, selected for spontaneous development of NIDDM (diabetes type II). Kidney samples from both GK and control Wistar 48-week-old rats showed accumulation of collagen in glomeruli and interstitial space. This accumulation was more pronounced in the GK samples.
TGaseII Hybridization Pattern
TGaseII-specific hybridization signal was associated with fibrotic phenotype (diffuse or focal) in kidneys of both healthy and diabetic rats. Non-fibrotic kidney samples demonstrate a diffuse, low but detectable TGaseII-specific hybridisation signal over proximal tubules. By 12 months of age, a stronger signal is concentrated over interstitial cells and epithelial lining of distended tubules that show clear signs of atrophy. In chronic renal failure sample (kidney from two years old rat), a strong TGaseII expression is visible in tubular profiles lined by atrophic or proliferating epithelium cells within surrounding interstitium. Similarly, ZDF samples showed intensification of the hybridization signal along with the progression of the fibrosis (or aging).
B. Induced Models of Kidney Fibrosis
B1. Permanent Unilateral Ureteral Obstruction (UUO) Model
Unilateral ureteral obstruction (UUO) is a model of acute interstitial fibrosis occurring in healthy non-diabetic animals. Renal fibrosis develops within days following the obstruction.
To produce the model, rats were anaesthetized with Ketamin/Xylazine and the abdominal cavity was opened. After exposure, the right ureter was ligated with a suture at the level of the lower kidney pole. In sham-operated rats, the ureter was exposed but not ligated. The study was terminated at different timepoints following ureteral obstruction (e.g., at 24 hr, 5 days, 10 days, 15 days, 20 days and 25 days), and the kidneys were removed and examined.
Histological examination revealed that permanent UUO results in a rapid activation of collagen synthesis by interstitial cells in both medulla and cortex. By 20-25 days of UUO, significant amounts of interstitial collagen were deposited in the interstitial space, whereas glomerular accumulation of collagen was confined to the outer capsule.
In situ hybridization analysis of obstructed kidney samples indicated rapid and significant changes in the pattern of TGaseII expression dependent on the duration of UUO. After 24 hours of UUO, a very strong hybridization signal was detected over cortical tubular epithelium, whereas medullar epithelial cells showed much weaker signal. At 5 and 10 days of UUO, the “foci” of strong hybridization signal were associated with the areas of developing tubulointerstitial fibrosis where the signal was located to both epithelial and interstitial cells. Similar results were observed in mice exposed to the same UUO model.
B2. 5/6 Nephrectomy
5/6 nephrectomy is another useful animal model for chronic renal insufficiency (CRI) in which fibrosis is evident.
In summary, the results suggest that an increase in TGaseII expression and activity level accompanies fibrotic changes in the kidneys.
Example 4 Establishment of TGaseII Role in Renal Fibrosis Employing the UUO Model Using TGaseII Knock-Out Mice
TgaseII knock-out mice were obtained from Dr. Melino (De Laurenzi V, Melino G., Gene disruption of tissue transglutaminase, Mol Cell Biol. 2001 January; 21(1):148-55). The mice appeared phenotypically normal. Also no morphological changes were observed in kidneys obtained from these mice.
TGaseII KO mice were then used for the analysis of the development of kidney fibrosis following induction of UUO by assessing kidney collagen accumulation compared to normal mice The results clearly demonstrated that disruption of TGaseII results in statistically significant reduced collagen accumulation following both three (3) and seven (7) days of obstruction, as compared to collagen accumulation in obstructed kidneys of wild type mice, thus implicating TGaseII as a functionally important factor in kidney fibrosis. It is thus suggested that inhibition of TGaseII activity may have an anti-fibrotic therapeutic effect.
Example 5 Inhibition of TGaseII Activity Using Monoclonal Neutralizing Antibodies
Recombinant monoclonal human-anti-human antibodies against TGaseII were obtained by screening of a phage display library. 720 clones were screened for binding human TGaseII. Seven (7) clones were selected which showed intense binding to the enzyme. The activity assay demonstrated that these antibodies inhibited the crosslinking activity of human TGaseII enzyme at a range of concentrations (1-5 nM).
1. A double-stranded RNA compound having the structure:
wherein each of N and N′ is a ribonucleotide which may independently be modified or unmodified in its sugar residue;
wherein each of x and y is an integer from 19 to 40;
wherein the sequence of (N)x comprises the sequence 5′ UUCCAGAUCAUCUCGCUCU 3′ (SEQ ID NO: 22)
and wherein the sequence of (N′)y comprises the sequence 5′ AGAGCGAGAUGAUCUGGAA 3′ (SEQ ID NO:4).
3. The compound of claim 1, wherein x=y.
4. The compound of claim 1, wherein x=y=19.
5. The compound of claim 1, wherein both Z and Z′ are absent.
6. The compound of claim 1, wherein one of Z or Z′ is present.
7. The compound of claim 1, wherein all of the ribonucleotides in (N)x or (N′)y or both are unmodified in their sugar residues.
8. The compound of claim 1, wherein at least one ribonucleotide is modified in its sugar residue.
9. The compound of claim 8, wherein the modification comprises a modification at the 2′ position of the sugar residue.
10. The compound of claim 9, wherein the modification at the 2′ position of the sugar residue results in the presence of a moiety selected from the group comprising amino, fluoro, alkoxy and alkyl groups.
11. The compound of claim 10, wherein the alkoxy group is a methoxy (2′-O-methyl).
12. The compound of claim 1, wherein in alternating ribonucleotides in both the antisense strand and the sense strand their sugar residues are modified.
13. The compound of claim 12, wherein each of the modified alternating ribonucleotides is modified at the 2′ position of its sugar moiety.
14. A composition comprising a double-stranded siRNA oligoribonucleotide compound of claim 1 and a carrier.
15. The compound of claim 13, wherein each of the ribonucleotides at the 5′ terminus and at the 3′ terminus of the antisense strand is modified in its sugar residue, and each ribonucleotide at the 5′ terminus and at the 3′ terminus of the sense strand is unmodified in its sugar residue.
16. A double-stranded RNA compound having the structure:
5′UUCCAGAUCAUCUCGCUCU3′(SEQ ID NO: 22) antisense strand
3′AAGGUCUAGUAGAGCGAGA5′(SEQ ID NO: 4) sense strand
wherein each of A, C, U and G is an unmodified or 2′-O-Methyl sugar modified ribonucleotide and each consecutive ribonucleotide is joined to the next ribonucleotide by a phosphodiester bond; and
wherein alternating ribonucleotides in both the antisense strand and the sense strand are 2′-O-Methyl sugar modified ribonucleotides, a 2-O-Methyl sugar modified ribonucleotide is present at both the 5′ terminus and the 3′ terminus of the antisense strand and an unmodified ribonucleotide is present at both the 5′ terminus and the 3′ terminus of the sense strand.
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US11045780 US20050197310A1 (en) 2004-01-30 2005-01-27 Oligoribonucleotides and methods of use thereof for treatment of fibrotic conditions and other diseases
US12072941 US7939652B2 (en) 2004-01-30 2008-02-28 Oligoribonucleotides and methods of use thereof for treatment of fibrotic conditions and other diseases
US13092501 US8198258B2 (en) 2004-01-30 2011-04-22 Oligoribonucleotides and methods of use thereof for treatment of fibrotic conditions and other diseases
US11045780 Continuation US20050197310A1 (en) 2004-01-30 2005-01-27 Oligoribonucleotides and methods of use thereof for treatment of fibrotic conditions and other diseases
US13092501 Division US8198258B2 (en) 2004-01-30 2011-04-22 Oligoribonucleotides and methods of use thereof for treatment of fibrotic conditions and other diseases
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US13092501 Active US8198258B2 (en) 2004-01-30 2011-04-22 Oligoribonucleotides and methods of use thereof for treatment of fibrotic conditions and other diseases
US (3) US20050197310A1 (en)
EP (2) EP1758998B1 (en)
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DE (1) DE602005025347D1 (en)
WO (1) WO2005072057A3 (en)
US20100266574A1 (en) * 2005-06-10 2010-10-21 Orna Mor Oligoribonucleotides and Methods of Use Thereof for Treatment of Fibrotic Conditions and Other Diseases
US20050197310A1 (en) * 2004-01-30 2005-09-08 Orna Mor Oligoribonucleotides and methods of use thereof for treatment of fibrotic conditions and other diseases
US8299040B2 (en) * 2006-10-18 2012-10-30 Board Of Regents, The University Of Texas System Methods for treating cancer targeting transglutaminase
US20100273854A1 (en) * 2007-06-15 2010-10-28 Hagar Kalinski Compositions and methods for inhibiting nadph oxidase expression
US9051568B2 (en) 2008-04-25 2015-06-09 Rutgers, The State University Of New Jersey Anti-sense microrna expression vectors
WO2012108841A1 (en) * 2011-02-09 2012-08-16 Singapore Health Services Pte. Ltd. Transglutaminase-2 inhibitors and uses thereof
JP6129844B2 (en) * 2011-09-14 2017-05-17 ラナ セラピューティクス インコーポレイテッド Multimeric oligonucleotide compound
WO2013142514A1 (en) * 2012-03-19 2013-09-26 Isis Pharmaceuticals, Inc. Methods and compositions for modulating alpha-1-antitrypsin expression
WO1998004245A1 (en) 1996-07-25 1998-02-05 The Victoria University Of Manchester Use of transglutaminase modulators to promote wound healing
WO2003070197A2 (en) 2002-02-20 2003-08-28 Sirna Therapeutics, Inc. RNA INTERFERENCE MEDIATED INHIBITION OF TGF-BETA AND TGF-BETA RECEPTOR GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
GB2397818B (en) 2002-02-20 2005-03-09 Sirna Therapeutics Inc Rna interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US4968713A (en) 1989-07-31 1990-11-06 Merck & Co., Inc. Certain imidazole compounds as transglutaminase inhibitors
US5021440A (en) 1989-07-31 1991-06-04 Merck & Co., Inc. Imidazole compounds and their use as transglutaminase inhibitors
US8574827B2 (en) * 2002-10-29 2013-11-05 Rigel Pharmaceuticals, Inc. Modulators of angiogenesis and tumorigenesis
Bartel, P. D. (2004) "MicroRNAs: Genomics, Biogenesis, Mechanism, and Function" Cell 116:281-297.
Bernstein, E. et al. (2001) "Role for a Bidentate Ribonuclease in the Initiation Step of RNA Interference" Nature 409:363-366.
Chalk, M. A. et al. (2004) "Improved and Automated Prediction of Effective siRNA" Biochemical and Biophysical Research Communication319:264-274.
Chen, J.S.K. et al. (1999) "Tissue Transglutaminase: An Enzyme with a Split Personality" Int. Jour. Of Biochem. & Cell Biology 31:817-836.
Citron, B.A. et al. (2002) "Protein Crosslinking, Tissue Transglutaminase, Alternative Splicing and Neurodegeneration" Neurochemistry International 40:69-78.
Communication pursuant to Rule 71(3) EPC issued by the European Patent Office on Jun. 2, 2010 in connection with European Patent Application No. 05703145.2.
Dieterich, W. et al. (1997) "Identification of Tissue Transglutaminase as the Autoantigen of Celiac Disease" Nature Medicine 3(7):797-801.
Edited by Beers et al., (1999), "The Merck Manual of Diagnosis and Therapy", 17 ed. Merck Research Laboratories, Whitehouse Station, NJ, Chapter 222, pp. 1845-1847.
Elbashir SM, et al. (2001) Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 20(23):6877-6888.
Elbashir, S.M. et al. (2001) "Duplexes of 21-Nucleotides RNAs Mediate RNA Interference in Cultured Mammalian Cells" Nature 411:494-498.
Examination Report issued by the European Patent Office on Feb. 26, 2009 in connection with European Application No. 05703145.2.
Examination Report issued by the European Patent Office on Jun. 2, 2008 in connection with European Application No. 05703145.2.
Fesus L, et al. (2002) "Transglutaminase 2: an enigmatic enzyme with diverse functions," Trends in Biochem. Sci.; 27(10):534-539.
Fire, A. et al. (1998) "Potent and Specific Genetic Interference by Double-Stranded RNA in Caenorhabditis elegans" Nature 391:806-811.
Fraij BM, et al. (1992) A retinoic acid-inducible mRNA from human erythroleukemia cells encodes a novel tissue transglutaminase homologue. J. Biol. Chem. 267(31):22616-22623.
Greenberg, B. (2001) "Treatment of Heart Failure: State of the Art and Prospective" Journal of Cardiovascular Pharmacology 38:S59-S63.
Grenard, P. (2001) "Transglutaminase-Mediated Cross-Linking is Involved in the Stabilization of Extracellular Matrix in Human Liver Fibrosis" Journal of Hepatology 35:367-375.
Griffin, M. et al. (1979) "Chances in Transglutaminase Activity in an Experimental Model of Pulmonary Fibrosis Induced by Paraquat" Br. J. Exp. Path. 60:653-661.
Hannon, G.J. (2002) "RNA Interference" Nature 418:244-251.
Hwang, K.C. et al. (1996) "alpha1-Adrenergic Receptor Coupling With Gh in the Failing Human Heart" American heart Association, Inc. 94:718-726.
Hwang, K.C. et al. (1996) "α1-Adrenergic Receptor Coupling With Gh in the Failing Human Heart" American heart Association, Inc. 94:718-726.
International Preliminary Report on Patentability issued by the International Bureau of WIPO on Jun. 19, 2007 in connection with International Application No. PCT/IL05/00102.
International Search Report issued by the International Searching Authority (ISA/US) on May 18, 2007 in connection with International Application No. PCT/IL05/00102.
Johnson, T.S. et al. (1997) "The Role of Transglutaminase in the Rat Subtotal Nephrectomy Model of Renal Fibrosis" J. Clin.Invest. 99(12):2950-2960.
Johnson, T.S. et al. (1999) "Transglutaminase Transcription and Antigen Translocation in Experimental Renal Scarring" J. Am. Soc. Nephrol. 10:2146-2157.
Karpuj, M.V. et al. (2002) "Prolonged Survival and Decreased Abnormal Movements in Transgenic Model of Huntington Disease, with Administration of the Transglutaminase Inhibitor Cystamine" Nature Medicine 8(2):143-149.
Lesort, M. et al. (2000) "Tissue Transglutaminase: A Possible Role in Neurodegenerative Diseases" Progress in Neurobiology 61:439-463.
Levenkova, N. et al. (2004) "Gene Specific siRNA Selector" Bioinformatics 20(3):430-432.
Mastroberardino PG, et al., (2002) "‘Tissue’ transglutaminase ablation reduces neuronal death and prolongs survival in a mouse model of Huntington's disease," Cell Death and Differentiation, 9(9):873-880.
Mastroberardino PG, et al., (2002) "'Tissue' transglutaminase ablation reduces neuronal death and prolongs survival in a mouse model of Huntington's disease," Cell Death and Differentiation, 9(9):873-880.
McManus, T. M. (2002) "Gene Silencing in Mammals by Small Interfering RNAs" Nature Reviews 3:737-747.
Mirza, A. et al. (1997) "A Role for Tissue Transglutaminase in Hepatic Injury and Fibrogenesis, and its Regulation by NF-kB" The American Physiological Society 281-288.
Piacentini, M. et al. (2002) "Transglutaminase Overexpression Sensitizes Neuronal Cell Lines to Apoptosis by Increasing Mitochondrial Membrane Potential and Cellular Oxidative Stress" Journal of Neurochemistry 81:1061-1072.
Reich, S.J., et al. (2003) "Small Interfering RNA (siRNA)Targeting VEGF Effectively Inhibits Ocular Neovascularization in a Mouse Model" Molecular Vision 9:210-216.
Response to the Communication Under Rule 71(3) EPC and Request for Amendments pursuant to Rule 71(4) EPC submitted Sep. 7, 2010 in connection with European Patent Application No. 05703145.2.
Shin, D.M. et al. (2004) "Cell Type-Specific Activation of Intracellular Transglutaminase 2 by Oxidative Stress or ultraviolet Irradiation" The Journal of Biological Chemistry 279(15):15032-15039.
Shridas, P. et al. (2001) "Transglutaminase-Mediated Cross-Linking of alpha-Crystallin: Structural and Functional Consequences" FEBS Letters 499:245-250.
Shridas, P. et al. (2001) "Transglutaminase-Mediated Cross-Linking of α-Crystallin: Structural and Functional Consequences" FEBS Letters 499:245-250.
Sioud, M. et al. "Potential Design Rules and Enzymatic Synthesis of siRNAs" Methods in Molecular Biology 252:457-468, 2004.
Soutschek, J. et al. (2004) "Therapeutic Silencing of an Endogeneous Gene by Systemic Administration of Modified siRNAs" Nature 432:173-178.
Supplementary European Search Report issued by the European Patent Office on Jan. 22, 2008 in connection with European Application No. 05703145.2-2405.
Takeuchi, N. et al. (2000) "Crystallin Proteins in Lenses of Hereditary Cataractous Rat, ICR/f" Biol. Pharm. Bull. 23(3):283-290.
Tolentino, M.J. (2004) "Intravitreal Injection of Vascular Endothelial Growth Factor Small Interfering RNA Inhibits Growth and Leakage in a Nonhuman Primate, Laser-Induced Model of Choroidal Neovascularization" Retina, The Journal of Retinal and Vitreous Disease 24(1):132-138.
Ui-Tei, K. et al. (2004) "Guidelines for the Selection of Highly Effective siRNA Sequences for Mammalian and Chick RNA Interference" Nucleic Acids Research 32(3):936-948.
Vickers et al The Journal of Biological Chemistry vol. 278(9):7108-7118, Feb. 28, 2003. *
Wan, X.H., et al. (2002) "Enhanced Expression of Transglutaminase 2 in Anterior Polar Cataracts and its Induction by TGB-beta in vitro" British Journal of Ophthalmology 86:1293-1298.
Wan, X.H., et al. (2002) "Enhanced Expression of Transglutaminase 2 in Anterior Polar Cataracts and its Induction by TGB-β in vitro" British Journal of Ophthalmology 86:1293-1298.
Written Opinion of the International Searching Authority (ISA/US) issued on May 18, 2007 in connection with International Application No. PCT/IL05/00102.
Zhang, Z. et al. (2003) "COX-2-Dependent Cardiac Failure in Gh/tTG Transgenic Mice" Circulation Research 92:1153-1161.
Zhu J, et al., (1999) "Rapamycin inhibits hepatic stellate cell proliferation in vitro and limits fibrogenesis in an in vivo model of liver fibrosis," Gastroenterology, 5:1198-1204.
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US8198258B2 (en) 2012-06-12 grant
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