# Patent

## Title
Modified TGF-beta oligonucleotide for use in a method of preventing and/or treating an ophthalmic disease

## Abstract

The invention refers to an oligonucleotide consisting of 10 to 20 nucleotides of selected regions of the TGF-beta1, TGF-beta2 or TGF-beta3 nucleic acid sequence, which comprises modified nucleotides such as LNA, ENA, polyalkylene oxide-, 2′-fluoro, 2′-O-methoxy and/or 2′-O-methyl modified nucleotides. The invention further relates to pharmaceutical compositions comprising such oligonucleotide, wherein the composition or the oligonucleotide is used in a method for the prevention and/or treatment of glaucoma, posterior capsular opacification, dry eye, Marfan or Loeys-Dietz syndrome, riboblastoma, choroidcarcinoma, macular degeneration, such as age-related macular degeneration, diabetic macular endma, or cataract.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the priority of PCT/EP2014/056222, filed on Mar. 27, 2014, which claims priority to European Patent Application No. 13161474.5, filed on Mar. 27, 2013; European Patent Application No. 13173078.0, filed on Jun. 20, 2013; European Patent Application No. 13199826.2, filed on Dec. 30, 2013; European Patent Application No. 13199831.2, tiled on Dec. 30, 2013; and European Patent Application 13199838.7, filed on Dec. 30, 2013, the entire contents of each of which are hereby incorporated in total by reference.
SEQUENCE LISTING
This application incorporates by reference the Sequence Listing contained in an ASCII text file named “362346_00028_SeqList.txt” submitted via EFS-Web. The text file was created on Sept. 24, 2015, and is 100 kb in size.
The invention is directed to a TGF-beta oligonucleotide comprising a bridged nucleotide, polyalkylene oxide-, 2′-fluoro, 2′-O-methoxy and/or 2′-O-methyl modified nucleotide for use in a method of preventing and/or treating an ophthalmic disease.
Technical Background
Transforming growth factor beta (TGF-beta), a multifunctional growth factor that for example controls proliferation or cellular differentiation, is one of the most important ligands involved in the regulation of cell behavior in ocular tissues in physiological or pathological processes of development or tissue repair, although various other growth factors are also involved. Increased activity of this ligand may induce unfavorable inflammatory responses and tissue fibrosis. In mammals, three isoforms of TGF-beta, that is beta1, beta2, and beta3, are known. In most cases, TGF-beta enhances extracellular matrix production and suppresses cell proliferation. Moreover, TGF-beta is capable of inducing a number of growth factors, that is connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF), fibroblast growth factors (FGFs), and vascular endothelial growth factor (VEGF), as well as TGF-beta1 itself. All these factors have important roles in restoration of normal tissue following injury.
The aqueous humor that bathes the inner ocular structures (corneal endothelium, iris, crystalline lens, trabecular meshwork, and retina) contains various cytokines and growth factors. TGF-beta, in particular TGF-beta2, is the predominant cytokine. Physiologically, TGF-beta is mainly produced in the ciliary epithelium and lens epithelium as a latent, inactive, form consisting of mature TGF-beta, the latency-associated peptide (LAP) (small latent form), and the latent-TGF-beta-binding protein (LTBP). Heterogeneous expression patterns of each TGF-beta isoform in the crystalline lens have been reported in humans and animals. During the clinical course of various ocular diseases, the concentration of TGF-beta2 in the aqueous humor changes. For example, in an eye with proliferative vitreoretinopathy (PVR), a disorder of post-retinal detachment and retinal fibrosis, the concentration of TGF-beta2 in the vitreous humor increases in association with the progression of retinal fibrosis. The concentration of total and active TGF-beta2 is also higher in patients with diabetic retinopathy and open-angle glaucoma than in normal subjects. In diabetic retinopathy, chronic obstruction of retinal microvessels induces upregulation of VEGF and chemotaxis of macrophages, a potent source of TGF-beta. VEGF and TGF-beta cooperate to induce both retinal neovascularization and fibrosis around these new vessels, which may potentially cause retinal detachment or bleeding. Increased TGF-beta2 levels induce matrix expression and deposition in trabecular meshwork cells, leading to obstruction of the aqueous drainage route and an increase of intraocular pressure in a glaucomatous eye. In each of these examples, TGF-beta plays a role in disease pathogenesis. In eyes with pseudoexforiation syndrome, a kind of glaucoma with deposition of exforiative material on the lens, iris, or trabecular meshwork, the level of TGF-beta1 increases, but the exact role of TGF-beta1 in the pathogenesis of this disease is unknown (see Shizuya Saika, Laborartory Investigation (2006), 86, 106-115).
TGF-beta is one of the most potent regulators of the production and deposition of extracellular matrix. It stimulates the production and affects the adhesive properties of the extracellular matrix by two major mechanisms. First, TGF-beta stimulates fibroblasts and other cells to produce extracellular-matrix proteins and cell-adhesion proteins, including collagen, fibronectin, and integrins. Second, TGF-beta decreases the production of enzymes that degrade the extracellular matrix, including collagenase, heparinase, and stromelysin, and increases the production of proteins that inhibit enzymes that degrade the extracellular matrix, including plasminogen-activator inhibitor type 1 and tissue inhibitor of metalloprotease. The net effect of these changes is to increase the production of extracellular-matrix proteins and either to increase or to decrease the adhesive properties of cells in a cell-specific manner (see Blobe G C et al., May 2000, “Role of transforming growth factor beta in human disease”, N. Engl. J. Med. 342 (18), 1350-1358).
Targeting TGF-beta has been proposed as a potential therapeutic measure for example in glaucoma. Concerning various aspects of TGF-beta in the pathogenesis of glaucoma, therapies should be directed to modulate its production, activation, interaction with receptors, downstream intracellular regulatory mechanisms and/or the final structural and ECM changes (see Prendes M A et al., Br J Ophthalmol (2013), 97, 680-686).
Glaucoma (GCM), based upon chronically increased intraocular pressure, is a progressive optic neuropathy characterized by progressive loss of retinal ganglion cells, which manifests clinically with loss of optic disc neuroretinal rim tissue, defects in the retinal nerve fiber layer, and deficits on functional visual field testing (see Danesh-Meyer et al., Ophthalmol. 2006, 113: 603-611). Glaucoma is the second leading cause for blindness in the adult in the USA. Despite a multitude of treatment options, including surgical procedures in referactory patients, blindness remains a major threat. Primary open-angle glaucoma (POAG) is the most common form of glaucoma in the USA. Worldwide, in the year 2000 the number of people with POAG has been estimated at nearly 66.8 million with 6.7 million having bilateral blindness (see Quingley, Br J Ophthalmol. 1996 May; 80(5):389-393).
Cataract surgery is the most common ophthalmic surgical procedure. Alone in the USA, up to 3.000.000 cataract surgeries are performed per year. The US government spends currently more than USD 3 billion per year on treating cataract (Medicare patients only). The lens of the eye is removed by the procedure, and an intraocular lens is implanted. The lens capsule remains in situ, and the posterior part of the capsule frequently develops posterior capsule opacification (PCO) due to mechanical disruption and potential other factors associated with lens replacement. This condition occurs in 20 to 40% of PCO patients, YAG-laser posterior capsulotomy (rates depend on country, lens type used and surgical experience) is performed within the first two years, to remove the opacification (see Johansson B et al., Br J Ophthalmol (2010), 94, 450-455; Mathew R G et al., Ophthalmic Surg Lasers Imaging (2010), 41, 651-655). The use of the YAG-laser is associated with distinct risks, including retinal detachment (1-3%), cystoid macular oedema (up to 5%) and secondary glaucoma (see Billotte C and Berdeaux G, J Cataract Refract Surg (2004), 30(10), 2064-2071).
TGF-beta has been closely associated with the pathophysiology of both, GCM and PCO; so far, the effect of the TGF-beta protein has been inhibited by ALK5 inhibitors as for example described in WO 2009/146408 or antibodies directed to TGF-beta, i.e., one of its isoforms, which are disclosed for example in WO 2012/167143. None of these compounds has so far been successful in effective inhibiting TGF-beta in the eye, and thus, to be successful in the prevention and/or treatment of ophthalmic diseases such as GCM or PCO.
It is the objective of the present invention to provide an oligonucleotide, preferably an antisense oligonucleotide, which is specifically inhibiting the expression of TGF-beta1, TGF-beta2, and/or TGF-beta3 mRNA, TGF-beta1 and TGF-beta2 mRNA, or TGF-beta1 and TGF-beta3 mRNA, or TGF-beta2 and TGF-beta3 mRNA, and consequently is highly efficient for use in prevention and/or treatment of an ophthalmic disease without causing any (severe) side effects.
Summary of the Invention
The present invention refers to the use of a TGF-beta oligonucleotides, preferably a TGF-beta1, TGF-beta2, and/or TGF-beta3 antisense oligonucleotide for use in a method for treating an ophthalmic disease such as dry eye, glaucoma or posterior capsule opacification.
The TGF-beta oligonucleotide consists of 10 to 20, preferably 12 to 18 nucleotides of the TGF-beta1 nucleic acid sequence of SEQ ID NO. 1 (see FIG. 1), or of the TGF-beta2 nucleic acid sequence of SEQ ID NO. 2 (see FIG. 2), or of the TGF-beta3 nucleic acid sequence of SEQ ID NO. 3 (see FIG. 3), wherein one or more nucleotide(s) of the oligonucleotide is/are modified. Some of the oligonucleotides of the present invention correspond to TGF-beta1, TGF-beta2, and TGF-beta3, or to TGF-beta1 and TGF-beta2, or TGF-beta1 and TGF-beta3, or TGF-beta2 and TGF-beta3, and hybridize with one or more of these sequences.
In particular, oligonucleotides for use in the present invention comprise or consist of 10 to 20, more preferred of 12 to 18 nucleotides of the region of nucleic acid no. 1380 to 1510 of SEQ ID NO. 2, wherein one or more nucleotide(s) of the oligonucleotide is/are modified. These oligonucleotides are highly effective in the reduction and inhibition of TGF-beta2 expression and activity, respectively. A preferred oligonucleotide comprises or consists of SEQ ID NO. 5 (e.g., ASPH36: GACCAGATGCAGGA), SEQ ID NO. 6 (e.g., ASPH80: GCGACCGTGACCAGAT), SEQ ID NO. 7 (e.g., ASPH98: GCGCGACCGTGACC), SEQ ID NO. 8 (e.g., ASPH111: AGCGCGACCGTGA), or SEQ ID NO. 9 (e.g., ASPH121 or ASPH153: GACCGTGACCAGAT), SEQ ID NO. 10 (e.g., ASPH15: CTGCCCGCGGAT), SEQ ID NO. 11 (e.g., ASPH17: TCTGCCCGCGGAT), SEQ ID NO. 12 (e.g., ASPH26 or ASPH27: GGATCTGCCCGCGGA), SEQ ID NO. 13 (e.g., ASPH37: CTTGCTCAGGATCTGCC), SEQ ID NO. 14 (e.g., ASPH52 or 53: GCTCAGGATCTGCCCGCGGA), SEQ ID NO. 15 (e.g., ASPH112: GGATCGCCTCGAT), SEQ ID NO. 16 (e.g., ASPH119: CCGCGGATCGCC), or SEQ ID NO. 34 (e.g., ASPH30: CGATCCTCTTGCGCAT).
In another embodiment the invention refers to an oligonucleotide, comprising or consisting of 10 to 20, more preferred of 12 to 18 nucleotides of the region of nucleic acid no. 2740 to 2810 of the TGF-beta2 nucleic acid sequence of SEQ ID NO. 2 wherein one or more nucleotide(s) of the oligonucleotide is/are modified. These oligonucleotides are highly effective in the reduction and inhibition of TGF-beta2 expression and activity, respectively. A preferred oligonucleotide comprises or consists of SEQ ID NO. 60 (e.g., ASPH65: TCTGAACTAGTACCGCC), SEQ ID NO. 76 (e.g., ASPH82: AACTAGTACCGCCTTT), or SEQ ID NO. 106 (e.g., ASPH115: CTAGTACCGCCTT).
In a further embodiment the invention refers to an oligonucleotide, comprising or consisting of 10 to 20, more preferred of 12 to 18 nucleotides of the region of nucleic acid no. 1660 to 1680 of the TGF-beta2 nucleic acid sequence of SEQ ID NO. 2 wherein one or more nucleotide(s) of the oligonucleotide is/are modified. These oligonucleotides are highly effective in the reduction and inhibition of TGF-beta1 and/or TGF-beta2 expression and activity, respectively. A preferred oligonucleotide comprises or consists of SEQ ID NO. 17 (e.g., ASHP01 or ASPH02: ACCTCCTTGGCGTAGTA), SEQ ID NO. 18 (e.g., ASPH03 or ASPH04: CCTCCTTGGCGTAGTA), SEQ ID NO. 19 (e.g., ASPH05, ASPH06, or ASPH07: CTCCTTGGCGTAGTA), or SEQ ID NO.20 (e.g., ASPH08: TCCTTGGCGTAGTA).
In another embodiment the invention relates to an oligonucleotide, comprising or consisting of 10 to 20, more preferred of 12 to 18 nucleotides, most preferably 13 nucleotides of the region of nucleic acid no. 2390 to 2410 of the TGF-beta2 nucleic acid sequence of SEQ ID NO. 2 wherein one or more nucleotide(s) of the oligonucleotide is/are modified. These oligonucleotides are highly effective in the reduction and inhibition of TGF-beta1, TGF-beta2, and/or TGF-beta3 expression and activity, respectively. A preferred oligonucleotide comprises or consists of SEQ ID NO. 21 (e.g., ASPH9 or ASPH10: CAGAAGTTGGCAT).
In another embodiment the invention relates to an oligonucleotide, comprising or consisting of 10 to 20, more preferred of 12 to 18 nucleotides of the TGF-beta2 nucleic acid sequence of SEQ ID NO. 2 wherein one or more nucleotide(s) of the oligonucleotide is/are modified. These oligonucleotides are highly effective in the reduction and inhibition of TGF-beta1, TGF-beta2, and/or TGF-beta3, most preferably of TGF-beta2 expression and activity, respectively. A preferred oligonucleotide comprises or consists of one of SEQ ID NO. 22 to 59, 61 to 75, 77 to 105, 107 to 140 (e.g., ASHP11-ASPH14, ASPH16, ASPH18-ASPH25, ASPH28-ASPH35, ASPH38-ASPH51, ASPH60-ASPH64, ASPH66-ASPH79, ASPH81, ASPH83-ASPH97, ASPH99-ASPH110, ASPH113, ASPH114, ASPH116-ASPH118, ASPH120, ASPH122-ASPH152, ASPH154-ASPH183, or T-LNA (SEQ ID NO: 144)).
Preferred oligonucleotides of the present invention are ASPH01, ASPH03, ASPH05, ASPH17, ASPH22, ASPH26, ASPH27, ASPH35, ASPH36, ASPH37, ASPH45, ASPH47, ASPH48, ASPH65, ASPH69, ASPH71, ASPH80, ASPH82, ASPH98, ASPH105, ASPH115, ASPH190, ASPH191, ASPH192, and ASPH193, respectively.
Further preferred oligonucleotides of the present invention are ASPH1000 to ASPH1132 as shown in Table 1, which preferably inhibit the expression and/or activity of TGFbeta1 mRNA. Preferred oligonucleotides this group are for example ASPH1047, ASPH1051, ASPH1059, ASPH1106, ASPH1139, ASPH1150, ASPH1162, ASPH1163, ASPH1175, ASPH1178, and ASPH1181, respectively.
In an alternative embodiment oligonucleotides are preferably inhibiting the expression and/or activity of TGF-beta3 mRNA. Such oligonucleotides are for example ASPH2000, ASPH2001, ASPH2002, ASPH2003, ASPH2004, ASPH2005, ASPH2006, ASPH2007, ASPH2008, ASPH2009, ASPH2010, ASPH2011, ASPH2012, ASPH2013, ASPH2014, ASPH2015, ASPH2016, ASPH2017, ASPH2018, ASPH2019, ASPH2020, ASPH2021, ASPH2022, ASPH2023, ASPH2024, ASPH2025, ASPH2026, ASPH2027, ASPH2028, ASPH2029, ASPH2030, ASPH2031, ASPH2032, ASPH2033, ASPH2034, ASPH2035, ASPH2036, ASPH2037, ASPH2038, ASPH2039, ASPH2040, ASPH2041, ASPH2042, ASPH2043, ASPH2044, ASPH2045, ASPH2046, ASPH2047, ASPH2048, ASPH2049, ASPH2050, ASPH2051, ASPH2052, ASPH2053, ASPH2054, ASPH2055, ASPH2056, ASPH2057, ASPH2058, ASPH2059, ASPH2060, ASPH2061, ASPH2062, ASPH2063, ASPH2064, ASPH2065, and ASPH2066, respectively.
Oligonucleotides of the present invention show an unexpected strong and specific inhibition of TGF-beta1, TGF-beta2, or TGF-beta3, or TGF-beta1 and TGF-beta2. Alternatively, oligonucleotides of the present invention show strong and specific inhibition of TGF-beta1 and TGF-beta3, or TGF-beta1 and TGF-beta2, or TGF-beta2 and TGF-beta3, and in a further alternative TGF-beta1, TGF-beta2 and TGF-beta3.
Modifications of one or more nucleotides of the oligonucleotides of the present invention are selected from the group consisting of LNA, ENA, polyalkylene oxide such as triethylene glycol (TEG), 2′-fluoro, 2′-O-methoxy and 2′-O-methyl. The modifications are preferably located at the 5′- and/or 3′-end of the oligonucleotide. An oligonucleotide comprising such modified nucleotide is a modified oligonucleotide.
Modified nucleotides are for example arranged in a row, one directly next to the other, or in different patterns, where one or more unmodified nucleotides follow a modified nucleotide. For example an oligonucleotide starts with one or more modified nucleotides followed by one or more, e.g., one, two, three or four, unmodified or unlocked nucleotides followed again by one or more modified nucleotides. In one embodiment both ends of the oligonucleotide comprise an identical pattern of modified and unmodified or unlocked nucleotides. In another embodiment, the pattern of modifications at the 3′- and 5′-end differ including that one end does not comprise a modified nucleotide. Preferably the modified oligonucleotides comprise a series of 8 or 9 unlocked nucleotides.
Alternatively, a nucleotide at any other position in the oligonucleotide is modified, or at least one nucleotide at the 5′- and/or 3′-end of the oligonucleotide and at any other position in the oligonucleotide. For example ASPH1071, ASPH1100, ASPH1109, ASPH 1110, ASPH1111, ASPH1115, ASPH1126, ASPH1127 and ASPH1128 belong to a group of TGF-beta oligonucleotides, for example TGF-beta1 oligonucleotides, which comprises modified nucleosides such as LNA, ENA etc. in different patterns, e.g., separated from each other by an unlocked nucleotide. The oligonucleotides comprise either one type of modification, or one or more different modifications. Optionally, at least one phosphate linkage between two consecutive nucleotides (modified or unmodified) of the oligonucleotide is a phosphorothioate or a methylphosphonate. In a preferred embodiment, the oligonucleotides of the present invention are phosphorothioates.
Moreover, the present invention refers to TGF-beta antisense oligonucleotides, which interact and inhibit the expression of more than one TGF-beta isoform, even if the oligonucleotide is not 100% complementary to the TGF-beta1, TGF-beta2 and/or TGF-beta3 sequence. Such antisense oligonucleotides are for example ASPH1024, ASPH1096, ASPH1131 and ASPH1132, respectively. These oligonucleotides preferably interact with TGF-beta sequences of the same or different species such as human, monkey, rat or mouse as for example ASPH1131 and ASPH1132, respectively.
All the oligonucleotides of the different embodiments are for use in a method of the prevention and/or treatment of an ophthalmic disease such as dry eye, glaucoma, posterior capsular opacification (PCO), retinoblastoma, choroidcarcinoma, Marfan or Loeys-Dietz syndrome, macular degeneration, such as age-related macular degeneration, diabetic macular endma, or cataract.



FIGURES
FIG. 1 presents the nucleic acid sequence of human TGF-beta1 mRNA (NM_000660.4).
FIG. 2 shows the nucleic acid sequence of human TGF-beta2 mRNA (NM_003238.3).
FIG. 3 depicts the nucleic acid sequence of human TGF-beta3 mRNA (NM_003239.2).
FIG. 4 presents examples of nucleotide modifications.
FIG. 5a) to 5c) depict the inhibition of the expression of TGF-beta1 and TGF-beta2 mRNA in human A172 glioma cells. A172 cells were transfected with different modified oligonucleotides in a dose of 10 nM (in the presence of a transfecting agent), and the inhibition of the TGF-beta1 (white columns) and TGF-beta2 (black columns) mRNA expression was measured 24 h after transfection. FIG. 5a) refers to the results for the modified oligonucleotides ASPH01, ASPH02, ASPH03, ASPH04, ASPH05, ASPH06, ASPH07, ASPH08, ASPH09, ASPH10, ASPH11, ASPH12, ASPH13, ASPH14, ASPH15, ASPH16, ASPH17, ASPH18, ASPH19, ASPH20, ASPH21, ASPH22, ASPH24, ASPH25, ASPH26, ASPH27, ASPH29, ASPH30, ASPH31, ASPH32, ASPH33, ASPH34, ASPH35, ASPH36, ASPH37, ASPH38, ASPH39, ASPH40, ASPH41, ASPH42, ASPH43, ASPH44, ASPH45, ASPH46, ASPH47, ASPH48, ASPH49, ASPH50, ASPH51, ASPH52, ASPH53, and ASPH54; FIG. 5b) to the results for the modified oligonucleotides ASPH36, ASPH60, ASPH61, ASPH62, ASPH63, ASPH64, ASPH65, ASPH66, ASPH67, ASPH68, ASPH69, ASPH70, ASPH71, ASPH72, ASPH73, ASPH74, ASPH75, ASPH76, ASPH77, ASPH78, ASPH79, ASPH80, ASPH81, ASPH82, ASPH83, ASPH84, ASPH85, ASPH86, ASPH87, ASPH88, ASPH89, ASPH90, ASPH91, ASPH92, ASPH93, ASPH94, ASPH95, ASPH96, ASPH97, ASPH98, ASPH99, ASPH100, ASPH101, ASPH102, ASPH103, ASPH104, ASPH105, ASPH106, ASPH107, ASPH108, ASPH109, ASPH110, ASPH111, ASPH112, ASPH113, ASPH114, ASPH115, ASPH116, ASPH117, ASPH118, and ASPH119; and FIG. 5c) to the results for the modified oligonucleotides ASPH36, ASPH71, ASPH73, ASPH120, ASPH121, ASPH122, ASPH123, ASPH124, ASPH125, ASPH126, ASPH127, ASPH128, ASPH129, ASPH130, ASPH131, ASPH132, ASPH133, ASPH134, ASPH135, ASPH136, ASPH137, ASPH138, ASPH139, ASPH140, ASPH141, ASPH142, ASPH143, ASPH145, ASPH146, ASPH147, ASPH148, ASPH149, ASPH150, ASPH151, ASPH152, ASPH153, ASPH154, ASPH155, ASPH157, ASPH158, ASPH160, ASPH161, ASPH162, ASPH163, ASPH164, ASPH165, ASPH166, ASPH167, ASPH168, ASPH169, ASPH170, ASPH171, ASPH172, ASPH173, ASPH174, ASPH175, ASPH176, ASPH177, ASPH178, ASPH179, ASPH180, ASPH181, ASPH182, and ASPH183. Experiments are described in Example 1.
FIG. 6a) to 6c) depict the inhibition of the expression of TGF-beta1 and TGF-beta2 mRNA in human Panc-1 pancreatic cancer cells. Panc-1 cells were transfected with different modified oligonucleotides in a dose of 10 nM (in the presence of a transfecting agent), and the inhibition of the TGF-beta1 (white columns) and TGF-beta2 (black columns) mRNA expression was measured 24 h after transfection. FIG. 6a) refers to the results for the modified oligonucleotides ASPH01, ASPH02, ASPH03, ASPH04, ASPH05, ASPH06, ASPH07, ASPH08, ASPH12, ASPH14, ASPH17, ASPH18, ASPH20, ASPH21, ASPH22, ASPH24, ASPH25, ASPH26, ASPH27, ASPH29, ASPH30, ASPH31, ASPH32, ASPH33, ASPH35, ASPH36, ASPH37, ASPH38, ASPH39, ASPH40, ASPH41, ASPH42, ASPH43, ASPH44, ASPH45, ASPH46, ASPH47, ASPH48, ASPH49, ASPH50, ASPH51, and ASPH52; FIG. 6b) to the results for the modified oligonucleotides ASPH36, ASPH60, ASPH61, ASPH62, ASPH63, ASPH64, ASPH65, ASPH66, ASPH67, ASPH68, ASPH69, ASPH70, ASPH71, ASPH72, ASPH73, ASPH74, ASPH75, ASPH76, ASPH77, ASPH78, ASPH79, ASPH80, ASPH81, ASPH82, ASPH83, ASPH84, ASPH85, ASPH86, ASPH87, ASPH88, ASPH89, ASPH90, ASPH91, ASPH92, ASPH93, ASPH94, ASPH96, ASPH97, ASPH98, ASPH99, ASPH100, ASPH101, ASPH102, ASPH103, ASPH104, ASPH105, ASPH106, ASPH107, ASPH108, ASPH109, ASPH110, ASPH111, ASPH112, ASPH113, ASPH114, ASPH115, ASPH116, ASPH117, ASPH118, and ASPH119; and FIG. 6c) to the results for the modified oligonucleotides ASPH36, ASPH71, ASPH73, ASPH120, ASPH121, ASPH122, ASPH127, ASPH128, ASPH129, ASPH130, ASPH131, ASPH132, ASPH133, ASPH135, ASPH136, ASPH137, ASPH139, ASPH141, ASPH142, ASPH143, ASPH145, ASPH146, ASPH147, ASPH149, ASPH150, ASPH151, ASPH152, ASPH153, ASPH154, ASPH155, ASPH157, ASPH160, ASPH161, ASPH162, ASPH163, ASPH164, ASPH165, ASPH166, ASPH167, ASPH168, ASPH169, ASPH170, ASPH171, ASPH172, ASPH173, ASPH174, ASPH175, ASPH176, ASPH177, ASPH178, ASPH179, ASPH180, ASPH181, ASPH182, and ASPH183. Experiments are described in Example 2.
FIG. 7 shows the inhibition of the expression of TGF-beta1 and TGF-beta2 mRNA in Panc-1 cells. Panc-1 cells were treated with different modified oligonucleotides in a dose of 3.3 μM in the absence of any transfection reagent (gymnotic transfection or unassisted transfection), and the inhibition of the TGF-beta1 (white columns) and TGF-beta2 (black columns) mRNA expression was measured after 72 h. FIG. 7 presents the results for the modified oligonucleotides ASPH17, ASPH18, ASPH22, ASPH25, ASPH33, ASPH35, ASPH36, ASPH41, ASPH42, ASPH45, ASPH46, ASPH47, ASPH48, ASPH49, ASPH65, ASPH66, ASPH67, ASPH69, ASPH71, ASPH79, ASPH80, ASPH82, ASPH88, ASPH89, ASPH90, ASPH91, ASPH98, ASPH99, ASPH102, ASPH105, ASPH111, ASPH115, ASPH119, ASPH121, ASPH139, ASPH140, ASPH146, ASPH151, ASPH153, ASPH165, ASPH171, ASPH172, ASPH176, ASPH178, ASPH180, and ASPH183. Experiments are described in Example 4.
FIG. 8 and FIG. 9 present the inhibition of the expression of TGF-beta1 (FIG. 8a) and TGF-beta2 (FIG. 8b) mRNA as well as the inhibition of TGF-beta1 (FIG. 9a) and TGF-beta2 (FIG. 9b) protein in Panc-1 cells. Panc-1 cells were treated with different modified oligonucleotides in a dose of 10 μM via gymnotic transfection, i.e., in the absence of any transfecting reagent, and the inhibition of the TGF-beta1 and TGF-beta2 mRNA expression and protein was measured 4 days after transfection. FIG. 8a) and FIG. 8b) show the results for the modified oligonucleotides ASPH01, ASPH03, ASPH05, ASPH09, ASPH17, ASPH18, ASPH22, ASPH35, ASPH36, ASPH37, ASPH41, ASPH45, ASPH46, ASPH47, and ASPH48 on mRNA level, and FIG. 9a) and FIG. 9b) on protein level. Experiments are described in Example 5.
FIG. 10 depicts the dose-dependent effect of modified oligonucleotides ASPH05 and ASPH36 on TGF-beta1 and TGF-beta2 mRNA expression. Panc-1 cells were treated for 4 days with 15 μM, 10 μM, 7.5 μM, 5 μM, 2.5 μM, 1.25 μM, or 0.625 μM of either ASPH05 (dual TGF-beta1 and TGF-beta2 oligonucleotide) or ASPH36 (selective TGF-beta2 oligonucleotide) modified oligonucleotide in the absence of a transfection reagent. Remaining TGF-beta1 (FIG. 10a) or TGF-beta2 mRNA (FIG. 10b) was measured after 4 days. Experiments are described in Example 6.
FIG. 11 shows the inhibition of the expression of TGF-beta1 and TGF-beta2 mRNA in mouse SMA-560 glioma cells. SMA-560 cells were transfected with ASPH01, ASPH03, ASPH05, ASPH09, ASPH17, ASPH18, ASPH22, ASPH26, ASPH36, ASPH37, ASPH41, ASPH42, ASPH45, ASPH46, ASPH47, or ASPH48 in a dose of 10 nM (in the presence of a transfecting agent). Inhibition of the mouse TGF-beta1 (white columns) and TGF-beta2 (black columns) mRNA expression was determined 24 h after transfection. Experiments are described in Example 7.
FIG. 12 presents in vivo data referring to the treatment of female athymic nude mice with ASPH01, ASPH03, ASPH05, ASPH17, ASPH22, ASPH37, ASPH41, ASPH45, ASPH46, ASPH47, or ASPH48 at 14 mg/kg body weight by subcutaneous injection for 5 consecutive days. 24 h after the last treatment, mice were sacrificed and mouse TGF-beta 2 mRNA was quantified in kidney tissue lysates. Data—representing TGF-beta2 to GAPDH mRNA ratio—are shown as a box plot in which median values and min. and max. values are presented (data expressed as n=4, except ASPH46 group n=3). Experiments are described in Example 8.
FIG. 13 shows the inhibition of the expression of TGF-beta3 mRNA in Panc-1 cells. Panc-1 cells were treated with ASPH09 in a dose of 10 μM in the absence of any transfection reagent (gymnotic transfection or unassisted transfection), and the inhibition of the TGF-beta3 mRNA expression was measured after 4 days. ASPH09 is a panspecific oliogonucleotide inhibiting the expression of TGF-beta3 as well as of TGF-beta1 and TGF-beta2 (FIGS. 8a and 8b). Experiment is described in Example 9.
FIG. 14 depicts the inhibition of the expression of TGF-beta1 mRNA in human Panc-1 pancreatic cancer cells. Panc-1 cells were transfected with different modified oligonucleotides in a dose of 10 nM (in the presence of a transfecting agent), and the inhibition of the TGF-beta1 mRNA expression was measured 24 h after transfection. FIG. 14 refers to the results for the modified oligonucleotides ASPH05, ASPH09, ASPH1000, ASPH1001, ASPH1002, ASPH1003, ASPH1004, ASPH1005, ASPH1006, ASPH1007, ASPH1008, ASPH1009, ASPH1010, ASPH1011, ASPH1012, ASPH1013, ASPH1014, ASPH1015, ASPH1016, ASPH1017, ASPH1018, ASPH1019, ASPH1020, ASPH1021, ASPH1022, ASPH1023, ASPH1024, ASPH1026, ASPH1027, ASPH1028, ASPH1029, ASPH1030, ASPH1031, ASPH1032, ASPH1033, ASPH1034, ASPH1035, ASPH1036, ASPH 1038, ASPH1039, ASPH1040, ASPH1041, ASPH1042, ASPH1043, ASPH1044, ASPH1045, ASPH1046, ASPH1047, ASPH1048, ASPH1049, ASPH1050, ASPH1051, ASPH1052, ASPH1054, ASPH1055, ASPH1056, ASPH1057, ASPH1058, ASPH1059, ASPH1060, and ASPH1061. Experiments are described in Example 12.
FIG. 15 shows the inhibition of the expression of TGF-beta1 mRNA in mouse SMA-560 glioma cells. The cells were transfected with different modified oligonucleotides in a dose of 10 nM (in the presence of a transfecting agent), and the inhibition of the TGF-beta1 mRNA expression was measured 24 h after transfection. FIG. 15 refers to the results for the modified oligonucleotides ASPH09, ASPH1000, ASPH1001, ASPH1002, ASPH1003, ASPH1004, ASPH1005, ASPH1006, ASPH1007, ASPH1008, ASPH1009, ASPH1010, ASPH1011, ASPH1012, ASPH1013, ASPH1014, ASPH1015, ASPH1016, ASPH1017, ASPH1018, ASPH1019, ASPH 1020, ASPH1021, ASPH1022, ASPH1023, ASPH1024, ASPH1026, ASPH1027, ASPH1028, ASPH1029, ASPH1030, ASPH1031, ASPH1032, ASPH1033, ASPH1034, ASPH1035, ASPH1036, ASPH1037, ASPH1038, ASPH1039, ASPH1040, ASPH1041, ASPH1042, ASPH1043, ASPH1044, ASPH1045, ASPH1046, ASPH1047, ASPH1048, ASPH1049, ASPH1050, ASPH1051, ASPH1052, ASPH1053, ASPH1054, ASPH1055, ASPH1056, ASPH1057, ASPH1058, ASPH1059, ASPH1060, ASPH1061, and ASPH1062. Experiments are described in Example 13.
FIG. 16 depicts the inhibition of the expression of TGF-beta1 and TGF-beta2 mRNA in human A172 cells. The cells were transfected with different modified oligonucleotides in a dose of 10 nM (in the presence of a transfecting agent), and the inhibition of the TGF-beta1 and TGF-beta2 mRNA expression was measured 24 h after transfection. FIG. 16 refers to the results for the modified oligonucleotides ASPH05, ASPH09, ASPH1000, ASPH1001, ASPH1002, ASPH1004, ASPH1005, ASPH1006, ASPH1007, ASPH1008, ASPH1009, ASPH1010, ASPH1011, ASPH1012, ASPH1013, ASPH1014, ASPH1015, ASPH1016, ASPH1017, ASPH1018, ASPH1019, ASPH1020, ASPH1021, ASPH1022, ASPH1023, ASPH1024, ASPH1026, ASPH1027, ASPH1028, ASPH1029, ASPH1030, ASPH1031, ASPH1032, ASPH1033, ASPH1034, ASPH1035, ASPH1036, ASPH1038, ASPH1039, ASPH1040, ASPH1041, ASPH1042, ASPH1043, ASPH1044, ASPH1045, ASPH1046, ASPH1047, ASPH1048, ASPH1049, ASPH1050, ASPH1051, ASPH1052, ASPH1053, ASPH1054, ASPH1056, ASPH1057, ASPH1058, ASPH1059, ASPH1060, ASPH1061, and ASPH1062. Experiments are described in Example 14.
FIG. 17 shows the inhibition of the expression of TGF-beta1 and TGF-beta2 mRNA in Panc-1 cells. Panc-1 cells were treated with different modified oligonucleotides in a dose of 3.3 μM in the absence of any transfection reagent (gymnotic transfection or unassisted transfection or gymnotic delivery), and the inhibition of the TGF-beta1 (black columns) and TGF-beta2 (white columns) mRNA expression was measured after 72 h. FIG. 17 refers to the results for the modified oligonucleotides ASPH05, ASPH09, ASPH1000, ASPH1001, ASPH1002, ASPH1004, ASPH1006, ASPH1007, ASPH1008, ASPH1009, ASPH1010, ASPH1011, ASPH1012, ASPH1013, ASPH1014, ASPH1015, ASPH1017, ASPH1018, ASPH1019, ASPH1020, ASPH1021, ASPH1022, ASPH1024, ASPH1026, ASPH1027, ASPH1028, ASPH1029, ASPH1032, ASPH1033, ASPH1034, ASPH1035, ASPH1036, ASPH1037, ASPH1038, ASPH1039, ASPH1040, ASPH1041, ASPH1042, ASPH1043, ASPH1044, ASPH1045, ASPH1046, ASPH1047, ASPH1049, ASPH1050, ASPH1051, ASPH1052, ASPH1053, ASPH1054, ASPH1055, ASPH1056, ASPH1057, ASPH1058, ASPH1059, ASPH1060, ASPH1061, and ASPH1062. Experiments are described in Example 15.
FIG. 18 depicts the inhibition of the expression of TGF-beta1, TGF-beta2 and TGF-beta3 mRNA in human A172 cells. The cells were transfected with different modified oligonucleotides in a dose of 10 nM (in the presence of a transfecting agent), and the inhibition of the TGF-beta1 (black column), TGF-beta2 (white column) and TGF-beta3 (striped column) mRNA expression was measured 24 h after transfection. FIG. 18 refers to the results for the modified oligonucleotides ASPH09, ASPH1047, ASPH1051, ASPH1059, ASPH1063, ASPH1064, ASPH1065, ASPH1066, ASPH1067, ASPH1068, ASPH1069, ASPH1070, ASPH1071, ASPH1072, ASPH1073, ASPH1074, ASPH1075, ASPH1076, ASPH1077, ASPH1078, ASPH1079, ASPH1080, ASPH1081, ASPH1082, ASPH1083, ASPH1084, ASPH1085, ASPH1086, ASPH1087, ASPH1088, ASPH1089, ASPH1090, ASPH1091, ASPH1092, ASPH1093, ASPH1094, ASPH1095, ASPH1097, ASPH1098, ASPH1099, ASPH1100, ASPH1101, ASPH1102, ASPH1103, ASPH1104, ASPH1105, ASPH1106, ASPH1107, ASPH1108, ASPH1109, ASPH1110, ASPH1111, ASPH1112, ASPH1113, ASPH114, ASPH1115, ASPH1116, ASPH1117, ASPH1118, ASPH1119, ASPH1120, ASPH1121, ASPH1122, ASPH1123, ASPH1124, ASPH1125, ASPH1126, ASPH1127, ASPH1128, ASPH1129, ASPH1130, ASPH1131, and ASPH1132. Experiments are described in Example 16.
FIG. 19a shows the inhibition of the expression of TGF-beta1, TGF-beta2 and TGF-beta3 mRNA in human Panc-1 and RenCa cells. The cells were transfected with different modified oligonucleotides in a dose dose of 3.3 μM in the absence of any transfection reagent (gymnotic transfection or unassisted transfection or gymnotic delivery), and the inhibition of the TGF-beta1 (black column), TGF-beta2 (white column) and TGF-beta3 (striped column) mRNA expression was measured 72 h after transfection. FIG. 19a refers to the results for the modified oligonucleotides ASPH1063, ASPH1064, ASPH1065, ASPH1066, ASPH1067, ASPH1068, ASPH1069, ASPH1070, ASPH1071, ASPH1072, ASPH1073, ASPH1074, ASPH1075, ASPH1076, ASPH1077, ASPH1078, ASPH1079, ASPH1080, ASPH1081, ASPH1082, ASPH1083, ASPH1084, ASPH1085, ASPH1086, ASPH1087, ASPH1088, ASPH1089, ASPH1090, ASPH1091, ASPH1092, ASPH1093, ASPH1094, ASPH1095, ASPH1097, ASPH1098, ASPH1099, ASPH1100, ASPH1101, ASPH1102, ASPH1103, ASPH1104, ASPH1105, ASPH1106, ASPH1107, ASPH1108, ASPH1109, ASPH1110, ASPH1111, ASPH1112, ASPH1113, ASPH114, ASPH1115, ASPH1116, ASPH1117, ASPH1118, ASPH1119, ASPH1120, ASPH1121, ASPH1122, ASPH1123, ASPH1124, ASPH1125, ASPH1126, ASPH1127, ASPH1128, ASPH1129, ASPH1130, ASPH1131, and ASPH1132. FIG. 19b presents the inhibiting effect of these oligonucleotides in RenCa cells.
FIG. 20 presents a sequence alignment of ASPH1024 and ASPH1096, which are 100% homologous to a human sequence of TGF-beta1, with a human sequence of TGF-beta2 and TGF-beta3, respectively. ASPH1024 has three mismatches with the human sequence of TGF-beta2 (FIG. 20a) and two mismatches with human sequence of TGF-beta3 (FIG. 20b). ASPH1096 has one mismatch with the human sequence of TGF-beta2 (FIG. 20a), and one mismatch with the human sequence of TGF-beta3 (FIG. 20b). Both oligonucleotides show inhibition of different human TGF-beta isoforms (TGF-beta1, TGF-beta2, and TGF-beta3). For example ASPH1024 inhibits the expression and activity of TGF-beta1 and TGF-beta2 (see FIG. 17) and ASPH1096 inhibits the expression and activity of TGF-beta1, TGF-beta2 and TGF-beta3 as depicted in FIG. 18 for example. ASPH009, which is 100% homologous to the human sequence of TGF-beta1, TGF-beta2, and TGF-beta3 was used as a control.
FIG. 21 shows a sequence alignment of ASPH1131 and ASPH1132, which are 100% homologous to a human sequence of TGF-beta1 and TGFβbeta3, with a human sequence of TGF-beta2. Each of ASPH1131 and ASPH1132 has one mismatch with the human sequence of TGF-beta2. Both oligonucleotides strongly inhibit the expression of all three human isoforms as depicted in FIG. 18 for example.
FIG. 22 depicts a sequence alignment of ASPH1131 and ASPH1132, which are 100% homologous to a murine sequence of TGF-beta1 and TGFbeta3, with a murine sequence of TGF-beta2. Each of ASPH1131 and ASPH1132 has two mismatches with the murine sequence of TGF-beta2. While ASPH1131 potently inhibits murine TGF-beta2 and TGF-beta3, ASPH1132 very potently suppresses all murine TGF-beta isoforms as depicted in FIG. 19b for example.
FIG. 23 shows TGF-beta2 mRNA expression in the kidney of mice bearing subcutaneous human pancreatic carcinoma Panc-1 tumors. Mice were treated with 1, 3, 10, and 30 mg/kg of ASPH47 after indicated treatment schedules for 5 days: Q1Dx1-d6 (single SC injection, termination 5 days later), Q1Dx5-d6 (daily SC injection for 5 days, termination 24 hours later), and Q1Dx5-d10 (daily SC injection for 5 days, termination 5 days later). TGF-beta2 and house-keeping gene GAPDH mRNA expression was detected by bDNA assay. Data—representing TGF-beta2 to GAPDH mRNA ratio—are shown as a box plot in which median values and min. and max. values are presented (data expressed as n=10, except n=9 for vehicle and 3 mg/kg Q1Dx1 d6 groups).
FIG. 24 depicts TGF-beta2 mRNA expression in the kidneys of mice bearing human pancreatic carcinoma Panc-1 tumors. Mice were treated with subcutaneous injections of various oligonucleotides for 5 consecutive days using indicated treatment doses: daily injection of 1, 5, 15 or 50 mg/kg oligonucleotides for five consecutive days. TGF-beta2 and house-keeping gene GAPDH mRNA expression was detected by bDNA. Data—representing TGF-beta2 to GAPDH mRNA ratio—are shown as a box plot in which median values and min. and max. values are presented (data expressed as n=5).
FIG. 25 presents TGF-beta2 mRNA expression in subcutaneous human renal cell carcinomas 786-O tumors. Mice were treated with a daily injection of 50 mg/kg oligonucleotides for five consecutive days. The tumors were collected 24 hours after the last treatment and snap frozen. TGF-beta2 and house-keeping gene GAPDH mRNA expression was detected by bDNA assay. Data—representing TGF-beta2 to GAPDH mRNA ratio—are shown as a box plot in which median values and min. and max. values are presented (data expressed as n=10, except for ASPH71 group n=9).
FIG. 26 depicts the inhibiting effect of oligonucleotides of the present invention on the expression of TGF-beta1 and TGF-beta2 protein. Panc-1 cells were transfected with 20, 6.67, 2.22, 0.74, 0.25, 0.08 or 0.009 μM of the modified oligonucleotides ASPH47 (FIG. 26a), ASPH1047 (FIG. 26b), ASPH1106 (FIG. 26c), ASPH1132 (FIG. 26d), or ASPH47 in combination with ASPH1047 (FIG. 26e). Negative control is the scrambled oligonucleotide (scrLNA) of SEQ ID No. 145 (FIG. 26 f) in concentrations of 40, 13.33, 4.44, 1.48, 0.49, 0.16, 0.05, or 0.02 μM. TGF-beta1 and TGF-beta2 protein levels in cell supernatants were determined by ELISA, wherein results for TGF-beta1 are indicated in diamonds, and results for TGF-beta2 in squares.
FIG. 27 shows the inhibition of the expression of TGF-beta1, TGF-beta2 and TGF-beta3 mRNA in human Panc-1 cells and mouse RenCa cells. Panc-1 and RenCa cells were treated with different modified oligonucleotides in a dose of 1.1 μM in the absence of any transfection reagent (gymnotic transfection or unassisted transfection or gymnotic delivery), and the inhibition of the TGF-beta1 (black columns), TGF-beta2 (white columns), and TGF-beta3 (striped columns) mRNA expression was measured after 72 h. FIG. 17 refers to the results for the modified oligonucleotides ASPH190, ASPH191, ASPH192, ASPH193, ASPH194, ASPH195, ASPH196, ASPH197, ASPH198, ASPH199, ASPH200, ASPH201, ASPH202, ASPH203, ASPH204, ASPH205, ASPH206, ASPH207, ASPH208, ASPH209, ASPH210, ASPH211, ASPH212, ASPH213, ASPH214, ASPH215, ASPH216, ASPH217, ASPH218, ASPH219, ASPH220, ASPH221, ASPH222, and ASPH223, respectively. FIG. 27a presents the inhibitory effect of these TGF-beta oligonucleotides in Panc-1 cells and FIG. 27b in RenCa cells.
FIG. 28 presents the inhibiting effect of oligonucleotides of the present invention on the expression of TGF-beta1, TGF-beta2, and TGF-beta3. Panc-1 cells (FIG. 28a) or RenCa cells (FIG. 28b) were transfected with 3.3 μM of different TGF-beta specific oligonucleotides in the absence of a transfecting agent. The expression of TGF-beta1 (black column), TGF-beta2 (white column) and TGF-beta3 (striped column) mRNA was determined 72 h after transfection.
FIG. 29 depicts the the inhibiting effect of oligonucleotides of the present invention on the expression of TGF-beta1, TGF-beta2, and TGF-beta3. A172 glioma cells were transfected with 10 nM of different TGF-beta specific oligonucleotides in the presence of transfecting agent. The expression of TGF-beta1 (black column), TGF-beta2 (white column) and TGF-beta3 (striped column) mRNA was determined 24 h after transfection.
FIGS. 30a and 30b present a compared analysis of time-dependent plasma (30a) and kidney (30b) concentration (PK profiles; with values expressed in μg/mL or μg/gr) and downregulation of TGF-β2 mRNA (PD profile) in kidney following single subcutaneous bolus administration of 50 mg/kg of ASPH_0047 to Balb/c mice.
FIG. 31 depicts TGF-β2 mRNA downregulation in established subcutaneous tumors (FIG. 30A-D) or kidney (FIG. 30E-F) in immunodeficient mouse following subcutaneous repeated administration of ASPH_0047 or control oligonucleotide. TGF-beta2 and GAPDH mRNA expression was detected by bDNA. Results are expressed as TGF-beta2/GAPDH mRNA ratio, and each individual tested sample is represented with line indicating median values.
FIG. 32 shows the effect of systemic treatment of Balb/c mice with ASPH_0047 (selective TGF-b2 antisense oligonucleotide) on lung metastasis in orthotopic and in i.v. mouse Renca renal carcinoma model. Level of lung metastasis was determined by either number of metastasis or based on lung weight. Results are shown as a box plot in which median values, upper and lower quartiles, and 90th and 10th percentiles are presented.
FIG. 33 presents human Panc-1 pancreatic cancer cells were treated with 3.3 μM of the indicated oligonucleotides in the absence of transfecting agent (gymnotic transfection or gymnotic delivery). The expression of TGF-beta1 (black column), TGF-beta2 (white column) and TGF-beta3 (striped column) mRNA was determined 72 h after transfection.
FIG. 34 depicts the effect of systemic treatment of Balb/c mice with ASPH_0047 on lung metastasis in orthotopic mouse 4T1 mammary carcinoma model. Data for each individual animal is represented with median values indicated as bold black line.



Detailed Description
The present invention is directed to oligonucleotides, in particular antisense oligonucleotides, which comprise at least one modified nucleotide and are suitable to interact with TGF-beta mRNA for use in a method for preventing and/or treating an ophthalmic disease such as dry eye, glaucoma, posterior capsule opacification, retinoblastoma or choroidcarcinoma. The oligonucleotides comprise or consist of 10 to 20, more preferred 12 to 18 nucleotides of the TGF-beta2 nucleic acid according to SEQ ID NO. 2 or of the TGF-beta1 nucleic acid according to SEQ ID NO. 1, or of the nucleic acid sequence of TGF-beta3 nucleic acid according to SEQ ID NO. 3. Most preferred the oligonucleotide comprises or consists of 12, 13, 14, 15, 16, 17, or 18 nucleotides. The oligonucleotides are preferably selected from the region of nucleic acid no. 1380 to 1510 (preferably no. 1380 to 1450 and/or no. 1480 to 1510), 1660 to 1680, or 2390 to 2410 of SEQ ID NO. 2. The oligonucleotide is a single or double stranded RNA or DNA, including siRNA, microRNA, apatmer or spiegelmer. Preferably, the oligonucleotide is an antisense oligonucleotide.
A nucleotide forms the building block of an oligonucleotide, and is for example composed of a nucleobase (nitrogenous base, e.g., purine or pyrimidine), a five-carbon sugar (e.g., ribose, 2-deoxyribose, arabinose, xylose, lyxose, allose, altorse, glucose, mannose, gulose, idose, galactose, talose or stabilized modifications of those sugars), and one or more phosphate groups. Examples of modified phosphate groups are phosphorothioate or methylphosphonate. Each compound of the nucleotide is modifiable, and is naturally occurring or none naturally occurring. The latter are for example locked nucleic acid (LNA), a 2′-O,4′-C-ethylene-bridged nucleic acid (ENA), polyalkylene oxide- (such as triethylene glycol (TEG)), 2′-fluoro, 2′-O-methoxy and 2′-O-methyl modified nucleotides as described for example by Freier & Altmann (Nucl. Acid Res., 1997, 25, 4429-4443) and Uhlmann (Curr. Opinion in Drug & Development (2000, 3 (2): 293-213)), which are shown in FIG. 4.
A LNA is a modified RNA nucleotide, wherein the ribose moiety is modified with an extra bridge connecting the 2′ oxygen and 4′ carbon (2′-4′ribonucleoside). The bridge “locks” the ribose in the 3′-endo (North) conformation, which is often found in the A-form duplexes. LNA nucleosides and nucleotides, respectively, comprise for example the forms of thio-LNA, oxy-LNA, or amino-LNA, in alpha-D- or beta-L-configuration, and are mixable and combineable, respectively, with (unmodified) DNA or RNA residues in the oligonucleotide.
The oligonucleotides of the present invention, i.e., modified oligonucleotides, comprise at least one modified nucleotide, preferably LNA and/or ENA, at the 5′- and/or 3′-end of the oligonucleotide. In a preferred embodiment, the oligonucleotide comprises 1, 2, 3, or 4 LNAs or ENAs at the 5′-end, and 1, 2, 3, or 4 LNAs or ENAs at the 3′-end. In another preferred embodiment, the oligonucleotide comprises 1, 2, 3, or 4 LNAs or ENAs at the 5′-end or 3′-end, and a polyalkylene oxide such as TEG at the 3′- or 5′-end. The modified oligonucleotides show a significantly increased inhibition on TGF-beta expression and activity, respectively, which results in an improved prevention and/or treatment of a malignant or benign tumor, an immunologic disease, fibrosis, eye disease such as glaucoma or posterior capsular opacification (PCO), CNS disease hair loss etc. The oligonucleotides of the present invention target TGF-beta linked diseases either by hybridization with TGF-beta mRNA, preferably TGF-beta1, TGF-beta2, or TGF-beta3, alternatively, TGF-beta1, TGF-beta2, and/or TGF-beta3 mRNAs, i.e., TGF-beta1 and TGF-beta2, or TGF-beta1 and TGF-beta3, or TGF-beta2 and TGF-beta3, or TGF-beta1, TGF-beta2 and TGF-beta3 mRNAs, or any other direct or indirect effect on the TGF-beta system. An oligonucleotide inhibiting the expression of TGF-beta1, TGF-beta2, and TGF-beta3 is defined as pan-specific oligonucleotide.
In a preferred embodiment, the oligonucleotides of the present invention are for use in a method for preventing and/or treating an ophthalmic disease such as dry eye, glaucoma or posterior capsule opacification.
Preferably two or more oligonucleotides are combined, wherein at least one oligonucleotide specifically inhibits TGF-beta1 and at least one oligonucleotide specifically inhibits TGF-beta2, or wherein at least one oligonucleotide specifically inhibits TGF-beta1 and at least one oligonucleotide specifically inhibits TGF-beta3, or wherein at least one oligonucleotide specifically inhibits TGF-beta2 and at least one oligonucleotide specifically inhibits TGF-beta3, or wherein at least one oligonucleotide specifically inhibits TGF-beta1, at least one oligonucleotide specifically inhibits TGF-beta2, and at least one oligonucleotide specifically inhibits TGF-beta3.
In another embodiment, one oligonucleotide inhibits two TGF-beta isoforms such as TGF-beta1 and TGF-beta2, TGF-beta2 and TGF-beta3, or TGF-beta1 and TGF-beta3. An oligonucleotide inhibiting the expression of all three isoforms—TGF-beta1, TGF-beta2, and TGF-beta3—is defined as pan-specific oligonucleotide.
In a further embodiment three or more oligonucleotides are combined, wherein at least one oligonucleotide specifically inhibits TGF-beta1, another oligonucleotide specifically inhibits TGF-beta2, and a further oligonucleotide specifically inhibits TGF-beta3, and optionally one or more additional oligonucleotides inhibiting TGF-beta1, TGF-beta2 or TGF-beta3, and/or optionally any other factor.
The oligonucleotides of the present invention have for example an IC50 in the range of 0.1 to 20 μM, preferably in the range of 0.2 to 15 μM, more preferably in the range of 0.4 to 10 μM, and even more preferred in the range of 0.5 to 5 μM.
The present invention further refers to a pharmaceutical composition comprising an oligonucleotide according to the invention as active ingredient. The pharmaceutical composition comprises at least one oligonucleotide of the present invention and optionally further an antisense compound, an antibody, a chemotherapeutic compound, an anti-inflammatory compound, an antiviral compound and/or an immuno-modulating compound. Pharmaceutically acceptable binding agents and adjuvants optionally comprise part of the pharmaceutical composition.
In one embodiment, the oligonucleotide and the pharmaceutical composition, respectively, is formulated as dosage unit in form of capsules, tablets and pills etc., respectively, which contain for example the following compounds: microcrystalline cellulose, gum or gelatin as binders; starch or lactose as excipients; stearates as lubricants, various sweetening or flavouring agents. For capsules the dosage unit may contain a liquid carrier like fatty oils. Likewise coatings of sugar or enteric agents may be part of the dosage unit.
In a preferred embodiment the oligonucleotide or the pharmaceutical composition comprising such oligonucleotide of the present invention is formulated as eye drops or eye ointment, which optionally comprise a dye such as BBG, BBR, or trypanblue, polyvinylpyrrolidone, polyethyleneglycol (PEG), preferably PEG200, PEG400, or PEG1000, hydrogenphosphate of calium or sodium.
The oligonucleotide and/or the pharmaceutical composition is administrable via different routes. These routes of administration include, but are not limited to, electroporation, epidermal, impression into skin, intra-arterial, intra-articular, intracranial, intradermal, intra-lesional, intra-muscular, intranasal, intra-ocular, intracameral, intrathecal, intraperitoneal, intraprostatic, intrapulmonary, intraspinal, intratracheal, intratumoral, intravenous, intravesical, placement within cavities of the body, nasal inhalation, oral, pulmonary inhalation (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer), subcutaneous, subdermal, topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), or transdermal.
For parenteral, subcutaneous, intradermal or topical administration the oligonucleotide and/or the pharmaceutical composition include for example a sterile diluent, buffers, regulators of toxicity and antibacterials. In a preferred embodiment, the oligonucleotide or pharmaceutical composition is prepared with carriers that protect against degradation or immediate elimination from the body, including implants or microcapsules with controlled release properties. For intravenous administration the preferred carriers are for example physiological saline or phosphate buffered saline. An oligonucleotide and/or a pharmaceutical composition comprising such oligonucleotide for oral administration includes for example powder or granule, microparticulate, nanoparticulate, suspension or solution in water or non-aqueous media, capsule, gel capsule, sachet, tablet or minitablet. An oligonucleotide and/or a pharmaceutical composition comprising for parenteral, intrathecal, intracameral or intraventricular administration includes for example sterile aqueous solutions which optionally contain buffer, diluent and other suitable additive such as penetration enhancer, carrier compound and other pharmaceutically acceptable carrier or excipient.
A pharmaceutically acceptable carrier is for example liquid or solid, and is selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutically acceptable carriers include, but are not limited to, a binding agent (e.g. pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); filler (e.g. lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricant (e.g., magnesium stearate, talcum, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrate (e.g., starch, sodium starch glycolate, etc.); or wetting agent (e.g., sodium lauryl sulphate, etc.). Sustained release oral delivery systems and/or enteric coatings for orally administered dosage forms are described in U.S. Pat. Nos. 4,704,295; 4,556,552; 4,309,406; and 4,309,404. An adjuvant is included under these phrases.
In one embodiment, the oligonucleotide or pharmaceutical composition is administered via a medical device, preferably a small pump such as a mini-pump, which is for example directly implanted into or onto the eye. Such device is for example connected to the eye motion muscle to deliver a therapeutic load, i.e., an oligonucleotide or pharmaceutical composition into the eye.
Besides being used in a method of human disease prevention and/or treatment, the oligonucleotide and/or the pharmaceutical composition according to the present invention is also used in a method for prevention and/or treatment of other subjects including veterinary animals, reptiles, birds, exotic animals and farm animals, including mammals, rodents, and the like. Mammals include for example horses, dogs, pigs, cats, or primates (for example, a monkey, a chimpanzee, or a lemur). Rodents include for example rats, rabbits, mice, squirrels, or guinea pigs.
The oligonucleotide or the pharmaceutical composition according to the invention is used in a method for the prevention and/or treatment of many different diseases, preferably benign or malignant tumors, immunologic diseases, bronchial asthma, heart disease, fibrosis (e.g., liver fibrosis, idiopathic pulmonary fibrosis, liver cirrhosis, kidney cirrhosis, scleroderma), diabetes, wound healing, disorders of the connective tissue (e.g., in heart, blood vessel, bone, joint, eye such as the Marfan or Loeys-Dietz syndrome), psoriasis, eye diseases (e.g., glaucoma, posterior capsular opacification (PCO) also known as secondary cataract, retinoblastoma, choroidcarcinoma, Marfan or Loeys-Dietz syndrome, macular degeneration, such as age-related macular degeneration, diabetic macular endma, or cataract), CNS disease (e.g., Alzheimer's disease, Parkinson's disease), coronary atherosclerosis (coronary intervention or coronary artery bypass graft (CABG) surgery or hair loss. A tumor is for example selected from the group of solid tumors, blood born tumors, leukemias, tumor metastasis, hemangiomas, acoustic neuromas, neurofibromas, trachomas, pyogenic granulomas, astrocytoma such as anaplastic astrocytoma, acoustic neuroma, blastoma, Ewing's tumor, craniopharyngioma, ependymoma, medulloblastoma, glioma, glioblastoma, hemangloblastoma, Hodgkins-lymphoma, medullablastoma, leukaemia, melanoma such as primary and/or metastatic melanoma, mesothelioma, myeloma, neuroblastoma, neurofibroma, non-Hodgkins lymphoma, pinealoma, retinoblastoma, sarcoma, seminoma, trachomas, Wilm's tumor, bile duct carcinoma, bladder carcinoma, brain tumor, breast cancer, bronchogenic carcinoma, carcinoma of the kidney, cervical cancer, choriocarcinoma, cystadenocarcinome, embryonal carcinoma, epithelial carcinoma, esophageal cancer, cervical carcinoma, colon carcinoma, colorectal carcinoma, endometrial cancer, gallbladder cancer, gastric cancer, head cancer, liver carcinoma, lung carcinoma, medullary carcinoma, neck cancer, non-small-cell bronchogenic/lung carcinoma, ovarian cancer, pancreas carcinoma, papillary carcinoma, papillary adenocarcinoma, prostate cancer, small intestine carcinoma, prostate carcinoma, rectal cancer, renal cell carcinoma (RCC, e.g., clear cell RCC, papillary RCC, chromophobe RCC), oncocytoma kidney cancer, transitional cell kidney cancer, skin cancer, small-cell bronchogenic/lung carcinoma, squamous cell carcinoma, sebaceous gland carcinoma, testicular carcinoma, and uterine cancer. The oligonucleotide or the pharmaceutical composition of the present invention is not only used in a method for the prevention and/or treatment of a tumor, but likewise on a metastasis.
The present invention is preferably directed to the prevention and/or treatment of ophthalmic diseases such as, but not limited to, glaucoma, posterior capsular opacification, dry eye, macular degeneration, e.g., age-related macular degeneration, diabetic macular endma, cataract, proliferative vitreoretinopathy, Marfan or Loeys-Dietz syndrome, and any other ocular disease linkable to TGF-beta, in particular TGF-beta1, TGF-beta2, and/or TGF-beta3, and/or being associated with fibrosis, inflammation, degeneration, aging or similar.
The antisense oligonucleotides of the present invention are characterized in that they show an unexpected low toxicity (see for example Table 7) and thus, are well tolerated by different organisms. They oligonucleotides show a reasonable distribution in the organism, wherein highest concentrations are measured in the kidney, liver, skin and spleen.
The present invention provides numerous oligonucleotides, which are highly efficient in the reduction and inhibition, respectively, of TGF-beta, in particular TGF-beta1, TGF-beta2 and/or TGF-beta3 expression due to the specific selection of the sequence of the oligonucleotide and the modification of the nucleotide. The following Table 1 shows numerous preferred modified oligonucleotides according to the present invention (modified nucleosides are indicated in bold letters). Each oligonucleotide is defined as ASPH and a number, which is defined by a specific sequence and modification of the nucleosides:













SEQ





ID





NO.
Sequence (5′ → 3′)
Modification
ASPH


















5
GACCAGATGCAGGA
LNA 3 + 3
36


 


6
GCGACCGTGACCAGAT
LNA 3 + 3
80


 


7
GCGCGACCGTGACC
LNA 3 + 3
98


 


8
AGCGCGACCGTGA
LNA 2 + 3
111


 


9
GACCGTGACCAGAT
LNA 2 + 2
121


 


9
GACCGTGACCAGAT
LNA 3 + TEG
153


 


10
CTGCCCGCGGAT
LNA 2 + 2
15


 


11
TCTGCCCGCGGAT
LNA 3 + 2
17


 


12
GGATCTGCCCGCGGA
LNA 4 + 3
26


 


12
GGATCTGCCCGCGGA
LNA 3 + 4
27


 


13
CTTGCTCAGGATCTGCC
LNA 4 + 4
37


 


14
GCTCAGGATCTGCCCGCGG
2′ O-meth 4 + 4
52



A




 


14
GCTCAGGATCTGCCCGCGG
2′ fluoro 4 + 4
53



A




 


15
GGATCGCCTCGAT
LNA 3 + 2
112


 


16
CCGCGGATCGCC
LNA 2 + 2
119


 


17
ACCTCCTTGGCGTAGTA
LNA 3 + 3
01


 


17
ACCTCCTTGGCGTAGTA
LNA 4 + 4
02


 


18
CCTCCTTGGCGTAGTA
LNA 3 + 3
03


 


18
CCTCCTTGGCGTAGTA
LNA 4 + 4
04


 


19
CTCCTTGGCGTAGTA
LNA 3 + 3
05


 


19
CTCCTTGGCGTAGTA
LNA 4 + 3
06


 


19
CTCCTTGGCGTAGTA
LNA 3 + 4
07


 


20
TCCTTGGCGTAGTA
LNA 3 + 3
08


 


21
CAGAAGTTGGCAT
LNA 3 + 2
09


 


21
CAGAAGTTGGCAT
LNA 2 + 3
10


 


22
AAGTGGGCGGGAT
—
11


 


22
AAGTGGGCGGGAT
LNA 4 + 4
12


 


22
AAGTGGGCGGGAT
2′ O-meth 4 + 4
13


 


22
AAGTGGGCGGGAT
2′ fluoro 4 + 4
14


 


23
GCGGGATGGCAT
LNA 2 + 2
16


 


24
GAAATCACCTCCG
LNA 2 + 3
18


 


25
AAGTGGGCGGGAT
LNA 2 + 3
19


 


26
TGTAGCGCTGGGT
LNA 2 + 3
20


 


27
CGAAGGAGAGCCA
LNA 3 + 2
21


 


28
TCGCGCTCGCAGGC
LNA 3 + 3
22


 


29
AAGTGGGCGGGATG
LNA 3 + 3
23


 


30
ATGTAGCGCTGGGT
LNA 3 + 3
24


 


31
CGAAGGAGAGCCAT
LNA 3 + 3
25


 


32
GAAAGTGGGCGGGAT
LNA 4 + 3
28


 


33
CGAAGGAGAGCCATT
LNA 4 + 3
29


 


34
CGATCCTCTTGCGCAT
LNA 4 + 4
30


 


35
AAGTGGGCGGGATGGC
LNA 4 + 4
31


 


36
GATGGAAATCACCTCCG
LNA 4 + 4
32


 


37
AAACCTCCTTGGCGTAG
LNA 4 + 4
33


 


38
TAGAAAGTGGGCGGGAT
LNA 4 + 4
34


 


39
GGCGGGATGGCAT
LNA 2 + 3
35


 


40
GGGTCTGTAGAAAGTG
LNA 4 + 4
38


 


41
GAAGGAGAGCCATTC
LNA 3 + 4
39


 


42
CCAGGTTCCTGTCTT
LNA 3 + 4
40


 


43
TCTGATCACCACTGG
LNA 3 + 4
41


 


44
TTTCTGATCACCACTGG
LNA 4 + 4
42


 


45
GTCTGTAGGAGGGCA
LNA 4 + 3
43


 


46
AGTCTGTAGGAGGGCA
LNA 4 + 4
44


 


47
TCTGTAGGAGGGC
LNA 2 + 3
45


 


48
CAGATGCCAGTTTTAAC
LNA 4 + 4
46


 


49
CAAAGTATTTGGTCTCC
LNA 4 + 4
47


 


50
CCTTAAGCCATCCATGA
LNA 4 + 4
48


 


51
GTACTGGCCAGCTAA
LNA 4 + 3
49


 


52
GCCTCGATCCTCTTGCGCAT
2′ O-meth 4 + 4
50


 


52
GCCTCGATCCTCTTGCGCAT
2′ fluoro 4 + 4
51


 


53
AAACCTCCTTGGCGTAGTAC
2′ O-meth 4 + 4
54


 


53
AAACCTCCTTGGCGTAGTAC
2′ fluoro 4 + 4
55


 


54
GAAAGTGGGCGGGATGGCA
2′ O-meth 4 + 4
56



T




 


54
GAAAGTGGGCGGGATGGCA
2′ fluoro 4 + 4
57



T




 


55
GAATTGCTCGCTTAGGG
LNA 3 + 3
60


 


56
CGTCGCGGTTGCGTTCA
LNA 3 + 3
61


 


57
CGTGGCCTACACCCTGG
LNA 3 + 3
62


 


58
TTCTAAAGCAATAGGCC
LNA 3 + 3
63


 


59
AGAATGGTTAGAGGTTC
LNA 3 + 3
64


 


60
TCTGAACTAGTACCGCC
LNA 3 + 3
65


 


61
CCCATTAATATGACCTC
LNA 3 + 3
66


 


62
TTTAGTTAGAACCCTAA
LNA 3 + 3
67


 


63
CCTCAGATATAGATAAC
LNA 3 + 3
68


 


64
TACTATTATGGCATCCC
LNA 3 + 3
69


 


65
TGCCCACTTGCATACTA
LNA 3 + 3
70


 


66
AGCGTAATTGGTCATCA
LNA 3 + 3
71


 


67
CGTTGGCAGAACATAGA
LNA 3 + 3
72


 


68
GGGATACTGTCTAGACC
LNA 3 + 3
73


 


69
ATTGGCAACTCGTTTGA
LNA 3 + 3
74


 


70
CGTCAGGCTAATATTC
LNA 3 + 3
75


 


71
GGATGACTCCCTAGAC
LNA 3 + 3
76


 


72
GTCGCGGTTGCGTTCA
LNA 3 + 3
77


 


73
CTCGGTACTCGGTCGG
LNA 3 + 3
78


 


74
GGTTCGGTCCTGCCTT
LNA 3 + 3
79


 


75
AATAGGCCGCATCCAA
LNA 3 + 3
81


 


76
AACTAGTACCGCCTTT
LNA 3 + 3
82


 


77
TCGGTCATATAATAAC
LNA 3 + 3
83


 


78
AGACCGTCAGGCTAA
LNA 3 + 3
84


 


79
GTCGCGGTTGCGTTC
LNA 3 + 3
85


 


80
TTCCACTGCGGCGCT
LNA 3 + 3
86


 


81
AAGGAGCGGTTCGGT
LNA 3 + 3
87


 


82
CTCGGGTGCGGAGTG
LNA 3 + 3
88


 


83
CTGACTTTGGCGAGT
LNA 3 + 3
89


 


84
GATAGGAACGGTACG
LNA 3 + 3
90


 


85
CACTTTGGATTCCCG
LNA 3 + 3
91


 


86
GTCGCGGTTGCGTT
LNA 3 + 3
92


 


87
TACACCCTGGCGGG
LNA 3 + 3
93


 


88
CTCGGTACTCGGTC
LNA 3 + 3
94


 


89
AGGAGCGGTTCGGT
LNA 3 + 3
95


 


90
GTCTCGGGTGCGGA
LNA 3 + 3
96


 


91
TACGGGACGGGCAG
LNA 3 + 3
97


 


92
CGTCGCTCCTCTCG
LNA 3 + 3
99


 


93
TAGCGCTGGGTTGG
LNA 3 + 3
100


 


94
AAGCAATAGGCCGC
LNA 3 + 3
101


 


95
TACGGGCATGCTCC
LNA 3 + 3
102


 


96
AGGCGCGGGATAGG
LNA 3 + 3
103


 


97
TTTGGATTCCCGCC
LNA 3 + 3
104


 


98
ACCACTAGAGCACC
LNA 3 + 3
105


 


99
GCGTTGGCAGAACA
LNA 3 + 3
106


 


100
TTGCTCGCTTAGG
LNA 2 + 3
107


 


101
GTCGCGGTTGCGT
LNA 3 + 2
108


 


102
GGCGCTCGGTACT
LNA 2 + 3
109


 


103
ATCTGAACTCGGC
LNA 3 + 2
110


 


104
CGGTTGGTCTGTT
LNA 2 + 3
113


 


105
TCCACCCTAGATC
LNA 2 + 3
114


 


106
CTAGTACCGCCTT
LNA 2 + 3
115


 


107
GGTCGGCAGTCAA
LNA 3 + 2
116


 


108
CTTGCGACACCC
LNA 2 + 2
117


 


109
GAGCGGTTCGGT
LNA 2 + 2
118


 


110
ACACAGTAGTGCAT
LNA 2 + 2
120


 


111
GGGTCTGTAGAAAG
LNA 2 + 2
122


 


111
GGGTCTGTAGAAAG
LNA 3 + TEG
154


 


112
GGTTGGAGATGTTA
LNA 2 + 2
123


 


112
GGTTGGAGATGTTA
LNA 3 + TEG
155


 


113
TGGGTTGGAGATGT
LNA 2 + 2
124


 


113
TGGGTTGGAGATGT
LNA 3 + TEG
156


 


114
GCTGGGTTGGAGAT
LNA 2 + 2
125


 


114
GCTGGGTTGGAGAT
LNA 3 + TEG
157


 


115
GCGCTGGGTTGGAG
LNA 2 + 2
126


 


115
GCGCTGGGTTGGAG
LNA 3 + TEG
158


 


116
AGCGCTGGGTTGGA
LNA 2 + 2
127


 


116
AGCGCTGGGTTGGA
LNA 3 + TEG
159


 


117
TAGCGCTGGGTTGG
LNA 2 + 2
128


 


117
TAGCGCTGGGTTGG
LNA 3 + TEG
160


 


118
GTAGCGCTGGGTTG
LNA 2 + 2
129


 


118
GTAGCGCTGGGTTG
LNA 3 + TEG
161


 


119
GATGTAGCGCTGGG
LNA 2 + 2
130


 


119
GATGTAGCGCTGGG
LNA 3 + TEG
162


 


120
CCATTCGCCTTCTG
LNA 2 + 2
131


 


120
CCATTCGCCTTCTG
LNA 3 + TEG
163


 


121
GAGAGCCATTCGCC
LNA 2 + 2
132


 


121
GAGAGCCATTCGCC
LNA 3 + TEG
164


 


122
AGCAGGGACAGTGT
LNA 2 + 2
133


 


122
AGCAGGGACAGTGT
LNA 3 + TEG
165


 


123
GCAGGAGATGTGGG
LNA 2 + 2
134


 


123
GCAGGAGATGTGGG
LNA 3 + TEG
166


 


124
CGGTTGGTCTGTTG
LNA 2 + 2
135


 


124
CGGTTGGTCTGTTG
LNA 3 + TEG
167


 


125
CCGGTTGGTCTGTT
LNA 2 + 2
136


 


125
CCGGTTGGTCTGTT
LNA 3 + TEG
168


 


126
GCCGGTTGGTCTGT
LNA 2 + 2
137


 


126
GCCGGTTGGTCTGT
LNA 3 + TEG
169


 


127
AGTTGGCATTGTAC
LNA 2 + 2
138


 


127
AGTTGGCATTGTAC
LNA 3 + TEG
170


 


128
GGTTAGAGGTTCTA
LNA 2 + 2
139


 


128
GGTTAGAGGTTCTA
LNA 3 + TEG
171


 


129
ATGGTTAGAGGTTC
LNA 2 + 2
140


 


129
ATGGTTAGAGGTTC
LNA 3 + TEG
172


 


130
AGAATGGTTAGAGG
LNA 2 + 2
141


 


130
AGAATGGTTAGAGG
LNA 3 + TEG
173


 


131
AGAGAATGGTTAGA
LNA 2 + 2
142


 


131
AGAGAATGGTTAGA
LNA 3 + TEG
174


 


132
CGTTGTCGTCGTCA
LNA 2 + 2
143


 


132
CGTTGTCGTCGTCA
LNA 3 + TEG
175


 


133
ACCAAGGCTCTCTT
LNA 2 + 2
144


 


133
ACCAAGGCTCTCTT
LNA 3 + TEG
176


 


134
GCTTCTTGTCTCTC
LNA 2 + 2
145


 


134
GCTTCTTGTCTCTC
LNA 3 + TEG
177


 


135
GGAACGGTACGTAC
LNA 2 + 2
146


 


135
GGAACGGTACGTAC
LNA 3 + TEG
178


 


136
TAGGAACGGTACGT
LNA 2 + 2
147


 


136
TAGGAACGGTACGT
LNA 3 + TEG
179


 


137
GGGATAGGAACGGT
LNA 2 + 2
148


 


137
GGGATAGGAACGGT
LNA 3 + TEG
180


 


138
CGCGGGATAGGAAC
LNA 2 + 2
149


 


138
CGCGGGATAGGAAC
LNA 3 + TEG
181


 


139
AGGCGCGGGATAGG
LNA 2 + 2
150


 


139
AGGCGCGGGATAGG
LNA 3 + TEG
182


 


140
GTCAAGCTGGATGG
LNA 2 + 2
151


 


140
GTCAAGCTGGATGG
LNA 3 + TEG
183


 


141
TCTGTAGGAGGGC
ENA 2 + 3
184


 


142
GACCAGATGCAGGA
ENA 3 + 3
185


 


143
CTCCTTGGCGTAGTA
ENA 3 + 3
186


 


144
CCTCCTTGGCGTAGTA
ENA 3 + 3
187


 


145
CAGATGCCAGTTTTAAC
ENA 4 + 4
188


 


146
AGCGTAATTGGTCATCA
ENA 3 + 3
189


 


147
AGTATTTGGTCTCC
LNA 3 + 3
190 or





M12-





ASPH47


 


148
AAGTATTTGGTCTC
LNA 3 + 3
191 or





M9-





ASPH47


 


149
AAGTATTTGGTCTCC
LNA 3 + 3
192 or





M8-





ASPH47


 


150
CAAAGTATTTGGTCTCC
LNA 3 + 3
193


 


151
AGCTCGTCCCTCCTCCC
LNA 3 + 3
1000


 


152
GAGGGCTGGTCCGGAAT
LNA 3 + 3
1001


 


153
CGAGGGCTGGTCCGGAA
LNA 3 + 3
1002


 


154
GAGGGCGGCATGGGGGA
LNA 3 + 3
1003


 


155
GCGGGTGCTGTTGTACA
LNA 3 + 3
1004


 


156
CGCGGGTGCTGTTGTAC
LNA 3 + 3
1005


 


157
GTCGCGGGTGCTGTTGT
LNA 3 + 3
1006


 


158
GGTCGCGGGTGCTGTTG
LNA 3 + 3
1007


 


159
CCGGTCGCGGGTGCTGT
LNA 3 + 3
1008


 


160
CCCGGTCGCGGGTGCTG
LNA 3 + 3
1009


 


161
AGCACGCGGGTGACCTC
LNA 3 + 3
1010


 


162
TTAGCACGCGGGTGACC
LNA 3 + 3
1011


 


163
GGGCTCGTGGATCCACT
LNA 3 + 3
1012


 


164
CCTTGGGCTCGTGGATC
LNA 3 + 3
1013


 


165
TGGCATGGTAGCCCTTG
LNA 3 + 3
1014


 


166
CGAGGGCTGGTCCGGA
LNA 3 + 3
1015


 


167
GCGGGTGCTGTTGTAC
LNA 3 + 3
1016


 


168
GCACGCGGGTGACCTC
LNA 3 + 3
1017


 


169
CCTTGGGCTCGTGGAT
LNA 3 + 3
1018


 


170
GGCATGGTAGCCCTTG
LNA 3 + 3
1019


 


171
GGGTGCTGTTGTAC
LNA 3 + 3
1020


 


172
TCGCGGGTGCTGTT
LNA 3 + 3
1021


 


173
GTCGCGGGTGCTGT
LNA 3 + 3
1022


 


174
CTCGTGGATCCACT
LNA 3 + 3
1023


 


175
ATGGTAGCCCTTGG
LNA 3 + 3
1024


 


176
TGGCATGGTAGCCC
LNA 3 + 3
1025


 


177
GAAGTTGGCATGGT
LNA 3 + 3
1026


 


178
TCGCGGGTGCTGT
LNA 2 + 3
1027


 


179
CACCCGGTCGCGG
LNA 2 + 3
1028


 


180
CCACCCGGTCGCG
LNA 2 + 3
1029


 


181
CGCCAGGAATTGT
LNA 3 + 2
1030


 


182
GGCTCGTGGATCC
LNA 2 + 3
1031


 


183
TGGGCTCGTGGAT
LNA 2 + 3
1032


 


184
GCATGGTAGCCCT
LNA 2 + 3
1033


 


185
AGTTGGCATGGTA
LNA 2 + 3
1034


 


186
TTGCAGGAGCGCA
LNA 2 + 3
1035


 


187
ATTAGCACGCGGGTGAC
LNA 3 + 3
1036


 


188
ACCATTAGCACGCGGGT
LNA 3 + 3
1037


 


189
CACCATTAGCACGCGGG
LNA 3 + 3
1038


 


190
CCACCATTAGCACGCGG
LNA 3 + 3
1039


 


191
TCCACCATTAGCACGCG
LNA 3 + 3
1040


 


192
TCCACCTTGGGCTTGCG
LNA 3 + 3
1041


 


193
TTAGCACGCGGGTGAC
LNA 3 + 3
1042


 


194
ACCATTAGCACGCGGG
LNA 3 + 3
1043


 


195
CACCATTAGCACGCGG
LNA 3 + 3
1044


 


196
CACCATTAGCACGCG
LNA 3 + 3
1045


 


197
GCGGCACGCAGCACG
LNA 3 + 3
1046


 


198
TCGATGCGCTTCCG
LNA 3 + 3
1047


 


199
TAGCACGCGGGTGA
LNA 3 + 3
1048


 


200
ATTAGCACGCGGGT
LNA 3 + 3
1049


 


201
CATTAGCACGCGGG
LNA 3 + 3
1050


 


202
ACCATTAGCACGCG
LNA 3 + 3
1051


 


203
CACCATTAGCACGC
LNA 3 + 3
1052


 


204
CCACCATTAGCACG
LNA 3 + 3
1053


 


205
TCCACCATTAGCAC
LNA 3 + 3
1054


 


206
GACCTTGCTGTACT
LNA 3 + 3
1055


 


207
GGACCTTGCTGTAC
LNA 3 + 3
1056


 


208
AGGACCTTGCTGTA
LNA 3 + 3
1057


 


209
CGGCACGCAGCACG
LNA 3 + 3
1058


 


210
ACCTTGGGCTTGCG
LNA 3 + 3
1059


 


211
TTAGCACGCGGGT
LNA 3 + 2
1060


 


212
ACCATTAGCACGC
LNA 3 + 2
1061


 


213
CGGCACGCAGCAC
LNA 3 + 2
1062


 


214
CACCAGCTCCATGTCGA
LNA 3 + 3
1063


 


215
TCGCGGGTGCTGTTGTA
LNA 3 + 3
1064


 


216
GTGTCCAGGCTCCAAAT
LNA 3 + 3
1065


 


216
GTGTCCAGGCTCCAAAT
LNA 4 + 2
1066


 


217
GCTCGTCCCTCCTCCC
LNA 3 + 3
1067


 


218
ACCAGCTCGTCCCTCC
LNA 3 + 3
1068


 


219
GGAGGCCCCGCCCCTG
LNA 3 + 3
1069


 


220
CATGGGGGAGGCGGCG
LNA 3 + 3
1070


 


220
CATGGGGGAGGCGGCG
3LNA + 9N + 1LNA +
1071




1N + 2LNA



 


221
ACCAGCTCCATGTCGA
LNA 3 + 3
1072


 


222
GGTCGCGGGTGCTGTT
LNA 3 + 3
1073


 


223
GGACCTTGCTGTACTG
LNA 3 + 3
1074


 


223
GGACCTTGCTGTACTG
LNA 4 + 2
1075


 


224
TCCACCTTGGGCTTGC
LNA 3 + 3
1076


 


225
AGCTCGTCCCTCCTC
LNA 3 + 3
1077


 


226
CCAGCTCGTCCCTCC
LNA 3 + 3
1078


 


227
GAGGGCTGGTCCGGA
LNA 3 + 3
1079


 


228
TCCCGAGGGCTGGTC
LNA 3 + 3
1080


 


229
CGGCATGGGGGAGGC
LNA 2 + 4
1081


 


230
CAGCTCCATGTCGAT
LNA 3 + 3
1082


 


231
ACCAGCTCCATGTCG
LNA 3 + 3
1083


 


232
TCGCGGGTGCTGTTG
LNA 3 + 3
1084


 


233
GTCGCGGGTGCTGTT
LNA 3 + 3
1085


 


234
GGTCGCGGGTGCTGT
LNA 3 + 3
1086


 


235
AGCACGCGGGTGACC
LNA 3 + 3
1087


 


236
TAGCACGCGGGTGAC
LNA 3 + 3
1088


 


237
CATTAGCACGCGGGT
LNA 3 + 3
1089


 


238
TCCACCATTAGCACG
LNA 3 + 3
1090


 


239
CCAGGAATTGTTGCT
LNA 4 + 2
1091


 


240
TTGGGCTCGTGGATC
LNA 3 + 3
1092


 


241
CTTGGGCTCGTGGAT
LNA 3 + 3
1093


 


242
TTGGCATGGTAGCCC
LNA 3 + 3
1094


 


243
GAAGTTGGCATGGTA
LNA 3 + 3
1095


 


244
AGAAGTTGGCATGGT
LNA 3 + 3
1096


 


245
TGTCCAGGCTCCAAA
LNA 4 + 2
1097


 


246
AGGACCTTGCTGTAC
LNA 3 + 3
1098


 


247
CACCTTGGGCTTGCG
LNA 4 + 2
1099


 


247
CACCTTGGGCTTGCG
1LNA + 1N + 2LNA +
1100




8N + 1LNA + 1N +





1LNA



 


248
AGCTCGTCCCTCCT
LNA 3 + 3
1101


 


249
CAGCTCGTCCCTCC
LNA 3 + 3
1102


 


250
ACCAGCTCGTCCCT
LNA 3 + 3
1103


 


251
CCCGAGGGCTGGTC
LNA 3 + 3
1104


 


252
GCGGCATGGGGGAG
LNA 2 + 4
1105


 


253
GTCTTGCAGGTGGA
LNA 3 + 3
1106


 


254
TCGATGCGCTTCCG
LNA 2 + 4
1107


 


254
TCGATGCGCTTCCG
LNA 2 + 3
1108


 


254
TCGATGCGCTTCCG
2LNA + 8N + 2LNA +
1109




1N + 1LNA



 


254
TCGATGCGCTTCCG
2LNA + 9N + 1LNA +
1110




1N + 1LNA



 


254
TCGATGCGCTTCCG
2LNA + 8N + 1LNA +
1111




2N + 1LNA



 


255
GGACAGGATCTGGC
LNA 4 + 2
1112


 


256
ACCTCCCCCTGGCT
LNA 3 + 3
1113


 


257
ACCATTAGCACGCG
LNA 4 + 2
1114


 


257
ACCATTAGCACGCG
3LNA + 8N + 1LNA +
1115




1N + 1LNA



 


258
CAGCAGTTCTTCTC
LNA 2 + 4
1116


 


259
TACAGCTGCCGCAC
LNA 3 + 3
1117


 


260
AGTTGGCATGGTAG
LNA 3 + 3
1118


 


260
AGTTGGCATGGTAG
LNA 4 + 2
1119


 


261
AAGTTGGCATGGTA
LNA 3 + 3
1120


 


262
GAAGTTGGCATGGT
LNA 4 + 2
1121


 


263
TCCAGGCTCCAAAT
LNA 3 + 3
1122


 


264
ACCTTGCTGTACTG
LNA 3 + 3
1123


 


264
ACCTTGGGCTTGCG
LNA 4 + 2
1124


 


264
ACCTTGGGCTTGCG
LNA 3 + 2
1125


 


264
ACCTTGGGCTTGCG
3LNA + 8N + 1LNA +
1126




1N + 1LNA



 


264
ACCTTGGGCTTGCG
2LNA + 9N + 1LNA +
1127




1N + 1LNA



 


264
ACCTTGGGCTTGCG
2LNA + 8N + 2LNA +
1128




1N + 1LNA



 


265
TTGCAGGAGCGCAC
LNA 3 + 3
1129


 


266
GCAGAAGTTGGCAT
LNA 4 + 2
1130


 


267
CGGGTGCTGTTGTA
LNA 3 + 3
1131


 


267
CGGGTGCTGTTGTA
LNA 2 + 4
1132


 


268
CCCAGCGGCAACGGAAA
LNA 3 + 3
1133


 


269
CAAGAGGTCCCCGCGCC
LNA 3 + 3
1134


 


270
GCGTCCCCGGCGGCAAA
LNA 3 + 3
1135


 


271
GGTCGGCGACTCCCGAG
LNA 3 + 3
1136


 


272
TCGGAGAGAGATCCGTC
LNA 3 + 3
1137


 


273
ATCCCACGGAAATAACC
LNA 3 + 3
1138


 


274
CTCAGTATCCCACGGAA
LNA 3 + 3
1139


 


275
ACTGCCGAGAGCGCGAA
LNA 3 + 3
1140


 


276
CTGATGTGTTGAAGAAC
LNA 3 + 3
1141


 


277
TGAGGTATCGCCAGGAA
LNA 3 + 3
1142


 


278
ACTGCCGCACAACTCCG
LNA 3 + 3
1143


 


279
CGGCCCACGTAGTACAC
LNA 3 + 3
1144


 


280
CCCAGCGGCAACGGAA
LNA 3 + 3
1145


 


281
TCGCGCCAAGAGGTCC
LNA 3 + 3
1146


 


282
GGTCGGCGACTCCCGA
LNA 3 + 3
1147


 


283
GTCGGAGAGAGATCCG
LNA 3 + 3
1148


 


284
TCAGTATCCCACGGAA
LNA 3 + 3
1149


 


285
CGAGAGCGCGAACAGG
LNA 3 + 3
1150


 


286
ACTGCCGAGAGCGCGA
LNA 3 + 3
1151


 


287
GGCGTCAGCACCAGTA
LNA 3 + 3
1152


 


288
GGTTTCCACCATTAGC
LNA 3 + 3
1153


 


289
GAGGTATCGCCAGGAA
LNA 3 + 3
1154


 


290
AACCACTGCCGCACAA
LNA 3 + 3
1155


 


291
CGGCCCACGTAGTACA
LNA 3 + 3
1156


 


292
CGGCGGCTCGTCTCA
LNA 3 + 3
1157


 


293
CCCAGCGGCAACGGA
LNA 3 + 3
1158


 


294
TCGCGCCAAGAGGTC
LNA 3 + 3
1159


 


295
CGTCGCGCCAAGAGG
LNA 3 + 3
1160


 


296
GGAGCAAGCGTCCCC
LNA 3 + 3
1161


 


297
GTGCGCCCGAGGTCT
LNA 3 + 3
1162


 


298
GTCTAGGATGCGCGG
LNA 3 + 3
1163


 


299
CAGTATCCCACGGAA
LNA 3 + 3
1164


 


300
CCGAGAGCGCGAACA
LNA 3 + 3
1165


 


301
GGCGTCAGCACCAGT
LNA 3 + 3
1166


 


302
GTTGCTGAGGTATCG
LNA 3 + 3
1167


 


303
ACCACTGCCGCACAA
LNA 3 + 3
1168


 


304
CGGCCCACGTAGTAC
LNA 3 + 3
1169


 


305
CTCGGCGACTCCTT
LNA 3 + 3
1170


 


306
AGCGGCAACGGAAA
LNA 3 + 3
1171


 


307
TCGCGCCAAGAGGT
LNA 3 + 3
1172


 


308
TCCCCGGCGGCAAA
LNA 3 + 3
1173


 


309
TGCGCCCGAGGTCT
LNA 3 + 3
1174


 


310
GTCTAGGATGCGCG
LNA 3 + 3
1175


 


311
GGTCGGAGAGAGAT
LNA 3 + 3
1176


 


312
CACGGAAATAACCT
LNA 3 + 3
1177


 


313
AGAGCGCGAACAGG
LNA 3 + 3
1178


 


314
ATAGTCCCGCGGCC
LNA 3 + 3
1179


 


315
TAGTAGTCGGCCTC
LNA 3 + 3
1180


 


316
ATAGATTTCGTTGT
LNA 3 + 3
1181


 


317
GAGGTATCGCCAGG
LNA 3 + 3
1182


 


318
GCCGCACAACTCCG
LNA 3 + 3
1183


 


319
TCGCGCCAAGAGG
LNA 2 + 3
1184


 


320
AAGCGTCCCCGGC
LNA 3 + 2
1185


 


321
GACGCCGTGTAGG
LNA 3 + 2
1186


 


322
GTCGGCGACTCCC
LNA 2 + 3
1187


 


323
TGCGCCCGAGGTC
LNA 3 + 2
1188


 


324
GTCGGAGAGAGAT
LNA 3 + 2
1189


 


325
TCCCACGGAAATA
LNA 3 + 2
1190


 


326
TGCCGAGAGCGCG
LNA 2 + 3
1191


 


327
TAGTCCCGCGGCC
LNA 3 + 2
1192


 


328
TAGTAGTCGGCCT
LNA 3 + 2
1193


 


329
CATAGATTTCGTT
LNA 2 + 3
1194


 


330
TTTAACTTGAGCC
LNA 3 + 2
1195


 


331
GAGGTATCGCCAG
LNA 3 + 2
1196


 


332
ACTCCGGTGACAT
LNA 2 + 3
1197


 


333
GCCCACGTAGTAC
LNA 2 + 3
1198


 


334
TCGGCGACTCCC
LNA 2 + 2
1199


 


335
GTCGGCGACTCC
LNA 2 + 2
1200


 


336
CAGGAAGCGCTGGCAAC
LNA 3 + 3
2000


 


337
GGTGCATGAACTCACTG
LNA 3 + 3
2001


 


338
GTCCCCTAATGGCTTCC
LNA 3 + 3
2002


 


339
ATCTGTCCCCTAATGGC
LNA 3 + 3
2003


 


340
CCGGGTGCTGTTGTAAA
LNA 3 + 3
2004


 


341
CCTGGATCATGTCGAAT
LNA 3 + 3
2005


 


342
CCCTGGATCATGTCGAA
LNA 3 + 3
2006


 


343
GTAGCACCTGCTTCCAG
LNA 3 + 3
2007


 


344
GGGCTTTCTAAATGAC
LNA 3 + 3
2008


 


345
TGACTCCCAGCAGGCC
LNA 3 + 3
2009


 


346
GTGCATGAACTCACTG
LNA 3 + 3
2010


 


347
GGTGCATGAACTCACT
LNA 3 + 3
2011


 


348
ATCTGTCCCCTAATGG
LNA 3 + 3
2012


 


349
CGGGTGCTGTTGTAAA
LNA 3 + 3
2013


 


350
CCGGGTGCTGTTGTAA
LNA 3 + 3
2014


 


351
CCTGGATCATGTCGAA
LNA 3 + 3
2015


 


352
CCCTGGATCATGTCGA
LNA 3 + 3
2016


 


353
TTTGAATTTGATTTCC
LNA 3 + 3
2017


 


354
GGGCCTGAGCAGAAGT
LNA 3 + 3
2018


 


355
GGGGGCTTTCTAAAT
LNA 3 + 3
2019


 


356
TTTGTTTACACTTCC
LNA 3 + 3
2020


 


357
CCAGCTAAAGGTGGG
LNA 3 + 3
2021


 


358
ATGGCTGGGTCCCAA
LNA 3 + 3
2022


 


359
GAGTTTTTCCTTAGG
LNA 3 + 3
2023


 


360
AGGGGTGGCAAGGCA
LNA 3 + 3
2024


 


361
TGACTCCCAGCAGGC
LNA 3 + 3
2025


 


362
GAAGCGCTGGCAACC
LNA 3 + 3
2026


 


363
GTGCATGAACTCACT
LNA 3 + 3
2027


 


364
GTGGTGCAAGTGGAC
LNA 3 + 3
2028


 


365
CTAATGGCTTCCACC
LNA 3 + 3
2029


 


366
CCCCTAATGGCTTCC
LNA 3 + 3
2030


 


367
ATCTGTCCCCTAATG
LNA 3 + 3
2031


 


368
GATCTGTCCCCTAAT
LNA 3 + 3
2032


 


369
AGATCTGTCCCCTAA
LNA 3 + 3
2033


 


370
GGTGCTGTTGTAAAG
LNA 3 + 3
2034


 


371
CCGGGTGCTGTTGTA
LNA 3 + 3
2035


 


372
GATCATGTCGAATTT
LNA 3 + 3
2036


 


373
CCTGGATCATGTCGA
LNA 3 + 3
2037


 


374
CCCTGGATCATGTCG
LNA 3 + 3
2038


 


375
GATTTCCATCACCTC
LNA 3 + 3
2039


 


376
TTGAATTTGATTTCC
LNA 3 + 3
2040


 


377
AGCAGTTCTCCTCCA
LNA 3 + 3
2041


 


378
GCCTGAGCAGAAGTT
LNA 3 + 3
2042


 


379
GGGCAAGGGCCTGAG
LNA 3 + 3
2043


 


380
CCCACACTTTCTTTA
LNA 3 + 3
2044


 


381
TAGCACCTGCTTCCA
LNA 3 + 3
2045


 


382
CGGGGGCTTTCTAA
LNA 3 + 3
2046


 


383
CCATTCATGCTTTC
LNA 3 + 3
2047


 


384
AAGCGCTGGCAACC
LNA 3 + 3
2048


 


385
ACCAGAGCCCTTTG
LNA 3 + 3
2049


 


386
CCCCTAATGGCTTC
LNA 3 + 3
2050


 


387
GTCCCCTAATGGCT
LNA 3 + 3
2051


 


388
ATCTGCCCCTAAT
LNA 3 + 3
2052


 


389
AGATCTGTCCCCTA
LNA 3 + 3
2053


 


390
CGGGTGCTGTTGTA
LNA 3 + 3
2054


 


391
ATCATGTCGAATTT
LNA 3 + 3
2055


 


392
CCCTGGATCATGTC
LNA 3 + 3
2056


 


393
CCTTTGAATTTGAT
LNA 3 + 3
2057


 


394
TTGCGGAAGCAGTA
LNA 3 + 3
2058


 


395
GCCTGAGCAGAAGT
LNA 3 + 3
2059


 


396
GGGGGCTTTCTAA
LNA 2 + 3
2060


 


397
AGCGCTGGCAACC
LNA 2 + 3
2061


 


398
CCCCTAATGGCTT
LNA 2 + 3
2062


 


398
CCCCTAATGGCTT
LNA 3 + 2
2063


 


399
TCCCCTAATGGCT
LNA 3 + 2
2064


 


400
TCATGTCGAATTT
LNA 2 + 3
2065


 


401
ATCATGTCGAATT
LNA 3 + 2
2066









Table 1 shows the nucleic acid sequences of selected oligonucleotides of the present invention as well as the modifications of the nucleotides, wherein LNA 4+4 means 4×LNAs at the 5′- and 3′-end of the oligonucleotide are modified, wherein LNA 4+3 means 4×LNAs at the 5′-end and 3×LNAs at the 3′-end of the oligonucleotide are modified, wherein LNA 3+4 means 3×LNAs at the 5′-end and 4×LNAs at the 3′-end of the oligonucleotide are modified, wherein LNA 3+3 means 3×LNAs at the 5′- and 3′-end of the oligonucleotide are modified, wherein LNA 3+2 means 3×LNAs at the 5′-end and 2×LNAs at the 3′-end of the oligonucleotide are modified, wherein LNA 2+3 means 2×LNAs at the 5′-end and 3×LNAs at the 3′-end of the oligonucleotide are modified, wherein LNA 2+2 means 2×LNAs at the 5′- and 3′-end of the oligonucleotide are modified. Alternatively, some oligonucleotides comprise ENA 4+4, i.e., 4×ENA at the 5′- and 3′-end of the oligonucleotide are modified, or ENA 3+3, i.e, 3×ENA at the 5′- and 3′-end of the oligonucleotide are modified. Further oligonucleotides comprise 2′O-meth 4+4, wherein the oligonucleotide comprises 4×2′O-methyl modified nucleotides at the 5′- and 3′-end of the oligonucleotide, or comprises 2′ fluoro 4+4, wherein the oligonucleotide comprises 4×2′ fluoro modified nucleotides at the 5′- and 3′-end. Oligonucleotides comprising LNA 3+TEG comprise 3×LNAs at the 5′-end and one triethylene glycol (TEG) at the 3′-end of the oligonucleotide. Some oligonucleotides comprise LNAs which are not arranged in a row but are separated by an unlocked nucleoside having for example the sequences 3LNA+9N+1LNA+1N+2LNA, 1LNA+1N+2LNA+8N+1LNA+1N+1LNA, 2LNA+8N+2LNA+1N+1LNA, 2LNA+9N+1LNA+1N+1LNA, 2LNA+8N+1LNA+2N+1LNA, 3LNA+8N+1LNA+1N+1LNA, 3LNA+8N+1LNA+1N+1LNA, 2LNA+9N+1LNA+1N+1LNA, or 2LNA+8N+2LNA+1N+1LNA, wherein “N” is a nucleoside without locked modification. “ASPH” in combination with a number refers to the different oligonucleotides and their different modifications as described in Table 1. These modified oligonucleotides were tested e.g. in experiments shown in the examples. The antisense oligonucleotides of the present invention can be described differently, e.g., ASPH47, ASPH0047, ASPH_47 or ASPH_0047 referring to the same oligonucleotide.
In Table 2 further preferred oligonucleotides of the present invention are shown, which are variations of the sequence and/or the LNA pattern of ASPH47 (SEQ ID NO. 49).













SEQ





ID





NO.
Sequence
Modification
ASPH


















402
AGTATTTGGTCTCC
LNA 2 + 3
194


 


402
AGTATTTGGTCTCC
1LNA + 1N + 1LNA + 8N +
195




3LNA



 


402
AGTATTTGGTCTCC
3LNA + 8N + 1LNA + 1N +
196




1LNA



 


402
AGTATTTGGTCTCC
LNA 3 + 2
197


 


403
AAGTATTTGGTCTC
LNA 4 + 2
198


 


403
AGTATTTGGTCTCCA
3LNA + 8N + 1LNA + 1N +
199




2LNA



 


403
AGTATTTGGTCTCCA
3LNA + 8N + 2LNA + 1N +
200




1LNA



 


403
AGTATTTGGTCTCCA
2LNA + 1N + 1LNA + 8N +
201




3LNA



 


403
AGTATTTGGTCTCCA
1LNA + 1N + 2LNA + 8N +
202




3LNA



 


403
AGTATTTGGTCTCCA
LNA 3 + 2
203


 


403
AGTATTTGGTCTCCA
LNA 2 + 3
204


 


403
AGTATTTGGTCTCCA
LNA 2 + 4
205


 


404
AAGTATTTGGTCTCC
3LNA + 8N + 1LNA + 1N +
206




2LNA



 


404
AAGTATTTGGTCTCC
3LNA + 8N + 2LNA + 1N +
207




1LNA



 


404
AAGTATTTGGTCTCC
2LNA + 1N + 1LNA + 8N +
208




3LNA



 


404
AAGTATTTGGTCTCC
1LNA + 1N + 2LNA + 8N +
209




3LNA



 


404
AAGTATTTGGTCTCC
LNA 3 + 2
210


 


404
AAGTATTTGGTCTCC
LNA 2 + 3
211


 


49
CAAAGTATTTGGTCT
LNA 3 + 3
212



CC




 


49
CAAAGTATTTGGTCT
LNA 2 + 2
213



CC




 


49
CAAAGTATTTGGTCT
1LNA + 2N + 2LNA + 8N +
214



CC
3LNA



 


49
CAAAGTATTTGGTCT
1LNA + 3N + 1LNA + 8N +
215



CC
3LNA



 


49
CAAAGTATTTGGTCT
1LNA + 2N + 2LNA + 8N +
216



CC
4LNA



 


49
CAAAGTATTTGGTCT
1LNA + 2N + 2LNA + 8N +
217



CC
1LNA + 1N + 2LNA



 


49
CAAAGTATTTGGTCT
1LNA + 1N + 3LNA + 8N +
218



CC
3LNA



 


49
CAAAGTATTTGGTCT
1LNA + 1N + 2LNA + 8N +
219



CC
3LNA



 


49
CAAAGTATTTGGTCT
1LNA + 2N + 3LNA + 8N +
220



CC
2LNA



 


49
CAAAGTATTTGGTCT
1LNA + 2N + 3LNA + 8N +
221



CC
1LNA + 1N + 1LNA



 


49
CAAAGTATTTGGTCT
LNA 3 + TEG
222



CC-TEG




 


49
CAAAGTATTTGGTCT
LNA 4 + TEG
223



CC-TEG




 


405
CAAAGTATTTGGTCT
LNA 4 + 3
M1-



C

ASPH47


 


406
CAAAGTATTTGGTCT
LNA 4 + 2
M2-





ASPH47


 


407
CAAAGTATTTGGTC
LNA 4 + 1
M3-





ASPH47


 


408
AAAGTATTTGGTCTC
LNA 3 + 4
M4-



C

ASPH47


 


409
AAAGTATTTGGTCTC
LNA 3 + 3
M5-





ASPH47


 


410
AAAGTATTTGGTCT
LNA 3 + 2
M6-





ASPH47


 


411
AAAGTATTTGGTC
LNA 3 + 1
M7-





ASPH47


 


412
AAGTATTTGGTCT
LNA 2 + 2
M10-





ASPH47


 


413
AAGTATTTGGTC
LNA 2 + 1
M11-





ASPH47


 


414
AGTATTTGGTCTC
LNA 1 + 3
M13-





ASPH47


 


415
AGTATTTGGTCT
LNA 1 + 2
M14-





ASPH47


 


416
AGTATTTGGTC
LNA 1 + 1
M15-





ASPH47









The description of the modifications in Table 2 corresponds to the description provided in Table 1; in addition, LNA nucleosides are indicated in the sequence in bold letters, and triethylene glycol is abbreviated as TEG in Table 2.
For the purpose of clarity and a concise description, features are described herein as part of the same or separate embodiments, however, it will be appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described.
The following examples will serve to further illustrate the present invention without, at the same time, however, constituting any limitation thereof. On the contrary, it is to be clearly understood that the scope of the present invention refers to various other embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the invention.
EXAMPLES
In the following examples, the effect of the oligonucleotides listed in Tables 1 and 2 have been tested in view of the reduction and inhibition, respectively, of TGF-beta1 and/or TGF-beta2 expression. SEQ ID NO. 144 (T-LNA: CGGCATGTCTATTTTGTA, wherein 3× nucleotides at the 5′- and 3′-end are LNAs) and SEQ ID NO. 145 (scr-LNA: CGTTTAGGCTATGTACTT, wherein 3× nucleotides at the 5′- and 3′-end are LNAs) are used as control oligonucleotides, wherein SEQ ID NO. 145 (negative control) is the scrambled form of SEQ ID NO. 144 (positive control). The cells were either transfected in the presence of a transfecting agent (e.g., Lipofectamine), or in the absence of any transfecting agent (gymnotic transfection or unassisted transfection or gymnotic delivery). As in case of a gymnotic transfection the entry of the oligonucleotide into the cell solely depends on the interaction of the oligonucleotide and the cell, and no compound supports the entry, gymnotic transfection reflects better conditions of the in vivo experimental settings.
Example 1
Human A172 glioma cells were transfected with 10 nM of ASPH01, ASPH02, ASPH03, ASPH04, ASPH05, ASPH06, ASPH07, ASPH08, ASPH09, ASPH10, ASPH11, ASPH12, ASPH13, ASPH14, ASPH15, ASPH16, ASPH17, ASPH18, ASPH19, ASPH20, ASPH21, ASPH22, ASPH24, ASPH25, ASPH26, ASPH27, ASPH29, ASPH30, ASPH31, ASPH32, ASPH33, ASPH34, ASPH35, ASPH36, ASPH37, ASPH38, ASPH39, ASPH40, ASPH41, ASPH42, ASPH43, ASPH44, ASPH45, ASPH46, ASPH47, ASPH48, ASPH49, ASPH50, ASPH51, ASPH52, ASPH53, and ASPH54 (see FIG. 5a); ASPH36, ASPH60, ASPH61, ASPH62, ASPH63, ASPH64, ASPH65, ASPH66, ASPH67, ASPH68, ASPH69, ASPH70, ASPH71, ASPH72, ASPH73, ASPH74, ASPH75, ASPH76, ASPH77, ASPH78, ASPH79, ASPH80, ASPH81, ASPH82, ASPH83, ASPH84, ASPH85, ASPH86, ASPH87, ASPH88, ASPH89, ASPH90, ASPH91, ASPH92, ASPH93, ASPH94, ASPH95, ASPH96, ASPH97, ASPH98, ASPH99, ASPH100, ASPH101, ASPH102, ASPH103, ASPH104, ASPH105, ASPH106, ASPH107, ASPH108, ASPH109, ASPH110, ASPH111, ASPH112, ASPH113, ASPH114, ASPH115, ASPH116, ASPH117, ASPH118, and ASPH119 (see FIG. 5b), or ASPH36, ASPH71, ASPH73, ASPH120, ASPH121, ASPH122, ASPH123, ASPH124, ASPH125, ASPH126, ASPH127, ASPH128, ASPH129, ASPH130, ASPH131, ASPH132, ASPH133, ASPH134, ASPH135, ASPH136, ASPH137, ASPH138, ASPH139, ASPH140, ASPH141, ASPH142, ASPH143, ASPH145, ASPH146, ASPH147, ASPH148, ASPH149, ASPH150, ASPH151, ASPH152, ASPH153, ASPH154, ASPH155, ASPH157, ASPH158, ASPH160, ASPH161, ASPH162, ASPH163, ASPH164, ASPH165, ASPH166, ASPH167, ASPH168, ASPH169, ASPH170, ASPH171, ASPH172, ASPH173, ASPH174, ASPH175, ASPH176, ASPH177, ASPH178, ASPH179, ASPH180, ASPH181, ASPH182, and ASPH183 (see FIG. 5c), and the controls of SEQ ID NO. 144 and 145, respectively, in the presence of a transfecting agent. The expression of TGF-beta1 and TGF-beta2 mRNA was determined 24 h after transfection. Significant reduction of the expression of TGF-beta1 and TGF-beta2 mRNA is demonstrated in FIG. 5a) to 5c). The dual TGF-beta1 and TGF-beta2 reactive oligonucleotides ASPH01, ASPH02, ASPH03, ASPH04, ASPH05, ASPH06, ASPH07, ASPH08 and ASPH09 show a significant reduction of the expression of both TGF-beta1 and TGF-beta2 mRNA, while the selective TGF-beta2 oligonucleotides significantly inhibit TGF-beta2 mRNA expression.
Example 2
Human Panc-1 pancreatic cancer cells were transfected with 10 nM of ASPH01, ASPH02, ASPH03, ASPH04, ASPH05, ASPH06, ASPH07, ASPH08, ASPH12, ASPH14, ASPH17, ASPH18, ASPH20, ASPH21, ASPH22, ASPH24, ASPH25, ASPH26, ASPH27, ASPH29, ASPH30, ASPH31, ASPH32, ASPH33, ASPH35, ASPH36, ASPH37, ASPH38, ASPH39, ASPH40, ASPH41, ASPH42, ASPH43, ASPH44, ASPH45, ASPH46, ASPH47, ASPH48, ASPH49, ASPH50, ASPH51, and ASPH52 (see FIG. 6a); ASPH36, ASPH60, ASPH61, ASPH62, ASPH63, ASPH64, ASPH65, ASPH66, ASPH67, ASPH68, ASPH69, ASPH70, ASPH71, ASPH72, ASPH73, ASPH74, ASPH75, ASPH76, ASPH77, ASPH78, ASPH79, ASPH80, ASPH81, ASPH82, ASPH83, ASPH84, ASPH85, ASPH86, ASPH87, ASPH88, ASPH89, ASPH90, ASPH91, ASPH92, ASPH93, ASPH94, ASPH96, ASPH97, ASPH98, ASPH99, ASPH100, ASPH101, ASPH102, ASPH103, ASPH104, ASPH105, ASPH106, ASPH107, ASPH108, ASPH109, ASPH110, ASPH111, ASPH112, ASPH113, ASPH114, ASPH115, ASPH116, ASPH117, ASPH118, and ASPH119 (see FIG. 6b), or ASPH36, ASPH71, ASPH73, ASPH120, ASPH121, ASPH122, ASPH127, ASPH128, ASPH129, ASPH130, ASPH131, ASPH132, ASPH133, ASPH135, ASPH136, ASPH137, ASPH139, ASPH141, ASPH142, ASPH143, ASPH145, ASPH146, ASPH147, ASPH149, ASPH150, ASPH151, ASPH152, ASPH153, ASPH154, ASPH155, ASPH157, ASPH160, ASPH161, ASPH162, ASPH163, ASPH164, ASPH165, ASPH166, ASPH167, ASPH168, ASPH169, ASPH170, ASPH171, ASPH172, ASPH173, ASPH174, ASPH175, ASPH176, ASPH177, ASPH178, ASPH179, ASPH180, ASPH181, ASPH182, and ASPH183 (see FIG. 6c) and the controls of SEQ ID NO. 144 and 145, respectively, in the presence of a transfecting agent. The expression of TGF-beta1 and TGF-beta2 mRNA was determined 24 h after transfection. Significant reduction of the expression of TGF-beta1 and TGF-beta2 mRNA is demonstrated in FIG. 6a) to 6c). The dual TGF-beta1 and TGF-beta2 reactive oligonucleotides ASPH01, ASPH02, ASPH03, ASPH04, ASPH05, ASPH06, ASPH07, and ASPH08 show again a significant reduction of the expression of both TGF-beta1 and TGF-beta2 mRNA, while the selective TGF-beta2 oligonucleotides significantly inhibit TGF-beta2 mRNA expression.
Example 3
In further experiments the inhibitory effect of ASPH01, ASPH03, ASPH05, ASPH17, ASPH18, ASPH22, ASPH26, ASPH27, ASPH33, ASPH36, ASPH37, ASPH41, ASPH42, ASPH45, ASPH46, ASPH47, ASPH48, ASPH49, ASPH64, ASPH65, ASPH66, ASPH69, ASPH71, ASPH80, ASPH82, ASPH88, ASPH89, ASPH90, ASPH98, ASPH99, ASPH102, ASPH105, ASPH115, ASPH121, ASPH140, ASPH153, ASPH165, ASPH171, ASPH178, ASPH181, ASPH184, ASPH185, ASPH186, ASPH187, ASPH188, ASPH189, and of the controls of SEQ ID NO.144 and SEQ ID NO. 145, respectively, was tested in A172 cells. A172 cells were transfected with these modified oligonucleotides in doses of 20 nM, 4 nM, 0.8 nM, 0.16 nM, and 0.04 nM, respectively, in the presence of a transfecting agent. The remaining TGF-beta2 mRNA was measured 24 h after transfection. TGF-beta2 values were normalized to GAPDH and oligonucleotide concentrations resulting in 50% reduction of TGF-beta2 mRNA (=IC50 values) were calculated. All IC50 values were referenced to the IC50 value of ASPH_036 (ASPH36) that was 0.33 nM and the results are shown as fold-difference of the IC50 value of ASPH_036 Table 3:















Fold IC50 referenced



Oligonucleotide
to ASPH_036



















ASPH_080
0.591



ASPH_069
0.673



ASPH_065
0.773



ASPH_105
0.882



ASPH_036
1.000



ASPH_046
1.142



ASPH_098
1.182



ASPH_071
1.237



ASPH_026
1.242



ASPH_047
1.303



ASPH_088
1.455



ASPH_185
1.456



ASPH_115
1.545



ASPH_153
1.665



ASPH_181
1.918



ASPH_027
2.000



ASPH_089
2.091



ASPH_102
2.091



ASPH_041
2.182



ASPH_018
2.212



ASPH_049
2.455



ASPH_022
2.485



ASPH_188
2.639



ASPH_189
2.660



ASPH_042
2.848



ASPH_178
3.147



ASPH_048
3.182



ASPH_066
3.182



ASPH_033
3.182



ASPH_045
3.636



ASPH_121
3.644



ASPH_171
3.871



ASPH_005
3.954



ASPH_003
4.111



ASPH_082
4.818



ASPH_037
5.303



ASPH_099
5.545



ASPH_090
6.727



ASPH_165
7.175



ASPH_186
7.655



ASPH_017
8.455



ASPH_001
9.242



ASPH_187
9.990



ASPH_064
10.091



ASPH_140
11.482



ASPH_184
12.224



SEQ ID NO 144
17.212



SEQ ID NO 145
n.a










All the modified oligonucleotides show an IC50 in a low nanomolar to picomolar range, which is markedly lower than IC50 to the control oligonucleotide of SEQ ID NO. 144; the IC50 of the control of SEQ ID NO. 145 was not calculable.
Example 4
Panc-1 cells were treated with 3.3 μM of each of ASPH17, ASPH18, ASPH22, ASPH25, ASPH33, ASPH35, ASPH36, ASPH41, ASPH42, ASPH45, ASPH46, ASPH47, ASPH48, ASPH49, ASPH65, ASPH66, ASPH67, ASPH69, ASPH71, ASPH79, ASPH80, ASPH82, ASPH88, ASPH89, ASPH90, ASPH91, ASPH98, ASPH99, ASPH102, ASPH105, ASPH111, ASPH115, ASPH119, ASPH121, ASPH139, ASPH140, ASPH146, ASPH151, ASPH153, ASPH165, ASPH171, ASPH172, ASPH176, ASPH178, ASPH180, and ASPH183, or the controls of SEQ ID NO. 144 and 145, respectively, in the absence of a transfecting agent (gymnotic transfection). The inhibitory effect of the modified oligonucleotides on expression of TGF-beta1 and TGF-beta2 mRNA, respectively, was determined 72 h after treatment start. Under gymnotic transfection experimental conditions, the oligonucleotides enter the cells and strongly inhibit the expression of TGF-beta2 mRNA. The results of the experiments are shown in FIG. 7.
Example 5
In further experiments Panc-1 cells were transfected with 10 μM of modified oligonucleotides ASPH01, ASPH03, ASPH05, ASPH09, ASPH17, ASPH18, ASPH22, ASPH35, ASPH36, ASPH37, ASPH41, ASPH45, ASPH46, ASPH47, and ASPH48, or the controls of SEQ ID NO. 144 and 145, respectively, in the absence of a transfecting agent (gymnotic transfection or gymnotic delivery). The oligonucleotides were added to the cells for 2 days, after which oligonucleotide containing incubation medium was changed, and further incubation for 2 days was carried out. Expression of TGF-beta1 mRNA (see FIG. 8a) and TGF-beta2 mRNA (see FIG. 8b) was then measured and normalized to HPRT1 (Hypoxanthin-Phosphoribosyl-Transferase1). Cell supernatants were analysed for TGF-beta1 (see FIG. 9a) and TGF-beta2 (see FIG. 9b) protein by ELISA. Under gymnotic delivery experimental conditions, the double reactive oligonucleotides ASPH01, ASPH03, ASPH05, and ASPH09 significantly inhibit the expression of TGF-beta1 and TGF-beta2 on mRNA, and likewise on the protein level. All the other oligonucleotides significantly inhibit the expression of TGF-beta2 on mRNA and protein level.
Example 6
In another experiment dose dependency of the inhibitory effect of modified oligonucleotides of the present invention was tested. Panc-1 cells were treated with 15 μM, 10 μM, 7.5 μM, 5 μM, 2.5 μM, 1.25 μM, or 0.625 μM ASPH05 or ASPH36, or the controls of SEQ ID NO. 144 and 145, respectively, without using a transfection reagent. The oligonucleotides were added to the cells for 2 days. Thereafter the incubation media containing the oligonucleotides were changed and cells were incubated for 2 further days. Thereafter (total treatment time: 4 days) the expression of TGF-beta1 (see FIG. 10a) and TGF-beta2 (see FIG. 10b) mRNA depending on the oligonucleotide concentration was measured. The dual TGF-beta1 and TGF-beta2 reactive oligonucleotide ASPH05 shows a marked dose dependent inhibition of both TGF-beta1 and TGF-beta2 mRNA expressions, and ASPH36 inhibits specifically the expression of TGF-beta2 mRNA in a dose-dependent manner.
Example 7
Mouse SMA-560 glioma cells were transfected with 10 nM ASPH01, ASPH03, ASPH05, ASPH09, ASPH17, ASPH18, ASPH22, ASPH26, ASPH36, ASPH37, ASPH41, ASPH42, ASPH45, ASPH46, ASPH47, or ASPH48, or the controls of SEQ ID NO. 144 and 145, respectively, in the presence of a transfecting agent. 24 h after transfection, the inhibition of the expression of TGF-beta1 (white columns) and TGF-beta2 (black columns) mRNA was determined. The dual TGF-beta1 and TGF-beta2 reactive oligonucleotide ASPH09 inhibits the expression of the mouse TGF-beta1 mRNA, and the other oligonucleotides tested strongly inhibit the expression of the mouse TGF-beta2 mRNA. The results are presented in FIG. 11.
Example 8
Female athymic nude mice (Hsd:Athymic Nude-Foxn1nu) were treated for 5 consecutive days with 14 mg/kg or 50 mg/kg of oligonucleotide ASPH01, ASPH03, ASPH05, ASPH17, ASPH22, ASPH37, ASPH41, ASPH45, ASPH46, ASPH47, or ASPH48, and control of SEQ ID NO. 145 or saline by subcutaneous injection. The day after the last treatment, the mice were sacrificed. Mouse TGF-beta2 mRNA was quantified in kidney tissue lysates. In FIG. 12, data—representing TGF-beta2 to GAPDH mRNA ratio—are shown as a box plot in which median values and min. and max. values are presented (data expressed as n=4, except ASPH46 group n=3). All the tested oligonucleotides inhibited the expression of TGF-beta2 mRNA in the kidney of these mice.
Example 9
In another experiments Panc-1 cells were transfected with 10 μM of modified oligonucleotide ASPH09 or the control of SEQ ID NO. 145 in the absence of a transfecting agent (gymnotic transfection). The oligonucleotides were added to the cells for 2 days, after which oligonucleotide containing incubation medium was changed, and further incubation for 2 days was carried out. Expression of TGF-beta3 mRNA (see FIG. 13) was then measured and normalized to HPRT1 (Hypoxanthin-Phosphoribosyl-Transferase1). Under gymnotic transfection experimental conditions, the triple reactive oligonucleotide ASPH09 significantly inhibits the expression of TGF-beta3 mRNA.
Example 10
Panc-1 cells were treated with 10 μM, 3.3 μM, 1.1 μM, 0.37 μM, and 0.12 μM of ASPH03, ASPH36, ASPH45, ASPH47, ASPH65, ASPH69, ASPH71, ASPH80, ASPH115, ASPH 121, ASPH153, ASPH185, and ASPH189, respectively, in the absence of a transfecting agent (gymnotic transfection). The inhibitory effect of the modified oligonucleotides on expression of TGF-beta2 mRNA, was determined 72 h after treatment start. TGF-beta2 values were normalized to GAPDH and oligonucleotide concentrations resulting in 50% reduction of TGF-beta2 mRNA (=IC50 values) were calculated. Under gymnotic transfection experimental conditions, the oligonucleotides enter the cells and strongly inhibit the expression of TGF-beta2 mRNA. The results of the experiments are shown in Table 4:














Name
IC50 (μM)



















ASPH_065
0.37



ASPH_071
0.371



ASPH_115
0.6



ASPH_069
0.655



ASPH_047
0.78



ASPH_080
0.81



ASPH_153
0.9



ASPH_045
1.21



ASPH_121
1.27



ASPH_036
1.5



ASPH_185
3.05



ASPH_003
3.62



ASPH_189
4.26










All the modified oligonucleotides show an IC50 in the low micromolar or even submicromolar range, showing that they have very high potency even without the requirement of a transfection reagent.
Example 11
Panc-1 cells were treated with 10 μM, 3.3 μM, 1.1 μM, 0.37 μM, and 0.12 μM of ASPH47, ASPH190, ASPH191, ASPH192, and ASPH193 in the absence of a transfecting agent (gymnotic transfection). The inhibitory effect of the modified oligonucleotides on expression of TGF-beta2 mRNA, was determined 72 h after treatment start. TGF-beta2 values were normalized to GAPDH and oligonucleotide concentrations resulting in 50% reduction of TGF-beta2 mRNA (=IC50 values) were calculated. Under gymnotic transfection experimental conditions, the oligonucleotides enter the cells and strongly inhibit the expression of TGF-beta2 mRNA. The results of the experiments are shown in Table 5:














Name
IC50 (μM)



















ASPH_047
0.76



ASPH_190
0.18



ASPH_191
0.97



ASPH_192
0.145



ASPH_193
0.144










All the modified oligonucleotides show an IC50 in the submicromolar to lower submicromolar range, showing that they have extremely high potency even without the requirement of a transfection reagent.
Example 12
Human Panc-1 pancreatic cancer cells were transfected with 10 nM of ASPH05, ASPH09, ASPH1000, ASPH1001, ASPH1002, ASPH1003, ASPH1004, ASPH1005, ASPH1006, ASPH 1007, ASPH1008, ASPH1009, ASPH1010, ASPH1011, ASPH1012, ASPH1013, ASPH1014, ASPH1015, ASPH1016, ASPH1017, ASPH1018, ASPH1019, ASPH1020, ASPH1021, ASPH1022, ASPH1023, ASPH1024, ASPH1026, ASPH1027, ASPH1028, ASPH1029, ASPH1030, ASPH1031, ASPH1032, ASPH1033, ASPH1034, ASPH1035, ASPH1036, ASPH 1038, ASPH1039, ASPH1040, ASPH1041, ASPH1042, ASPH1043, ASPH1044, ASPH1045, ASPH1046, ASPH1047, ASPH1048, ASPH1049, ASPH1050, ASPH1051, ASPH1052, ASPH1054, ASPH1055, ASPH1056, ASPH1057, ASPH1058, ASPH1059, ASPH1060, or ASPH1061 (see FIG. 14) and the control of SEQ ID NO. 145, in the presence of a transfecting agent. The expression of TGF-beta1 mRNA was determined 24 h after transfection. Significant reduction of the expression of TGF-beta1 in Panc-1 cells is demonstrated in FIG. 14.
Example 13
Mouse SMA-560 glioma cells were transfected with 10 nM of ASPH09, ASPH1000, ASPH1001, ASPH1002, ASPH1003, ASPH1004, ASPH1005, ASPH1006, ASPH1007, ASPH1008, ASPH1009, ASPH1010, ASPH1011, ASPH1012, ASPH1013, ASPH1014, ASPH1015, ASPH1016, ASPH1017, ASPH1018, ASPH1019, ASPH 1020, ASPH1021, ASPH1022, ASPH1023, ASPH1024, ASPH1026, ASPH1027, ASPH1028, ASPH1029, ASPH1030, ASPH1031, ASPH1032, ASPH1033, ASPH1034, ASPH1035, ASPH1036, ASPH1037, ASPH1038, ASPH1039, ASPH1040, ASPH1041, ASPH1042, ASPH1043, ASPH1044, ASPH1045, ASPH1046, ASPH1047, ASPH1048, ASPH1049, ASPH1050, ASPH1051, ASPH1052, ASPH1053, ASPH1054, ASPH1055, ASPH1056, ASPH1057, ASPH1058, ASPH1059, ASPH1060, ASPH1061, or ASPH1062 (see FIG. 15) and the control of SEQ ID NO. 145, in the presence of a transfecting agent. The expression of TGF-beta1 mRNA was determined 24 h after transfection. Significant reduction of the expression of TGF-beta1 in SMA-560 cells is demonstrated in FIG. 15.
Example 14
In these experiments human A172 glioma cells were transfected with 10 nM of ASPH05, ASPH09, ASPH1000, ASPH1001, ASPH1002, ASPH1004, ASPH1005, ASPH1006, ASPH1007, ASPH1008, ASPH1009, ASPH1010, ASPH1011, ASPH1012, ASPH1013, ASPH1014, ASPH1015, ASPH1016, ASPH1017, ASPH1018, ASPH1019, ASPH1020, ASPH1021, ASPH1022, ASPH1023, ASPH1024, ASPH1026, ASPH1027, ASPH1028, ASPH1029, ASPH1030, ASPH1031, ASPH1032, ASPH1033, ASPH1034, ASPH1035, ASPH1036, ASPH1038, ASPH1039, ASPH1040, ASPH1041, ASPH1042, ASPH1043, ASPH1044, ASPH1045, ASPH1046, ASPH1047, ASPH1048, ASPH1049, ASPH1050, ASPH1051, ASPH1052, ASPH1053, ASPH1054, ASPH1056, ASPH1057, ASPH1058, ASPH1059, ASPH1060, ASPH1061, or ASPH1062 (see FIG. 16), and the control of SEQ ID NO. 145, in the presence of a transfecting agent. The expression of TGF-beta1 and TGF-beta2 mRNA was determined 24 h after transfection. Significant reduction of the expression of TGF-beta1 mRNA is demonstrated in FIG. 16. The dual TGF-beta1 and TGF-beta2 reactive oligonucleotides ASPH05 shows a significant reduction of the expression of both TGF-beta1 and TGF-beta2 mRNA, while the selective TGF-beta1 oligonucleotides significantly inhibit TGF-beta1 mRNA expression.
Example 15
Panc-1 cells were treated with 3.3 μM of ASPH05, ASPH09, ASPH1000, ASPH1001, ASPH1002, ASPH1004, ASPH1006, ASPH1007, ASPH1008, ASPH1009, ASPH1010, ASPH1011, ASPH1012, ASPH1013, ASPH1014, ASPH1015, ASPH1017, ASPH1018, ASPH1019, ASPH1020, ASPH1021, ASPH1022, ASPH1024, ASPH1026, ASPH1027, ASPH1028, ASPH1029, ASPH1032, ASPH1033, ASPH1034, ASPH1035, ASPH1036, ASPH1037, ASPH1038, ASPH1039, ASPH1040, ASPH1041, ASPH1042, ASPH1043, ASPH1044, ASPH1045, ASPH1046, ASPH1047, ASPH1049, ASPH1050, ASPH1051, ASPH1052, ASPH1053, ASPH1054, ASPH1055, ASPH1056, ASPH1057, ASPH1058, ASPH1059, ASPH1060, ASPH1061, or ASPH1062, or the control of SEQ ID NO. 145 in the absence of a transfecting agent (gymnotic transfection or gymnotic delivery). The inhibitory effect of the modified oligonucleotides on expression of TGF-beta1 and TGF-beta2 mRNA, respectively, was determined 72 h after treatment start. Significant reduction of the expression of TGF-beta1 mRNA is demonstrated in FIG. 17. The dual TGF-beta1 and TGF-beta2 reactive oligonucleotides ASPH05 shows a significant reduction of the expression of both TGF-beta1 and TGF-beta2 mRNA, while the selective TGF-beta1 oligonucleotides significantly inhibit TGF-beta1 mRNA expression.
Example 16
Human A172 cells were treated with 10 nM (in the presence of a transfecting agent), of ASPH09, ASPH1047, ASPH1051, ASPH1059, ASPH1063, ASPH1064, ASPH1065, ASPH1066, ASPH1067, ASPH1068, ASPH1069, ASPH1070, ASPH1071, ASPH1072, ASPH1073, ASPH1074, ASPH1075, ASPH1076, ASPH1077, ASPH1078, ASPH1079, ASPH1080, ASPH1081, ASPH1082, ASPH1083, ASPH1084, ASPH1085, ASPH1086, ASPH1087, ASPH1088, ASPH1089, ASPH1090, ASPH1091, ASPH1092, ASPH1093, ASPH1094, ASPH1095, ASPH1097, ASPH1098, ASPH1099, ASPH1100, ASPH1101, ASPH1102, ASPH1103, ASPH1104, ASPH1105, ASPH1106, ASPH1107, ASPH1108, ASPH1109, ASPH1110, ASPH1111, ASPH1112, ASPH1113, ASPH114, ASPH1115, ASPH1116, ASPH1117, ASPH1118, ASPH1119, ASPH1120, ASPH1121, ASPH1122, ASPH1123, ASPH1124, ASPH1125, ASPH1126, ASPH1127, ASPH1128, ASPH1129, ASPH1130, ASPH1131, and ASPH1132, or the positive control ASPH1047. The expression of TGF-beta1 (black column), TGFβbeta2 (white column) and TGF-beta3 (striped column) mRNA was determined 24 h after transfection. Significant reduction of the expression of TGF-beta1 mRNA is demonstrated in FIG. 18. The pan-specific TGF-beta1, TGF-beta2 and TGF-beta3 reactive oligonucleotides ASPH0009, ASPH1096, ASPH1131, and ASPH1132 show a significant reduction of the expression of all three isoformes, while the selective TGF-beta1 oligonucleotides significantly inhibit TGF-beta1 mRNA expression.
Example 17
Either Panc-1 cells (FIG. 19a) or RenCa cells (FIG. 19b) were treated with 3.3 μM of ASPH09, ASPH1047, ASPH1051, ASPH1059, ASPH1063, ASPH1064, ASPH1065, ASPH1066, ASPH1067, ASPH1068, ASPH1069, ASPH1070, ASPH1071, ASPH1072, ASPH1073, ASPH1074, ASPH1075, ASPH1076, ASPH1077, ASPH1078, ASPH1079, ASPH1080, ASPH1081, ASPH1082, ASPH1083, ASPH1084, ASPH1085, ASPH1086, ASPH1087, ASPH1088, ASPH1089, ASPH1090, ASPH1091, ASPH1092, ASPH1093, ASPH1094, ASPH1095, ASPH1097, ASPH1098, ASPH1099, ASPH1100, ASPH1101, ASPH1102, ASPH1103, ASPH1104, ASPH1105, ASPH1106, ASPH1107, ASPH1108, ASPH1109, ASPH1110, ASPH1111, ASPH1112, ASPH1113, ASPH114, ASPH1115, ASPH1116, ASPH1117, ASPH1118, ASPH1119, ASPH1120, ASPH1121, ASPH1122, ASPH1123, ASPH1124, ASPH1125, ASPH1126, ASPH1127, ASPH1128, ASPH1129, ASPH1130, ASPH1131, and ASPH1132, respectively, or the positive control ASPH1047 in the absence of a transfecting agent (gymnotic transfection or gymnotic delivery). The expression of TGF-beta1 (black column), TGF-beta2 (white column) and TGF-beta3 (striped column) mRNA was determined 72 h after transfection. Significant reduction of the expression of TGF-beta1 mRNA is demonstrated in FIG. 18. The pan-specific TGF-beta1, TGF-beta2 and TGF-beta3 reactive oligonucleotides ASPH09, ASPH1096, ASPH1131, and ASPH1132 show a significant reduction of the expression of all three isoforms, while the selective TGF-beta1 oligonucleotides significantly inhibit TGF-beta1 mRNA expression.
Example 18
Mice bearing subcutaneous human pancreatic carcinoma Panc-1 tumors were treated with 1, 3, 10, and 30 mg/kg of ASPH47 under various treatment schedules: Q1Dx1-d6 (single SC injection, termination 5 days later), Q1Dx5-d6 (daily SC injection for 5 days, termination 24 hours later), and Q1Dx5-d10 (daily SC injection for 5 days, termination 5 days later). There was a dose dependent down-regulation of TGF-beta 2 mRNA in the kidney of these animals. TGF-beta 2 down-regulation was persistent up to 5 days after the last treatment with ASPH47, even after only single administration. TGF-beta 2 expression was detected by bDNA assay (branched DNA assay, which is a sandwich nucleic acid hybridization method that uses bDNA molecules to amplify signal from captured target RNA) and normalized to GAPDH. As shown in FIG. 23, data—representing TGF-beta2 to GAPDH mRNA ratio—are shown as a box plot in which median values and min. and max. values are presented (data expressed as n=10, except n=9 for vehicle and 3 mg/kg Q1Dx1 d6 groups).
Example 19
Mice bearing subsutaneous human pancreatic carcinoma Panc-1 tumors on both left and right flanks were treated with a daily injection of 1, 5, 15 or 50 mg/kg oligonucleotides for five consecutive days. The tumors were collected 24 hours after the last treatment and snap frozen. TGF-beta mRNA expression in tumors was detected by bDNA assay. Data—representing TGF-beta2 to GAPDH mRNA ratio—are shown as a box plot in which median values and min. and max. values are presented (data expressed as n=5). TGF-beta2 mRNA was down-regulated in tumors treated with various oligonucleotides (FIG. 24). There was no significant TGF-beta1 mRNA down-regulation in those groups (data not shown).
Example 20
Mice bearing subcutaneous human renal cell carcinoma 786-O tumors on both left and right flanks were treated with a daily injection of 50 mg/kg oligonucleotides for five consecutive days. The tumors were collected 24 hours after the last treatment and snap frozen. TGF-beta mRNA expression in tumors was detected by bDNA assay. There was significant down-regulation of TGF-beta2 mRNA in tumors treated with ASPH05, ASPH17, ASPH26, ASPH36, ASPH45, ASPH47, ASPH71, ASPH82, ASPH98, and ASPH105, respectively, (FIG. 25). Data—representing TGF-beta2 to GAPDH mRNA ratio—are shown as a box plot in which median values and min. and max. values are presented (data expressed as n=10, except for ASPH71 group n=9).
Example 21
Human Panc-1 cells were transfected with 20, 6.67, 2.22, 0.74, 0.25, 0.08 or 0.009 μM of the modified oligonucleotides ASPH47, ASPH1047, ASPH1106, ASPH1132, or ASPH47 in combination with ASPH1047; results are shown in FIG. 26a to 26e). Negative control is the scrambled oligonucleotide (scr LNA) of SEQ ID No. 145 (FIG. 26f). All cells were transfected in the absence of a transfecting agent (gymnotic transfection or gymnotic delivery). The modified oligonucleotides were added to the cells for 3 days, which were incubated at 37° C. Thereafter the oligonucleotide containing medium was exchanged with fresh oligonucleotide containing medium and cells were incubated for further 4 days at 37° C. TGF-beta1 and TGF-beta2 protein levels in cell supernatants were determined by ELISA. ASPH47 specifically inhibits the expression of TGF-beta2 in a dose-dependent manner and does not have any target inhibiting effect on TGF-beta1 (FIG. 26a). ASPH1047 specifically inhibits the expression of TGF-beta1 and does not have any target inhibiting effect on TGF-beta2 (FIG. 26b), or only a slight TGF-beta2 inhibiting effect at higher concentrations. Also ASPH1106 inhibits TGF-beta1 expression in a dose dependent manner (FIG. 26c). The multispecific ASPH 1132 shows a dose-dependent inhibition of the expression of TGF-beta1 and TGF-beta2 protein (FIG. 26d). If ASPH47 and ASPH1047 are combined, the expression of both, TGF-beta1 and TGF-beta2 protein is inhibited in a dose-dependent manner (FIG. 26e). The scrLNA of SQE ID No. 145 does not show any inhibiting effect on the expression of neither TGF-beta1 nor TGF-beta2, even if the concentrations were doubled (40, 13.33, 4.44, 1.48, 0.49, 0.16, 0.05, or 0.02 μM) in comparison to the individual concentrations of ASPH47, ASPH1047, ASPH1106, or ASPH1132. Results for TGF-beta1 are indicated in diamonds, and results for TGF-beta2 in squares in FIGS. 26a to 26f. 
Example 22
Either Panc-1 cells (FIG. 27a) or RenCa cells (FIG. 27b) were treated with 1.1 μM of ASPH190, ASPH191, ASPH192, ASPH193, ASPH194, ASPH195, ASPH196, ASPH197, ASPH198, ASPH199, ASPH200, ASPH201, ASPH202, ASPH203, ASPH204, ASPH205, ASPH206, ASPH207, ASPH208, ASPH209, ASPH210, ASPH211, ASPH212, ASPH213, ASPH214, ASPH215, ASPH216, ASPH217, ASPH218, ASPH219, ASPH220, ASPH221, ASPH222, and ASPH223, respectively, in the absence of a transfecting agent (gymnotic transfection or gymnotic delivery). The expression of TGF-beta1 (black column), TGF-beta2 (white column) and TGF-beta3 (striped column) mRNA was determined 72 h after transfection. Significant reduction of the expression of TGF-beta2 mRNA is demonstrated in FIGS. 27a and 27b. The negative control is scrambled LNA (scr LNA) of SEQ ID No. 145.
Example 23
Panc-1 cells were treated with 10 μM, 3.3 μM, 1.1 μM, 0.37 μM, and 0.12 μM of ASPH47, M1-ASPH47, M2-ASPH47, M3-ASPH47, M4-ASPH47, M5-ASPH47, M6-ASPH47, M7-ASPH47, M8-ASPH47, M9-ASPH47, M10-ASPH47, M11-ASPH47, M12-ASPH47, M13-ASPH47, M14-ASPH47, or M15-ASPH47 in the absence of a transfecting agent (gymnotic transfection or gymnotic delivery). The inhibitory effect of the modified oligonucleotides on expression of TGF-beta2 mRNA, was determined 72 h after treatment start. TGF-beta2 values were normalized to GAPDH and oligonucleotide concentrations resulting in 50% reduction of TGF-beta2 mRNA (=IC50 values) were calculated. Under gymnotic transfection experimental conditions, the oligonucleotides enter the cells and strongly inhibit the expression of TGF-beta2 mRNA. The results of the experiments are shown in Table 6:














oligos
IC50 (μM)









M1_ASPH_0047
0.3 



M2_ASPH_0047
0.49



M3_ASPH_0047
1.75



M4_ASPH_0047
0.95



M5_ASPH_0047
0.85



M6_ASPH_0047
1.49



M7_ASPH_0047
n.a.



M8_ASPH_0047
0.89



M9_ASPH_0047
1.05



M10_ASPH_0047
7.75



M11_ASPH_0047
n.a.



M12_ASPH_0047
1.58



M13_ASPH_0047
1.91



M14_ASPH_0047
n.a.



M15_ASPH_0047
n.a.



ASPH_0047
 0.348










Most of the modified oligonucleotides show an IC50 in the submicromolar to lower submicromolar range, showing that they have extremely high potency even without the requirement of a transfection reagent.
Example 24
Human Panc-1 pancreatic cancer cells (FIG. 28a) or mouse RenCa renal cell carcinoma cells (FIG. 28b) were treated with 3.3 μM of ASPH0009, ASPH1132, ASPH2000, ASPH2001, ASPH2002, ASPH2003, ASPH2004, ASPH2005, ASPH2006, ASPH2007, ASPH2009, ASPH2010, ASPH2012, ASPH2013, ASPH2014 ASPH2015, ASPH2016, ASPH2017, ASPH2018, ASPH2019, ASPH2020, ASPH2021, ASPH2023, ASPH2024, ASPH2025, ASPH23026, ASPH2027, ASPH2028, ASPH2029, ASPH2030, ASPH2031, ASPH2032, ASPH2033, ASPH2034, ASPH2035, ASPH2036, ASPH2037, ASPH2038, ASPH2039, ASPH2040, ASPH2041, ASPH2043, ASPH2044, ASPH2045, ASPH2046, ASPH2047, ASPH2048, ASPH2049, ASPH2050, ASPH2052, ASPH2053, ASPH2054, ASPH2055, ASPH2056, ASPH2057, ASPH2060, ASPH2061, ASPH2062, ASPH2063, ASPH2064, ASPH2065, or ASPH2066 in the absence of a transfecting agent (gymnotic transfection or gymnotic delivery). The expression of TGF-beta1 (black column), TGF-beta2 (white column) and TGF-beta3 (striped column) mRNA was determined 72 h after transfection. Significant reduction of the expression of TGF-beta3 mRNA is shown in FIGS. 28a and 28b. As anticipated from the sequences, the TGF-beta1, -beta2 and -beta3 reactive oligonucleotide ASPH_0009 (pan-selective) and ASPH_1132 that has 100% homology to mRNAs of human TGF-beta1 and -beta3 but has a mismatch to TGF-beta2 show significant reduction of the expression of all three isoforms. The selective TGF-beta3 oligonucleotides only significantly inhibit TGF-beta3 mRNA expression.
Example 25
Human A172 glioma cells were treated for 24 h with 10 nM (in the presence of a transfecting agent), of ASPH0009, ASPH1132, ASPH2000, ASPH2001, ASPH2002, ASPH2003, ASPH2004, ASPH2006, ASPH2007, ASPH2008, ASPH2009, ASPH2010, ASPH2011, ASPH2012, ASPH2013, ASPH2014, ASPH2016, ASPH2017, ASPH2018, ASPH2020, ASPH2021, ASPH2022, ASPH2023, ASPH2024, ASPH2025, ASPH2026, ASPH2027, ASPH2028, ASPH2029, ASPH2030, ASPH2031, ASPH2032, ASPH2033, ASPH2034, ASPH2035, ASPH2036, ASPH2037, ASPH2038, ASPH2039, ASPH2040, ASPH2041, ASPH2042, ASPH2043, ASPH2044, ASPH2045, ASPH2047, ASPH2049, ASPH2050, ASPH2051, ASPH2052, ASPH2053, ASPH2054, ASPH2056, ASPH2057, ASPH2058, ASPH2059, ASPH2060, ASPH2061, ASPH2062, ASPH2063, or ASPH2066. The expression of TGF-beta1 (black column), TGF-beta2 (white column) and TGF-beta3 (striped column) mRNA was then determined from cell extracts by bDNA assay. Significant reduction of the expression of TGF-beta3 mRNA is shown in FIG. 29. As anticipated from the sequences, the TGF-beta1, -beta2 and -beta3 reactive oligonucleotide) ASPH_0009 (pan-selective) and ASPH_1132 that has 100% homology to mRNAs of human TGF-beta1 and -beta3 but has a mismatch to TGF-beta2 show significant reduction of the expression of all three isoforms. The selective TGF-beta3 oligonucleotides only significantly inhibit TGF-beta3 mRNA expression.
Example 26
Target mRNA Downregulation in Rabbit Cells
Sequences of selected oligonucleotides were aligned with rabbit mRNA sequences of TGF-beta1 and 2. ASPH_0036 (TGF-beta2 selective antisense oligonucleotide, based on human mRNA sequence) showed 100% homology with rabbit TGF-beta2 mRNA, while ASPH_1059 (TGF-beta1 selective antisense oligonucleotide, based on human mRNA sequence) showed 100% homology with rabbit TGF-beta1 mRNA.
Rabbit Rab-9 skin fibroblasts were treated with 5 nM or 20 nM of either ASPH_0036 and ASPH_1059 in the presence of a transfecting agent for 24 hr. The expression of TGF-beta1 and TGF-beta2 mRNA was then determined in cell extracts by bDNA assay. Significant reduction of the expression of TGF-beta1 mRNA (51 and 77% at 5 and 20 nM, respectively) was achieved with ASPH_1059. Significant reduction of TGF-beta2 mRNA (79 and 80% at 5 and 20 nM, respectively) was achieved with ASPH_0036.
Example 27
Tissue Biodistribution and Target mRNA Downregulation Following Systemic Administration of ASPH_0047 in Balb/c Mice
Balb/C mice were treated with a single subcutaneous injection of ASPH_0047 (formulated in sterile physiological saline) at 5, 20 and 50 mg/kg animal body weight. Plasma and tissues were collected at the indicated times (from 3 individual animals), immediately snap-frozen and stored at −80° C. until analysis with an AEX-HPLC method (plasma/tissue PK) or for measurement of TGF-β2 and GAPDH mRNA levels by bDNA assay. TGF-β2 mRNA levels were expressed relative to GAPDH mRNA expression level in corresponding samples.
The data depict that a single subcutaneous bolus administration of 50 mg/kg ASPH_0047 resulted in rapid transfer of the drug from subcutaneous to circulating blood compartments (TMAX of  ̃5-30 min), biphasic pharmacokinetic profile in plasma, with rapid initial elimination phase (within the first 24 hrs), followed by long terminal half-life (FIG. 30a). It is further demonstrated that a marked long-lasting accumulation of the drug in various selected tissues. The major target organ (highest exposure/CMAX) is the kidney, then the liver, skin and spleen, and lowest in the brain (data not shown). As also depicted in FIG. 30b, ASPH_0047 remained in the kidney tissue with pharmacological relevant doses ( ̃50 μg/gr, equivalent to 10 μM) from 24 h and for up to 14 days, with consequent long-lasting and marked suppression of TGF-62 mRNA expression in the kidney tissue, with effective  ̃80% target mRNA downregulation observed for at least 14 days.
Example 28
Immunodeficient mice were injected subcutaneously with human 786-O renal cell carcinoma cells (FIG. 30A), pancreatic Panc1 cancer cells (FIG. 30B, C), or mouse SMA-560 glioma cells (FIG. 30D). When subcutaneous tumors reached the size of 100-300 mm3 (established tumors), animals were treated subcutaneously, Q1Dx5, with saline (Mock), control oligonucleotide (Control; 50 mg/kg), inactive oligonucleotides in this context (e.g., ASPH_0065 and ASPH_0071; 50 mg/kg) or ASPH_0047 at 50 mg/kg, or the indicated doses. Tumors (FIG. 30A-D) and kidneys (FIG. 30E-F) were collected 24 hr after the last administration. Tumors/kidneys were then further processed for determination of TGF-□2 and GAPDH mRNA levels by bDNA assay. In these experiments, control oligonucleotide was a 18-mer, 3+3 LNA gapmer scrambled sequence. Results are expressed as TGF-beta2/GAPDH mRNA ratio, and each individual tested sample is represented with median values indicated as red line. Under described experimental conditions (schedule and route of administration), systemic repeated administrations of ASPH_0047 in Balb/c mice led to a sequence-specific downregulation of TGF-beta 2 mRNA in established subcutaneous tumors and kidneys.
Example 29
Balb/c mice were injected with mouse Renca cells into renal subcapsule (FIG. 32A, B) or i.v. (FIG. 32C, D) on Day 0. Systemic treatment with vehicle or indicated oligonucleotides started on Day 7 (FIG. 32A; 50 mg/kg, s.c., twice weekly), on Day 1 (FIG. 32B; 12.5 mg/kg, s.c., twice weekly) for two consecutive weeks, or on Day 7 (FIGS. 32C and 32D; indicated doses, s.c., twice weekly) for 26-27 days. Number of lung metastasis was macroscopically evaluated, and level of lung metastasis was determined by either number of metastasis (FIG. 32A, C) or based on lung weight (FIG. 32B, D). Results are represented as box plot; with median values, upper and lower quartiles, and 90th and 10th percentiles. Under described experimental designs, Balb/c mice treated with ASPH_0047 showed a reduced number of lung metastasis or reduced lung weight (lung weight correlates to extent of lung metastasis) in mouse Renca RCC models.
Example 30
Human Panc-1 pancreatic cancer cells were treated with 3.3 μM of the indicated oligonucleotides in the absence of transfecting agent (gymnotic transfection or gymnotic delivery). The expression of TGF-beta1 (black column), TGF-beta2 (white column) and TGF-beta3 (striped column) mRNA was determined 72 h after transfection. Significant reduction of the expression of TGF-beta1 mRNA is shown in FIG. 32. The selective TGF-beta1 oligonucleotides only significantly inhibit TGF-beta1 mRNA expression while the control oligonucleotide LNA-scr does not affect expression of any TGF-beta isoform.
Example 31
Balb/c mice were injected with mouse 4T1 cells into mammary fat pad on Day 0. Systemic treatment with saline (Mock), pan-TGF-beta antibody (1D11), control oligonucleotide (LNA-scr), or ASPH_0047 started on Day 3 (30 mg/kg, s.c., twice weekly) and continued until D28, when animals were sacrificed. Number of lung metastasis was macroscopically evaluated, and level of lung metastasis was determined by either number of metastasis (left panel) or based on lung weight (right panel). Under described experimental design, treatment with ASPH_0047 reduced metastasis to the lungs, whereas the positive control, monoclonal TGF-beta antibody 1D11 had no effect on pulmonary metastasis in this model.
Example 32
CB17 SCID or Balb/c nude mice (n=3-5, except ASPH_0018 n=1 and ASPH_0037 n=2) were treated with 14-15 mg/kg of indicated LNA-modified oligonucleotides for four or five consecutive days (Q1Dx4-5). Plasma was collected 24 h after the last treatment and ALT levels were determined in plasma. Results are expressed as median values. Under this experimental condition, only 6/48 (12.5%) of tested oligonucleotides induced marked increase in plasma ALT (>300 units/l) indicating liver toxicity. The following Table 7 shows liver toxicity of systemically administered LNA-modified oligonucleotides:














Name
ALT (units/l)



















ASPH_0001
20.5



ASPH_0003
20.0



ASPH_0005
33.0



ASPH_0009
834.0



ASPH_0017
55.0



ASPH_0018
7723.0



ASPH_0022
28.5



ASPH_0026
77.0



ASPH_0027
75.0



ASPH_0035
25.0



ASPH_0036
131.5



ASPH_0037
161.0



ASPH_0041
655.0



ASPH_0045
27.5



ASPH_0046
3199.0



ASPH_0047
42.5



ASPH_0048
29.5



ASPH_0065
27.0



ASPH_0069
32.5



ASPH_0071
23.5



ASPH_0080
34.0



ASPH_0082
31.0



ASPH_0098
33.0



ASPH_0105
40.0



ASPH_0115
985.5



ASPH_0190
902.0



ASPH_0191
36.5



ASPH_0192
49.5



ASPH_0193
35.0



ASPH_0005_C1
25.5



ASPH_0005_C2
35.5



ASPH_0005_C3
25.0



ASPH_0036_C1
34.0



ASPH_0036_C2
26.0



ASPH_0036_C3
39.0



ASPH_0045_C1
38.5



ASPH_0045_C2
23.5



ASPH_0045_C3
65.0



ASPH_0047_C1
35.5



ASPH_0047_C2
30.0



ASPH_0047_C3
29.5



ASPH_0047_C4
52.5



ASPH_0047_C5
28.0



ASPH_0047_C6
33.5



ASPH_0047_C7
37.0



ASPH_0047_C8
32.0



ASPH_0047_C9
49.0



ASPH_0047_C10
32.5

## Claims

1. A method of inhibiting and/or treating an ophthalmic disease associated with TGF-beta2 expression, comprising:
administering to a subject in need thereof an antisense oligonucleotide consisting of 12 to 18 nucleotides of the TGF-beta2 nucleic acid sequence of SEQ ID NO. 2, wherein one or more nucleotide(s) of the oligonucleotide is/are modified, and wherein the modified nucleotide is a LNA, and/or an ENA, polyalkylene oxide-, 2′-fluoro-, 2′-O-methoxy-, and/or 2′0-methyl-modified nucleotide; and
said oligonucleotide consisting of GACCAGATGCAGGA (ASPH36, SEQ ID NO. 5).

2. The method of claim 1, wherein the modified nucleotide is located at the 5′- and/or 3′-end of the antisense oligonucleotide.

3. The method of claim 1, wherein the ophthalmic disease is selected from the group consisting of glaucoma, posterior capsular opacification, dry eye, Marfan or Loeys-Dietz syndrome, macular degeneration, retinoblastoma and choroidcarcinoma.

4. The method of claim 1, wherein the ophthalmic disease is macular degeneration and wherein the macular degeneration is an age-related macular degeneration, diabetic macular endma, or cataract.

5. A method of inhibiting and/or treating an ophthalmic disease associated with TGF-beta2 expression, comprising:
administering to a subject in need thereof a composition comprising an antisense oligonucleotide, said oligonucleotide consisting of GACCAGATGCAGGA (ASPH36, SEQ ID NO. 5).

6. A method of inhibiting and/or treating an ophthalmic disease associated with TGF-beta2 expression, comprising:
administering to a subject in need thereof an antisense oligonucleotide consisting of 12 to 18 nucleotides of the TGF-beta2 nucleic acid sequence of SEQ ID NO. 2, wherein one or more nucleotide(s) of the oligonucleotide is/are modified, wherein the modified nucleotide is a LNA, and/or an ENA, polyalkylene oxide-, 2′-fluoro-, 2′-O-methoxy-, and/or 2′0-methyl-modified nucleotide and wherein said oligonucleotide is selected from the group consisting of GACCAGATGCAGGA (ASPH36, SEQ ID NO. 5), CAAAGTATTTGGTCTCC (ASPH47, SEQ ID NO. 49), ACCTTGGGCTTGCG (ASPH1059, SEQ ID NO. 210) and CGGGTGCTGTTGTA (ASPH1132, SEQ ID NO. 267).

7. The method of claim 1, wherein said method is directed to treating an ophthalmic disease.

8. The method of claim 5, wherein said method is directed to treating an ophthalmic disease.

9. A method of treating an ophthalmic disease associated with TGF-beta2 expression, comprising:
administering to a subject in need thereof an antisense oligonucleotide consisting of 12 to 18 nucleotides of the TGF-beta2 nucleic acid sequence of SEQ ID NO. 2, wherein one or more nucleotide(s) of the oligonucleotide is/are modified, wherein the modified nucleotide is a LNA, and/or an ENA, polyalkylene oxide-, 2′-fluoro-, 2′-O-methoxy-, and/or 2′0-methyl-modified nucleotide and wherein said oligonucleotide is selected from the group consisting of GACCAGATGCAGGA (ASPH36, SEQ ID NO. 5), CAAAGTATTTGGTCTCC (ASPH47, SEQ ID NO. 49), ACCTTGGGCTTGCG (ASPH1059, SEQ ID NO. 210) and CGGGTGCTGTTGTA (ASPH1132, SEQ ID NO. 267).

10. The method of claim 9, wherein the ophthalmic disease is selected from the group consisting of glaucoma, posterior capsular opacification, dry eye, Marfan or Loeys-Dietz syndrome, macular degeneration, and choroidcarcinoma.

11. The method of claim 9, wherein the ophthalmic disease is macular degeneration and wherein the macular degeneration is an age-related macular degeneration, diabetic macular endma, or cataract.