Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-02-23
2004-03-30
Forman, B J (Department: 1634)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S025400, C514S04400A
Reexamination Certificate
active
06713616
ABSTRACT:
FIELD OF THE INVENTION
Described herein are methods for identifying and preparing high affinity nucleic acid ligands that bind human transforming growth factor &bgr;2 (TGF&bgr;2). The method utilized herein for identifying such nucleic acid ligands is called SELEX, an acronym for Systematic Evolution of Ligands by EXponential Enrichment. This invention includes high affinity nucleic acids of human TGF&bgr;2. Further disclosed are RNA ligands to TGF&bgr;2. Also included are oligonucleotides containing nucleotide derivatives modified at the 2′ position of the pyrimidines. Additionally disclosed are ligands to TGF&bgr;2 containing 2′-OCH
3
purine modifications that may have higher stability in serum and in animals. This invention also includes high affinity nucleic acid inhibitors of TGF&bgr;2. The oligonucleotide ligands of the present invention are useful in any process in which binding to TGF&bgr;2 is required. This includes, but is not limited to, their use as pharmaceuticals, diagnostics, imaging agents, and immunohistochemical reagents.
BACKGROUND OF THE INVENTION
Transforming growth factors betas (TGF&bgr;s) are part of a superfamily of proteins that includes inhibins, activins, bone morphogenetic and osteogenic proteins, growth/differentiation factors, Mullerian-inhibiting substance, decapentaplegic and 60A (Drosophila), daf-7 and unc-129 (
C. elegans
), and vg1 (Xenopus) (Schlunegger and Grutter (1992) Nature 358:430-434). Three TGF&bgr; isotypes exist in mammals that are called TGF&bgr;1, TGF&bgr;2, and TGF&bgr;3. There is about 80% sequence identity between any pair of mammalian TGF&bgr;s. TGF&bgr;s bind to at least 5 receptors, but only 2 or 3 of them (types I, II, and possibly V) are signaling receptors. The intracellular signaling pathways activated by TGF&bgr;s involve SMAD proteins and are being intensively studied (Padgett et al. (1998) Pharmacol Ther 78:47-52). The signaling receptors are found on a variety of cells. In turn, a variety of cells express TGF&bgr;s.
TGF&bgr;s are synthesized as precursors composed of latency-associated protein (LAP) at the amino terminus and mature TGF&bgr; at the carboxyl terminus. The precursor is cleaved and assembles as a homodimer. TGF&bgr;s are secreted from cells bound to LAP and latent TGF&bgr; binding proteins (LTBPs). Latent TGF&bgr;s are released from LAP and LTBP and become active by a relatively uncharacterized mechanism that may involve proteolysis by plasmin or regulation by thrombospondin (Crawford et al. (1998) Cell 93:1159-70). The mature, released TGF&bgr; homodimer has a combined molecular weight of ~25000 daltons (112 amino acids per monomer). TGF&bgr;1 and TGF&bgr;2 bind heparin and there are indications that basic amino acids at position 26 are required for heparin binding (Lyon et al. (1997) Jour. Biol. Chem. 272:18000-18006).
The structure of TGF&bgr;2 has been determined using x-ray crystallography (Daopin et al. (1992) Science 257:369-373; Schlunegger and Grutter (1992) Nature 358:430-434) and is very similar to the structure of TGF&bgr;1. TGF&bgr;s belong to a structural family of proteins called the “cysteine knot” proteins that includes vascular endothelial growth factor, nerve growth factor, human chorionic gonadotropin, and platelet-derived growth factor. These proteins are structurally homologous, but have only 10-25% primary sequence homology.
The biological activities of the TGF&bgr;s vary (Moses (1990) Growth Factors from Genes to Clinical Application 141-155; Wahl (1994) J. Exp. Med. 180:1587-1590). In some cases they inhibit cell proliferation (Robinson et al. (1991) Cancer Res. 113:6269-6274) and in other cases they stimulate it (Fynan and Reiss (1993) Crit. Rev. Oncogenesis 4:493-540). They regulate extracellular matrix formation and remodeling (Koli and Arteaga (1996) J. Mammary Gland Bio. and Neoplasia 1:373-380). They are also are very potent immunosuppressants (Letterio and Roberts (1998) Ann. Rev. Immunol. 16:137-161). TGF&bgr;s are thought to play a significant role in fibrotic diseases, preventing the immune system from rejecting tumors (Fakhrai et al. (1996) Proc. Natl. Acad. USA 93:2090-2914), cancer cell growth (Koli and Arteaga (1996) J. Mammary Gland Bio. and Neoplasia 1:373-380; Reiss and Barcellos-Hoff (1997) Breast Cancer Res. and Treatment 45:81-85; Jennings and Pietenpol (1998) J. Neurooncol. 36:123-140), and tumor metastasis. They may have ancillary roles in autoimmune and infectious diseases. Inhibition of TGF&bgr;2 by an expressed antisense RNA (Fakhrai et al. (1996) Proc. Natl. Acad. USA 93:2090-2914) and by exogenous antisense oligonucleotides (Marzo et al. (1997) Cancer Research 57:3200-3207) has been reported to prevent glioma formation in rats.
The gene for mouse TGF&bgr;2 has been deleted (Sanford et al. (1997) Development 124:2659-2670). Mice lacking TGF&bgr;2 function die near birth and have aberrant epithelial-mesencymal interactions that lead to developmental defects in the heart, eye, ear, lung, limb, craniofacial area, spinal cord, and urogenital tracts. These defects, for the most part, do not overlap abnormalities that have been observed in TGF&bgr;1 and TGF&bgr;3 knockout mice. TGF&bgr;s have also been overexpressed in cell lines or transgeneic mice (Koli and Arteaga (1996) J. Mammary Gland Bio. and Neoplasia 1:373-380; Bottinger et al. 1997 Kidney Int. 51:1355-1360; Bottinger and Kopp (1998) Miner Electrolyte Metab 24:154-160) with a variety of effects.
A method for the in vitro evolution of nucleic acid molecules with high affinity binding to target molecules has been developed. This method, Systematic Evolution of Ligands by EXponential enrichment, termed SELEX, is described in U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled “Systematic Evolution of Ligands by Exponential Enrichment,” now abandoned, U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled “Nucleic Acid Ligands,” now U.S. Pat. No. 5,475,096 and U.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled “Methods for Identifying Nucleic Acid Ligands,” now U.S. Pat. No. 5,270,163 (see also WO91/19813), each of which is herein specifically incorporated by reference. Each of these applications, collectively referred to herein as the SELEX Patent Applications, describe a fundamentally novel method for making a nucleic acid ligand to any desired target molecule.
The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection theme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield high affinity nucleic acid ligands to the target molecule.
The basic SELEX method may be modified to achieve specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, entitled “Method for Selecting Nucleic Acids on the Basis of Structure,” now abandoned, describes the use of SELEX in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA (See U.S. Pat. No. 5,707,796). U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled “Photoselection of Nucleic Acid Ligands,” now abandoned, describes a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule.
Gold Larry
Lochrie Michael
Pagratis Nikos
Forman B J
Gilead Sciences, Inc.
Swanson & Bratschun LLC
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