High affinity oligonucleotide ligands to growth factors

Details High affinity oligonucleotide ligands to growth factors High affinity oligonucleotide ligands to growth factors

1998-05-11

2001-03-27

Zitomer, Stephanie (Department: 1634)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S025400, C435S006120, C435S091200

Reexamination Certificate

active

06207816

ABSTRACT:

FIELD OF THE INVENTION
Described herein are methods for identifing and preparing high-affinity nucleic acid ligands to TGF&bgr;, PDGF, and hKGF. 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 acid ligands of TGFB, PDGF, and hKGF. Further disclosed are RNA and DNA ligands to TGF&bgr;1 and PDGF and RNA ligands to hKGF. Also included are oligonucleotides containing nucleotide derivatives chemically modified at the 2′- positions of pyrimidines. Additionally disclosed are RNA ligands to TGF&bgr;1 and hKGF containing 2′-NH
2
-modifications or 2′-F-modifications and RNA ligands to PDGF containing 2′-F modifications. This invention also includes high affinity nucleic acid inhibitors of TGF&bgr;1, PDGF, and hKGF. The oligonucleotides of the present invention are useful as pharmaceuticals or diagnostic agents.
BACKGROUND OF THE INVENTION
TGF&bgr;
The transforming growth factor −&bgr; (TGF&bgr;) polypeptides influence growth, differentiation, and gene expression in many cell types. The first polypeptide of this family that was characterized, TGF&bgr;1 has two identical 112 amino acid subunits which are covalently linked. TGF&bgr;1 is a highly conserved protein with only a single amino acid difference distinguishing human from mice forms. There are two other members of the TGF&bgr; gene family that are expressed in mammals. TGF&bgr;2 is 71% homologous to TGF&bgr;1(de Martin et al., (1987)
EMBO J.
6:3673-3677), whereas TGF&bgr;3 is 80% homologous to TGF&bgr;1 (Derynck et al., (1988)
EMBO J
7:3737-3743). The structural characteristics of TGF&bgr;1 as determined by nuclear magnetic resonance (Archer et al., (1993)
Biochemistry
32:1164-1171) agree with the crystal structure of TGF&bgr;2 (Daopin et al., (1992)
Science
257:369-374; Schlunegger and Grutter (1992)
Nature
38:430-434).
Even though the TGF&bgr;'s have similar three dimensional structures, they are by no means physiologically equivalent. There are at least three different extracellular receptors, type I, II and III, involved in transmembrane signaling of TGF&bgr; to cells carrying the receptors. For reviews, see Derynck (1994) TIBS 19:548-553 and Massague (1990) Annu. Rev. Cell Biol 6:597-641. In order for TGF&bgr;2 to effectively interact with the type II TGF&bgr; receptor, the type III receptor must also be present (Derynck (1994) TIBS 19:548-553). Vascular endothelial cells lack the type III receptor. Instead endothelial cells express a structurally related protein called endoglin (Cheifetz et al., (1992)
J. Biol. Chem.
267:19027-19030), which only binds TGF&bgr;1 and TGF&bgr;3 with high alnnity. Thus, the relative potency of the TGF&bgr;'s reflect the type of receptors expressed in a cell and organ system.
In addition to the regulation of the components in the multifactorial signaling pathway, the distribution of the synthesis of TGF&bgr; polypeptides also affects physiological function. The distribution of TGF&bgr;2 and TGF&bgr;3 is more limited (Derynck et al., (1988)
EMBO J
7:3737-3743) than TGF&bgr;1, e.g., TGF&bgr;3 is limited to tissues of mesenchymal origin, whereas TGF&bgr;1 is present in both mesenchymal and epithelial cells.
TGF&bgr;1 is a multifunctional cytokine critical for tissue repair. High concentrations of TGF&bgr;1 are delivered to the site of injury by platelet granules (Assoian and Sporn, (1986) J Cell Biol. 102:1217-1223.). TGF&bgr;1 initiates a series of events that promote healing including chemotaxis of cells such as leukocytes, monocytes and fibroblasts, and regulation of growth factors and cytokines involved in angiogenesis, cell division associated with tissue repair and inflammatory responses. TGF&bgr;1 also stimulates the synthesis of extracellular matrix components (Roberts et aL, (1986)
Proc. Natl. Acad Sci USA
83:4167-4171; Sporn et al., (1983)
Science
219:1329-1330; Massague, (1987)
Cell
49:437-438) and most importantly for understanding the pathophysiology of TGF&bgr;1, TGF&bgr;1 autoregulates its own synthesis (Kim et al., (1989)
J Biol Chem
264:7041-7045).
A number of diseases have been associated with TGF&bgr;1 overproduction. Fibrotic diseases associated with TGF&bgr;1 overproduction can be divided into chronic conditions such as fibrosis of kidney, lung and liver and more acute conditions such as dermal scarring and restenosis. Synthesis and secretion of TGF&bgr;1 by tumor cells can also lead to immune suppression such as seen in patients with aggressive brain or breast tumors (Arteaga et al., (1993)
J Clin Invest
92: 2569-2576). The course of Leishmanial infection in mice is drastically altered by TGF&bgr;1 (Barral-Netto et al., (1992)
Science
257:545-547). TGF&bgr;1 exacerbated the disease, whereas TGF&bgr;1 antibodies halted the progression of the disease in genetically susceptible mice. Genetically resistant mice became susceptible to Leishmanial infection upon administration of TGF&bgr;1.
The profound effects of TGF&bgr;1 on extracellular matrix deposition have been reviewed (Rocco and Ziyadeh, (1991) in Contemporary Issues in Nephrology v23, Hormones, autocoids and the kidney. ed. Jay Stein, Churchill Livingston, New York pp391-410; Roberts et al., (1988)
Rec. Prog. Hormone Res.
44:157-197) and include the stimulation of the synthesis and the inhibition of degradation of extracellular matrix components. Since the structure and filtration properties of the glomerulus are largely determined by the extracellular matrix composition of the mesangium and glomerular membrane, it is not surprising that TGF&bgr;1 has profound effects on the kidney. The accumulation of mesangial matrix in proliferative glomerulonephritus (Border et al., (1990)
Kidney Int.
37:689-695) and diabetic nephropathy (Mauer et al., (1984)
J. Clin Invest.
74:1143-1155) are clear and dominant pathological features of the diseases. TGF&bgr;1 levels are elevated in human diabetic glomerulosclerosis (advanced neuropathy) (Yamamoto et al., (1993)
Proc. Natl. Acad. Sci.
90:1814-1818). TGF&bgr;1 is an important mediator in the genesis of renal fibrosis in a number of animal models (Phan et al., (1990)
Kidney Int.
37:426; Okuda et al., (1990)
J. Clin Invest.
86:453). Suppression of experimentally induced glomerulonephritus in rats has been demonstrated by antiserum against TGF&bgr;1 (Border et al., (1 990)
Nature
346:371) and by an extracellular matrix protein, decorin, which can bind TGF&bgr;1 (Border et al., (1992)
Nature
360:361-363).
Too much TGF&bgr;1 leads to dermal scar-tissue formation. Neutralizing TGF&bgr;1 antibodies injected into the margins of healing wounds in rats have been shown to inhibit scarring without interfering with the rate of wound healing or the tensile strength of the wound (Shah et al, (1992)
Lancet
339:213-214). At the same time there was reduced angiogenesis, reduced number of macrophages and monocytes in the wound, and a reduced amount of disorganized collagen fiber deposition in the scar tissue.
TGF&bgr;1 may be a factor in the progressive thickening of the arterial wall which results from the proliferation of smooth muscle cells and deposition of extracellular matrix in the artery after balloon angioplasty. The diameter of the restenosed artery may be reduced 90% by this thickening, and since most of the reduction in diameter is due to extracellular matrix rather than smooth muscle cell bodies, it may be possible to open these vessels to 50% simply by reducing extensive extracellular matrix deposition. In uninjured pig arteries transfected in vivo with a TGF&bgr;1 gene, TGF&bgr;1 gene expression was associated with both extracellular matrix synthesis and hyperplasia (Nabel et al., (1993)
Proc. Natl. Acad. Sci USA
90-10759-10763). The TGF&bgr;1 induced hyperplasia was not as extensive as that induced with PDGF-BB, but the extracellular matrix was more extensive with TGF&bgr;1 transfectants. No extracellular matrix deposition was associated with FGF-1 (a sec

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