Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1999-08-25
2003-05-13
Nguyen, Dave T. (Department: 1636)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S320100, C435S455000, C435S375000, C435S471000, C536S023100, C536S063000
Reexamination Certificate
active
06562799
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to hypoxia-inducible DNA-binding proteins and more specifically to DNA binding proteins that are modified such that they are stable under non-hypoxic as well as hypoxic conditions.
BACKGROUND OF THE INVENTION
Mammals require molecular oxygen (O
2
) for essential metabolic processes including oxidative phosphorylation in which O
2
serves as electron acceptor during ATP formation. Systemic, local, and intracellular homeostatic responses elicited by hypoxia (the state in which O
2
demand exceeds supply) include erythropoiesis by individuals who are anemic or at high altitude (Jelkmann,
Physiol. Rev.
72:449-489, 1992), neovascularization in ischemic myocardium (White et al.,
Circ. Res.
71:1490-1500, 1992), and glycolysis in cells cultured at reduced O
2
tension (Wolfe et al.,
Eur. J. Biochem.
135:405-412, 1983). These adaptive responses either increase O
2
delivery or activate alternate metabolic pathways that do not require O
2
. Hypoxia-inducible gene products that participate in these responses include erythropoietin (EPO) (reviewed in Semenza,
Hematol. Oncol. Clinics N.
erythropoietin (EPO) (reviewed in Semenza,
Hematol. Oncol. Clinics N. Amer.
8:863-884, 1994), vascular endothelial growth factor (VEGF) (Shweiki et al.,
Nature
359:843-845, 1992; Banai et al.,
Cardiovasc. Res.
28:1176-1179, 1994; Goldberg & Schneider,
J. Biol. Chem.
269:4355-4359, 1994), and glycolytic enzymes (Firth et al.,
Proc. Natl. Acad. Sci. USA
91:6496-6500, 1994; Semenza et al.,
J. Biol. Chem.
269:23757-23763, 1994).
The molecular mechanisms that mediate genetic responses to hypoxia have been extensively investigated for the EPO gene, which encodes a growth factor that regulates erythropoiesis and thus blood O
2
-carrying capacity (Jelkmann, 1992, supra; Semenza, 1994, supra). Cis-acting DNA sequences required for transcriptional activation in response to hypoxia were identified in the EPO 3′-flanking region and a trans-acting factor that binds to the enhancer, hypoxia-inducible factor 1 (HIF-1), fulfilled criteria for a physiological regulator of EPO transcription. In particular, inducers of EPO expression (1% O
2
, cobalt chloride [CoCl
2
], and desferrioxamine [DFX]) also induced HIF-1 DNA binding activity with similar kinetics. In addition, inhibitors of EPO expression (actinomycin D, cycloheximide, and 2-aminopurine) blocked induction of HIF-1 activity. Furthermore, mutations in the EPO 3′-flanking region that eliminated HIF-1 binding also eliminated enhancer function (Semenza, 1994, supra). These results support a signal transduction pathway requiring ongoing transcription, translation, and protein phosphorylation participates in the induction of HIF-1 DNA-binding activity and EPO transcription in hypoxic cells (Semenza, 1994, supra).
EPO expression is cell type specific, but induction of HIF-1 activity by 1% O
2
, CoCl
2
, or DFX was detected in many mammalian cell lines (Wang & Semenza,
Proc. Natl. Acad. Sci. USA
90:4304-4308, 1993). The EPO enhancer directed hypoxia-inducible transcription of reporter genes transfected into non-EPO-producing cells (Wang & Semenza, 1993, supra; Maxwell et al.,
Proc. Natl. Acad. Sci. USA
90:2423-2427, 1993). RNAs encoding several glycolytic enzymes were induced by 1% O
2
, CoCl
2
, or DFX in EPO-producing Hep3B or nonproducing HeLa cells whereas cycloheximide blocked their induction and glycolytic gene sequences containing HIF-1 binding sites mediated hypoxia-inducible transcription in transfection assays (Firth et al., 1994, supra; Semenza et al., 1994, supra). These experiments support the role of HIF-1 in activating homeostatic responses to hypoxia.
Hypoxia inducible factor-1(HIF-1) is a mammalian transcription factor expressed uniquely in response to physiologically relevant levels of hypoxia (Wang, G. L., et al.,
Proc. Natl. Acad. Sci. USA
92:5510-5514, 1995; Wang, G. L., and Semenza, G. L.,
J. Biol. Chem.
270:1230-1237, 1995; U.S. Pat. No. 5,882,914). HIF-1 is a basic helix loop-helix protein that binds to cis-acting hypoxia-responsive elements of genes induced by hypoxia (Wang, G. L., and Semenza, G. L.,
Curr. Opin. Hematol.
3:156-162, 1992; Jiang, B. H., et al.,
J. Biol. Chem.
212:19253-19260, 1997). The genes that are activated by HIF-1 in cells subjected to hypoxia include EPO, vascular endothelial growth hormone (VEGF), heme oxygenase-1, inducible nitric oxide synthase, and glycolytic enzymes aldolase A, enolase 1, lactate dehydrogenase A, phosphofructokinase I, and phosphoglycerate kinase 1 (Semenza, G. L., et al.,
Kid. Int.
51:553-555, 1997). HIF-1 DNA binding activity and HIF-1 protein concentration increase exponentially as cells are subjected to decreasing O
2
concentrations (Jiang, B. H., et al.,
Am J. Physiol.
271:C 172-C1180, 1996).
HIF-1 also activates transcription of the VEGF gene in hypoxic cells (Forsythe et al., 1996; Iyer et al., 1998). When cultured cells are transfected with pCEP4/HIF-1alpha plasmid under conditions that allow expression of HIF-1alpha from a cytomegalovirus promoter and a reporter plasmid containing the hypoxia response element from the VEGF gene, reporter gene expression is increased in cells under non-hypoxic conditions and there is a dramatic superinduction under hypoxic conditions that is dependent upon the presence of an intact HIF-1 binding site (Forsythe et al., 1996). In embryonic stem cells from a knockout mouse, which lack HIF-1alpha expression, there is no expression of VEGF mRNA in response to hypoxia (Iyer et al., 1998).
HIF-1 is a heterodimer of two subunits, HIF-1alpha and HIF-1beta. The HIF-1alpha subunit is unique to HIF-1, whereas HIF-1beta (also known as aryl hydrocarbon receptor nuclear translocator, ARNT) can dimerize with other proteins. The concentration of HIF-1alpha and HIF-1beta RNA and HIF-1alpha and HIF-1beta polypeptide increases in cells exposed to hypoxic conditions (Wiener, C. M., et al.,
Biochem. Biophys. Res. Commun.
225:485-488, 1996; Yu, A. Y., et al.,
Am J. Physiol.
275:L818-L826, 1998).
Structural analysis of HIF-1alpha revealed that dimerization requires two domains, termed HLH and PAS. DNA binding is mediated by a basic domain (Semenza, G. L., et al.,
Kid. Int.
51:553-555, 1997). Two transactivation domains are contained in HIF-1alpha, located between amino acids 531 and 826. The minimal transactivation domains are at amino acid residues 531-575 and 786-826 (Jiang, B. H., et al., 1997, supra; Semenza, G. L., et al., 1997, supra). Amino acids 1-390 are required for optimal heterodimerization with HIF1beta (ARNT) and DNA binding. In addition, deletion of the carboxy terminus of HIF-1alpha (amino acids 391-826) decreased the ability of HIF-1 to activate transcription. However, HIF-1alpha (1-390) was expressed at high levels in both hypoxic and non-hypoxic cells in contrast to full-length HIF-1alpha (1-826) which was expressed at much higher levels in hypoxic relative to non-hypoxic cells (Jiang, B.-H., et al.,
J. Biol. Chem.
271:17771-17778, 1996). Thus, hypoxia has two independent effects on HIF-1alpha activity: (1) hypoxia increases the steady-state levels of HIF-1alpha protein by stabilizing it (i.e. decreasing its degradation); and (2) hypoxia increases the specific transcriptional activity of theprotein (i.e. independent of the protein concentration).
SUMMARY OF THE INVENTION
This invention is based on the discovery and isolation of unique variant forms of HIF-1alpha polypeptide that are stable under hypoxic and nonhypoxic conditions. The invention further includes chimeric proteins having HIF-1alpha DNA binding domain and dimerization domains and a heterologous transactivation domain. Given the structural and functional similarities between HIF-1alpha , HIF-2alpha (also known as EPAS 1, HLF, HRF, and MOP2), and HIF-3alpha (see Gu, Y.-Z., et al., Gene Expr. 7:205-213, 1998) it is understood that HIF-1alpha is described for illustrative purposes, but that all these HIFs are included herein.
A stable HIF-1alpha (sHIF-1alpha ) protein of the inve
Gray Cary Ware & Freidenrich LLP
Haile Lisa A.
Imbra Richard J.
Nguyen Dave T.
Nguyen Quang
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