Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2001-12-12
2004-12-21
Kunz, Gary (Department: 1647)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S006120, C435S007100, C435S007200, C514S04400A, C536S023500
Reexamination Certificate
active
06833237
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to genes and the proteins encoded thereby which are involved in neurotoxicity and/or are regulated by FK506. Polynucleotides were discovered using in vivo or in vitro models by determining which genes were differentially upregulated or downregulated when subjected to various stresses, such as hypoxia, and/or upon treatment of the model with FK506. Polynucleotides were also found by a functional selection (assay) of cDNA fragments specifically selected for their ability to confer cell resistance to various stresses which can result in neurotoxicity, such as hypoxia, glutamate or dopamine treatment. The invention includes such polynucleotides, corresponding genes, and proteins encoded thereby, as well as naturally-occurring variants of such polynucleotides, analogs, salts and functional derivatives of such proteins, DNA encoding such analogs, antibodies, antisense molecules and methods of use. Such methods of use include methods for protecting cells from neurotoxicity and ameliorating the effects of stroke, hypoxia and/or ischemia by regulating such genes or proteins.
BACKGROUND OF THE INVENTION
Brain injury such as trauma and stroke are among the leading causes of mortality and disability in the modern world.
Traumatic brain injury (TBI) is one of the most serious reasons for hospital admission and disability in modern society. Clinical experience suggests that TBI may be classified into primary damage occurring immediately after injury, and secondary damage, which occurs during several days post injury. Current therapy of TBI is either surgical or else mainly symptomatic. Stroke is the third leading cause of death and disability in developed countries, affecting more than half a million Americans each year. Stroke is an acute neurologic injury occurring as a result of an insult to the brain, thus interrupting its blood supply. Stroke induces neuronal cell death, which leads to the clinical outcomes of patients' death or disability ranging from total paralysis to milder dysfunction. Cerebral ischemia is the most common type of stroke, which may lead to irreversible neuronal damage at the core of the ischemic focus, whereas neuronal dysfunction in the penumbra may be reversible. Cells in the penumbra have an estimated time window for survival of up to 6 hours. The ability to intervene as soon as the patient is identified is essential for recovery. It is well established that ischemic tissue damage is multifactorial and involves at least excitotoxicity, reactive oxygen species, and inflammation—all leading to neuronal cell death.
Treatment strategies for stroke are aimed to induce rapid reperfusion and rescue of neurons in the penumbral area. Neuroprotective drugs are constantly being developed in an effort to rescue neurons in the penumbra from dying. However, potential cerebroprotective agents need to counteract all the above-mentioned destructive mechanisms. Therefore, current therapy in stroke focuses primarily on prevention, minimizing subsequent worsening of the infarction, and decreasing edema.
FK506 (tacrolimus) is a known immunosuppressive agent produced by
Streptomyces tsukubaesis
, a species discovered by the Fujisawa Pharmaceuticals' scientists in a soil sample from Tsukuba, Japan. See Kino et al, 1987, and U.S. Pat. No. 5,338,684. FK506 possesses neuroprotective activity by delaying or preventing hypoxia-induced death of neuronal cells. In addition, it can cause regrowth of damaged nerve cells. The specific molecular mechanism underlying the neuroprotective activity of FK506 is largely unknown although there are indications for suppression of activities of calcineurin and nitric oxide synthase as well as prevention of stroke induced generation of ceramide and Fas signaling. An additional model has been proposed involving steroid receptor complexes in context of FK506 neurotrophic actions. As a first step to novel drug discovery, these mechanisms should be delineated and key genes involved in these processes should be identified.
SUMMARY OF THE INVENTION
The polynucleotides of the present invention have been discovered by merging two technologies:
(1) microarray-based differential gene expression, evaluated in both in viva and in vitro models, and
(2) direct functional selection of genes with pro- or anti-apoptotic activities, performed in cell systems subjected to neurotoxic stress, such as hypoxia, glutamate or dopamine.
Differential profiling of gene expression was performed both in an in vivo model of permanent ischemia in rats either treated or untreated with FK506, performed by electrocoagulation of middle cerebral artery (MCA), and in an in vitro model of primary rat cerebellar neuron cultures exposed to hypoxia, with or without FK506 treatment. Polynucleotides were identified which were either upregulated or downregulated by either ischemia/hypoxia or the FK506 treatment or influenced by the combination of both treatments. Two proprietary cDNA microarrays, the “Apoptosis” and “Stroke” chips, were used in this study.
In addition, a direct functional selection of genes exhibiting pro-or anti-apoptotic activities induced by hypoxia, glutamate or dopamine was done on BE2C, an established human neuroblastoma cell line, upon introduction of expression cDNA library cloned into retroviral vector.
Accordingly, the present invention is directed to either novel polynucleotides whose expression (or function) in cells, in particular neural cells is modulated when cells are subjected to neurotoxic stress or whose activity is important for transduction of neurotoxic signals.
A total of 131 fragments, SEQ ID Nos: 1-131, were characterized as polynucleotides located in genes whose expression in neural cells is modulated when cells are subjected to neurotoxic stress or whose activity is important for neurotoxic signal transduction. Of these, 14 fragments which are incorporated in cDNA clones (all being KIAA clones) have been identified in our selection procedures as particularly preferred. This includes all of the polynucleotides of SEQ ID NOs:49, 50, 51, 65, 67, 85, 87 and 94-100, as well as the naturally-occurring full-length RNAs and corresponding full-length cDNAs and genes and natural antisense polynucleotides which include any one of these sequences, and corresponding polypeptides and proteins encoded by them.
Currently most preferred according to the present invention are the polynucleotides identified as SEQ ID NO: 94, which is a fragment of KIAA 0538 and SEQ ID NO: 65 which is a fragment of KIAA 0284. The former of these has been further identified as encoding a Ca2+-dependent Ras-GTPase Activator Protein. Elevated expression of Ras-GAP results in increased Ras inactivation and may contribute to cell death, in particular neuronal cell death.
The invention is further directed to naturally-occurring polynucleotides having at least 70% identity with any of the polynucleotides which include any one of SEQ ID Nos: 1-131, preferably SEQ ID Nos: 49, 50, 51, 65, 67, 85, 87 and 94-100, or which are capable of hybridizing under moderately stringent conditions to any of such polynucleotides, and whose expression or activity in naturally-occurring neural cells is modulated when the cells are subjected to neurotoxic stress.
The present invention is also directed to the polynucleotide comprising the sequence of any one of SEQ ID Nos: 1-48, 52-64, 66, 68-84, 86, 88-93, 101-131, which are novel polynucleotides and genes. The expression or activity of these polynucleotides in naturally-occurring neural cells is modulated when the cells are subjected to neurotoxic stress.
The present invention is also directed to fragments having at least 20 nucleotides of any of the polynucleotides of the present invention and to polynucleotide sequences complementary to any of such polynucleotides or fragment and to polypeptides encoded by any of the polynucleotides of the present invention.
In a more preferred embodiment, the isolated polynucleotide is a strand of a full-length cDNA.
According to one currently more preferred embodi
Feinstein Elena
Gorodin Svetlana
Kachalsky Sylvia G.
Mett Igor
Gucker Stephen
Kunz Gary
Quark Biotech, Inc.
White John P.
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