Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2000-07-14
2002-12-17
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
Reexamination Certificate
active
06495339
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to apoptosis, tumor necrosis factor-&agr; (TNF-&agr;) mediated signalling, cell cycle and tumor growth suppression.
Apoptosis is a morphologically distinct form of programmed cell death that is important in the normal development and maintenance of multicellular organisms. Dysregulation of apoptosis can take the form of inappropriate suppression of cell death, as occurs in the development of cancers, or in a failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain neurodegenerative disorders.
Some baculoviruses encode proteins termed “inhibitors of apoptosis proteins” (IAPs) because they inhibit the apoptosis that would otherwise occur when insect cells are infected by the virus. These proteins are thought to work in a manner that is independent of other viral proteins. The baculovirus IAP genes include sequences encoding a ring zinc finger-like motif (RZF), which may be involved in DNA binding, and two N-terminal domains that consist of a 70 amino acid repeat motif termed a BIR domain (Baculovirus IAP Repeat).
We have recently discovered a mammalian family of IAP polypeptides. These polypeptides include the human proteins HIAP-1, HIAP-2, and XIAP and their murine homologs. A related protein, NAIP, has also been found. The mammalian IAP levels have been shown to be increased both in cancer cells and cells which survive events known to induce apoptosis (e.g., ischemia). The IAPs have also been shown to block apoptosis triggered by diverse stimuli. These results are consistent with a role for the mammalian IAPs as inhibitors of apoptosis.
The IAP family is now known to include at least two Drosophila proteins, in addition to the original four mammalian homologues (Hay et al., Cell 83: 1253-1262, 1995). Although we and others have established that the IAPs can suppress apoptosis in tissue culture model systems their mechanism of action is still under investigation.
SUMMARY OF THE INVENTION
We have discovered a novel family of genes, the XAFs. Members of the XAF gene family encode proteins that interact with IAPs and are associated with apoptosis. Our discovery allows the development of diagnostic, prognostic, and therapeutic compounds and methods for the detection and treatment of diseases involving apoptosis.
In a first aspect, the invention features substantially pure nucleic acid encoding a XAF polypeptide.
In a second aspect, the invention features substantially pure nucleic acid corresponding to at least ten nucleotides of a nucleic acid encoding a XAF polypeptide, where the nucleic acid is antisense nucleic acid and the antisense nucleic acid is sufficient to decrease XAF biological activity. In various embodiments of this aspect, the antisense nucleic acid corresponds to at least fifteen nucleotides of a nucleic acid encoding a XAF polypeptide, at least thirty nucleotides of a nucleic acid encoding a XAF polypeptide, or at least 100 nucleotides of a nucleic acid encoding a XAF polypeptide. In other embodiments, the XAF biological activity is decreased by at least 20%, 40%, 60%, or 80%. In yet another embodiment of this aspect of the invention, the antisense nucleic acid is in a vector where the vector is capable of directing expression of the antisense nucleic acid in a vector-containing cell.
In a third aspect, the invention features a vector that includes a substantially pure nucleic acid encoding a XAF polypeptide, where the vector is capable of directing expression of the polypeptide in a vector-containing cell.
In another related aspect, the invention features a cell that contains a substantially pure nucleic acid encoding a XAF polypeptide. In a preferred embodiment of this aspect, the nucleic acid is expressed in the cell. In various preferred embodiments, the cell is present in a patient having a disease that is caused by excessive or insufficient cell death and the cell is selected from the group that includes a fibroblast, a neuron, a glial cell, an insect cell, an embryonic stem cell, a myocardial cell, and a lymphocyte.
In a fifth aspect, the invention features a transgenic animal generated from a cell genetically engineered to lack nucleic acid encoding a XAF polypeptide, where the transgenic animal lacks expression of the XAF polypeptide.
In a related aspect, the invention features a transgenic animal generated from a cell that contains a substantially pure nucleic acid that replaces DNA encoding a XAF polypeptide, where the nucleic acid is expressed in the transgenic animal.
In various embodiments of this aspect, the XAF polypeptide is from a mammal (e.g., a human or a rodent). In another embodiment, the nucleic acid is genomic DNA or cDNA, and is operably linked to regulatory sequences for expression of the polypeptide where the regulatory sequences include a promoter (e.g., a constitutive promoter, a promoter inducible by one or more external agents, or a cell-type specific promoter). In other preferred embodiments, the XAF polypeptide is selected from a group that includes XAF-1, XAF-2 N terminus, XAF-2L and XAF-2S. In another embodiment, the XAF-1 has the amino acid sequence of SEQ ID NO.: 2 or the nucleic acid sequence of SEQ ID NO.: 1, and may include a deletion of the nucleic acids encoding the carboxy terminal amino acids 173 to 317 of XAF-1 (SEQ ID NO.: 8); or a deletion of the nucleic acids encoding the amino terminal amino acids 1 to 172 of XAF-1 (SEQ ID NO.: 7). In another embodiment of this aspect of the invention, the XAF-2 N terminus polypeptide has the amino acid sequence of SEQ ID NO.: 4 or the nucleic acid sequence of SEQ ID NO.: 3. In another embodiment, the XAF-2L polypeptide has the amino acid sequence of SEQ ID NO.: 10 or the nucleic acid sequence of SEQ ID NO.: 9. In yet another embodiment, the XAF-2S polypeptide has the amino acid sequence of SEQ ID NO.: 12 or the nucleic acid sequence of SEQ ID NO.: 11.
In a seventh aspect, the invention features a method of identifying a compound that modulates apoptosis. The method includes: (a) providing a cell that has a XAF gene; (b) contacting the cell with a candidate compound; and (c) monitoring expression of the XAF gene, where an alteration in the level of expression of the XAF gene indicates the presence of a compound which modulates apoptosis. In one preferred embodiment of this aspect, the alteration that is an increase indicates the compound is increasing apoptosis, and the alteration that is a decrease indicates the compound is decreasing apoptosis. In various embodiments of this aspect, the cell is transformed and the cell is not able to induce apoptosis by expression of p53.
In a related aspect, the invention features another method of identifying a compound that is able to modulate apoptosis that includes: (a) providing a cell including a reporter gene operably linked to a promoter from a XAF gene; (b) contacting the cell with a candidate compound; and (c) measuring expression of the reporter gene, where a change in the expression in response to the candidate compound identifies a compound that is able to modulate apoptosis. In one preferred embodiment of this aspect, the alteration that is an increase indicates the compound is increasing apoptosis, and the alteration that is a decrease indicates the compound is decreasing apoptosis. In various embodiments of this aspect, the cell is transformed and the cell is not able to induce apoptosis by expression of p53.
In a ninth aspect, the invention features a method of identifying a compound that is able to inhibit XAF-mediated apoptosis that includes: (a) providing a cell expressing an apoptosis-inducing amount of XAF; (b) contacting the cell with a candidate compound; and (c) measuring the level of apoptosis in the cell, where a decrease in the level relative to a level in a cell not contacted with the candidate compound indicates a compound that able to inhibit XAF-mediated apoptosis. In various embodiments of this aspect, the cell is transformed and the cell is not able to induce apoptosis by expression of p53.
In a tenth aspect,
Korneluk Robert G.
Liston Peter
MacKenzie Alexander E.
Tamai Katsuyuki
Aegera Therapeutics Inc.
Bieker-Brady Kristina
Clark & Elbing LLP
McKelvey Terry
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