Methods and compounds for modulating male fertility

Details Methods and compounds for modulating male fertility Methods and compounds for modulating male fertility

2001-12-18

2004-09-28

Fredman, Jeffrey (Department: 1636)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S069100, C435S320100, C435S325000, C435S375000, C435S455000, C536S023100, C536S023500, C530S350000

Reexamination Certificate

active

06797473

ABSTRACT:

BACKGROUND OF THE INVENTION
The invention relates to apoptosis in cells, particularly cell involved in fertility.
Apoptosis is a fundamental process of cell death required for the elimination of unwanted cells in multicellular organisms and involves an ordered cascade of events leading to hallmark morphological changes including nuclear condensation, chromosome laddering, and membrane blebbing. In one specific example, apoptosis plays a prominent role during all stages of sperm development. Spermatogenesis is a process that results in the generation of mature sperm cells from primary germ cells, and some of the events affected by apoptosis include the elimination of unwanted cells and the prevention of the death of those cells destined to become functional sperm (Hsueh et al., Recent Prog. Horm. Res. 51: 433, 1996; Furuchi et al., Development 122: 1703, 1996).
Much of the current knowledge of the biochemical pathways involved in apoptosis comes from the study of viruses. The baculoviral proteins involved in apoptosis, CpIAP and OpIAP, are characterized by two amino terminal cys/his motifs (Xaa
3
—R—Xaa
20-23
—G—Xaa
11
—C—Xaa
2
—C—Xaa
16
—H—Xaa
6
—C—Xaa
3
) (SEQ ID NOs: 6-9) and a carboxy terminal C—Xaa
2
—C—Xaa
11
—C—Xaa—H—Xaa
3
—C—Xaa
2
—C—Xaa
6—C—Xaa
2
—C RING zinc finger motif (SEQ ID NO: 10) reviewed in Clem et al., Cell Death Differ. 3: 9, 1996). The two amino terminal motifs, termed Baculovirus IAP Repeat (BIR) domains, are the hallmark of the IAP family of proteins and are present as two or three copies in all IAP members discovered to date.
The first mammalian IAP (inhibitor of apoptosis protein), NAIP, was identified during a positional cloning effort seeking candidate genes for spinal muscular atrophy (Roy et al., Cell 80: 167, 1995). Following the identification of NAIP, three other human IAPs, HIAP-1, HIAP-2, and XIAP (Liston et al., Nature 379: 349, 1996), and their murine homologues MIAP-1, MIAP-2, and MIAP-3 (Farahani et al., Genomics 42: 514, 1997), have been reported. In addition, two Drosophila IAPs, DIAP-1 and DIAP-2, and one chicken IAP, ITA have been described (see Liston et al., Apoptosis 2: 423, 1997). A structural comparison of these IAPs is shown in FIG.
1
.
The physiological role of XIAP remains elusive. Recent work has demonstrated that XIAP directly inhibits two of the caspases, namely caspase-3 (also known as CPP32, Apopain, or YAMA) and caspase-7 (Devereaux et al., Nature 388: 300, 1997).
SUMMARY OF THE INVENTION
In general, the invention features methods and reagents useful for the treatment of excessive or insufficient apoptosis, particularly in testicular cells. The methods and reagents of the invention are useful in diagnosing and treating testicular cancers, cancers in non-testicular tissues, male infertility, and for achieving male birth control.
In a first aspect, the invention features a substantially pure nucleic acid molecule encoding a TIAP polypeptide. In one embodiment, the nucleic acid molecule has a sequence that is substantially identical to SEQ ID NO: 1. In another embodiment, the nucleic acid molecule has a sequence that hybridizes under high stringency conditions to SEQ ID NO: 1. In other embodiments, the TIAP polypeptide has a sequence that is substantially identical to SEQ ID NO: 2, or has a sequence that is at least 80% identical to SEQ ID NO: 2. In a preferred embodiment, the TIAP has the amino acid sequence of SEQ ID NO.: 2 or the nucleic acid sequence of SEQ ID NO.: 1. Preferably, identity is measured using sequence software analysis, such as the Sequence Analysis Software Package of the Genetics Computer Group. TIAP polypeptides encoded by the aforementioned nucleic acids are another aspect of the invention.
In a second aspect, the invention features a substantially pure nucleic acid molecule corresponding to at least fifteen nucleotides of a nucleic acid molecule encoding a TIAP polypeptide, where the nucleic acid molecule is an antisense nucleic acid molecule that is sufficient to decrease TIAP biological activity. In various embodiments, the antisense nucleic acid molecule corresponds to: at least thirty nucleotides of a nucleic acid molecule encoding a TIAP polypeptide, at least fifty nucleotides of a nucleic acid molecule encoding a TIAP polypeptide, or at least 100 nucleotides of a nucleic acid molecule encoding a TIAP polypeptide. In other preferred embodiments, the TIAP biological activity is decreased by at least 20%, at least 40%, at least 60%, or at least 80%. In yet another embodiment of the second aspect of the invention, the antisense nucleic acid molecule is in a vector that is capable of directing expression of the antisense nucleic acid molecule in a vector-containing cell.
In a third aspect, the invention features a vector including a substantially pure nucleic acid molecule encoding a TIAP polypeptide, the vector being capable of directing expression of the polypeptide in a vector-containing cell.
In a fourth aspect, the invention features a cell that contains a substantially pure nucleic acid molecule encoding a TIAP polypeptide. In one preferred embodiment, the nucleic acid molecule is expressed in the cell. In another embodiment, the cell is selected from a cell from the testis. In yet another embodiment, the cell is present in an animal having a condition that is associated with excessive or insufficient cell death.
In a fifth aspect, the invention features a transgenic animal generated from a cell genetically engineered to lack a nucleic acid molecule encoding a TIAP polypeptide, where the transgenic animal lacks expression of the TIAP polypeptide.
In a sixth aspect, the invention features a transgenic animal generated from a cell that contains a substantially pure nucleic acid molecule replacing DNA encoding a TIAP polypeptide, where the nucleic acid molecule is expressed in the transgenic animal.
In one embodiment of the first six aspects of the invention, the nucleic acid molecule is from an animal. In another embodiment, the nucleic acid molecule is genomic DNA or cDNA. In a preferred embodiment of the first six aspects of the invention, the nucleic acid molecule is operably linked to regulatory sequences for expression of the polypeptide and where the regulatory sequences include a promoter. Such a promoter may be from the TIAP gene, or may be selected from the group consisting of a constitutive promoter, a promoter that is inducible by one or more external agents, and a cell-type specific promoter.
In a seventh aspect, the invention features a method of identifying a compound that modulates TIAP biological activity that includes: (a) providing a cell including a TIAP gene; (b) contacting the cell with a candidate compound; and (c) measuring expression of the TIAP gene, where an alteration in the expression in response to the candidate compound relative to an cell not contacted with the candidate compound indicates a compound that modulates TIAP biological activity. In one embodiment of this aspect of the invention, the cell is transformed.
In an eighth aspect, the invention features a method of identifying a compound that modulates TIAP biological activity that includes: (a) providing a cell including a reporter gene operably linked to a promoter from a TIAP gene; (b) contacting the cell with a candidate compound; and (c) measuring expression of the reporter gene, where an alteration in the expression in response to the candidate compound relative to an cell not contacted with the candidate compound indicates a compound that modulates TIAP biological activity. In one embodiment of this aspect of the invention, the cell is transformed.
In a ninth aspect, the invention features a method of identifying a compound that is able to modulate TIAP biological activity in a cell that includes the steps of: (a) providing a expressing a normal amount of TIAP; (b) contacting the cell with a candidate compound; and (c) measuring level of apoptosis in the cell, where an alteration in the level relative to a level in a cell not contacted with the candidate compound indicates a compound that modulates TIAP

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