Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
2001-11-01
2004-12-07
Paras, Peter (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C435S325000, C435S354000
Reexamination Certificate
active
06828473
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to genetically-modified non-human mammals and genetically-modified animal cells containing a functionally disrupted PDE11A gene. The invention also features methods of screening for agents that modulate PDE11A and methods of modulating cAMP and cGMP signal transduction in cells that express PDE11A.
BACKGROUND OF THE INVENTION
Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of cyclic nucleotides, such as the second messengers cAMP (cyclic adenosine 3′5′-monophosphate) and cGMP (cyclic guanine 3′5′-monophosphate). Thus, PDEs play a pivotal regulatory role in a wide variety of signal transduction pathways (Beavo, Physiol. Rev. 75: 725-48, 1995). For example, PDEs mediate processes involved in vision (McLaughlin et al., Nat. Genet. 4: 130-34, 1993), olfaction (Yan et al., Proc. Natl. Acad. Sci. USA 92: 9677-81, 1995), platelet aggregation (Dickinson et al. Biochem. J. 323: 371-77, 1997), aldosterone synthesis (MacFarland et al., J. Biol. Chem. 266: 136-42, 1991), insulin secretion (Zhao et al., J. Clin. Invest. 102: 869-73, 1998), T cell activation (Li et al., Science 283: 848-51, 1999), and smooth muscle relaxation (Boolell et al., Int. J. Impot. Res. 8: 47-52, 1996; Ballard et al., J. Urol. 159: 2164-71, 1998).
PDEs form a superfamily of enzymes that are subdivided into 11 major families (Beavo, Physiol. Rev. 75: 725-48, 1995; Beavo et al., Mol. Pharmacol. 46: 399-05, 1994; Soderling et al., Proc. Natl. Acad. Sci. USA 95: 8991-96, 1998; Fisher et al., Biochem. Biophys. Res. Commun. 246: 570-77, 1998; Hayashi et al., Biochem. Biophys. Res. Commun. 250: 751-56, 1998; Soderling et al., J. Biol. Chem. 273: 15553-58, 1998; Fisher et al., J. Biol. Chem. 273: 15559-64, 1998; Soderling et al., Proc. Natl. Acad. Sci. USA 96: 7071-76, 1999; and Fawcett et al., Proc. Natl. Acad. Sci. USA 97: 3702-07, 2000).
Each PDE family is distinguished functionally by unique enzymatic characteristics and pharmacological profiles. In addition, each family exhibits distinct tissue, cell, and subcellular expression patterns (Beavo et al., Mol. Pharmacol. 46: 399-405, 1994; Soderling et al., Proc. Natl. Acad. Sci. USA 95: 8991-96, 1998; Fisher et al., Biochem. Biophys. Res. Commun. 246: 570-77, 1998; Hayashi et al., Biochem. Biophys. Res. Commun. 250: 751-56, 1998; Soderling et al., J. Biol. Chem. 273: 15553-58, 1998; Fisher et al., J. Biol. Chem. 273: 15559-64, 1998; Soderling et al., Proc. Natl. Acad. Sci. USA 96: 7071-76, 1999; Fawcett et al., Proc. Natl. Acad. Sci. USA 97: 3702-07, 2000; Boolell et al., Int. J. Impot. Res. 8: 47-52, 1996; Ballard et al., J. Urol. 159: 2164-71, 1998; Houslay, Semin. Cell Dev. Biol. 9: 161-67, 1998; and Torphy et al., Pulm. Pharmacol. Ther. 12: 131-35, 1999). Therefore, by administering a compound that selectively regulates the activity of one family or subfamily of PDE enzymes, it is possible to regulate cAMP and/or cGMP signal transduction pathways in a cell- or tissue-specific manner.
PDE11 is one of the most recently described families of PDEs; PDE11A is the sole member of this family so far identified (Fawcett et al., Proc. Natl. Acad. Sci. USA 97: 3702-07, 2000, hereinafter “Fawcett, 2000,” Yuasa et al., J. Biol. Chem. 275: 31469-79, 2000, hereinafter “Yuasa, 2000”). While PDE11A is known to be expressed in, e.g., testis, skeletal muscle, kidney, liver, various glandular tissue (e.g., pituitary, salivary, adrenal, mammary, and thyroid), pancreas, spinal cord, and trachea (Fawcett, 2000), little is known about PDE11A function. The present invention provides biological tools to study PDE11A function and methods to identify agents that regulate PDE11A activity for use in treating diseases and conditions that are linked to these PDE11A functions.
One embodiment of the present invention is the provision of a PDE11A knockout mouse, which provides an excellent opportunity to investigate genes involved in, inter alia, spermatogenesis, and, when compared with the wildtype mouse, to dissect out the components involved in spermatogenesis. One method for this type of analysis is microarray technology. With DNA microarray technology, it becomes possible to monitor large-scale gene expression over time. Prefabricated arrays of large numbers of especially designed oligonucleotide probes, e.g. as manufactured by Affymetrix (CA, USA), enable simultaneous hybridization-based analysis of thousands of genes.
SUMMARY OF THE INVENTION
The present invention features genetically-modified animal cells and genetically-modified non-human mammals containing a disrupted PDE11A gene, as well as assays for identifying PDE11A function in cells and tissues that express PDE11A, methods for identifying agents that modulate these PDE11A functions, and methods of treating or preventing diseases or conditions in mammals by modulating PDE11A function. For example, modulators of PDE11A activity are administered to a mammal to modulate spermatogenesis.
One aspect of the invention features a genetically-modified, non-human mammal, wherein the modification results in a functionally disrupted PDE11A gene. Preferably, the mammal is heterozygous for the modification. More preferably, the mammal is homozygous for the modification. In other preferred embodiments, the mammal is a rodent, more preferably, a mouse.
In a second aspect, the invention provides a genetically-modified animal cell, wherein the modification comprises a functionally disrupted PDE11A gene. Preferably, the animal cell is heterozygous for the modification. More preferably, the cell is homozygous for the modification. In other preferred embodiments, the cell is an embryonic stem (ES) cell, and/or the cell is human or murine.
The third aspect of the invention features a method of identifying an agent that modulates spermatogenesis, involving contacting an agent with a PDE11A polypeptide and measuring PDE11A activity, wherein a difference between the activity in the absence of the agent and in the presence of the agent is indicative that the agent can modulate spermatogenesis. Preferably, the method identifies an agent that inhibits PDE11A activity for use in decreasing spermatogenesis.
In a related fourth aspect, the invention also provides a method of identifying an agent that modulates spermatogenesis, which includes contacting an agent with a cell that expresses a PDE11A polypeptide and measuring PDE11A activity or PDE11A expression, wherein a difference between the activity or expression in the absence of the agent and in the presence of the agent is indicative that the agent can modulate spermatogenesis.
The fifth aspect provides a method of modulating spermatogenesis in a mammal, the method comprising administering an agent that modulates PDE11A activity. Preferably, the agent administered reduces PDE11A activity and reduces spermatogenesis.
Featured in the sixth aspect is a method of modulating cAMP and/or cGMP-mediated signal transduction in a mammal in testis, prostate, pituitary gland, bladder urothelium and/or bladder nerve fibers, neurons, skeletal muscle, cardiac myocytes, vascular smooth muscle, and/or vascular endothelial cells, which includes administering an agent that modulates PDE11A activity. In preferred embodiments, the agent selective for PDE11A, or is UK-336,017 (IC-351), UK-227,786 (E4021), or UK-235,187.
The invention also features in a seventh aspect, a method of treating hypertension, cardiac insufficiency, atherosclerosis, hyperprolactinemia, growth hormone insufficiency, incontinence, or disorders associated with skeletal muscle metabolism or contractility, wherein the method comprises administering an agent that modulates PDE11A activity. Preferably, the agent is selective for PDE11A. More preferably, the agent is UK-336,017 (IC-351), UK-227,786 (E4021), or UK-235,187.
In an eighth aspect, the invention provides biomarkers indicative of modulated spermatogenesis in a mammal; these biomarkers can be used to monitor spermatogenesis in a mammal as well as for monitoring diseases or disorders characterised by reduced sper
Burslem Martyn Frank
Harrow Ian Dennis
Lanfear Jeremy
Phillips Stephen Charles
Benson Gregg C.
Bertoglio Valarie
Martin Deborah A.
Paras Peter
Pfizer Inc.
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