Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1999-04-28
2001-03-27
LeGuyader, John L. (Department: 1633)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S009000, C800S022000, C435S320100, C435S325000, C435S455000
Reexamination Certificate
active
06207876
ABSTRACT:
1.0 BACKGROUND OF THE INVENTION
1.1 Field of the Invention
The present invention provides adenosine deaminase (ADA) deficient mice. Also provided are methods of using the mice as an animal models for the analysis of physiological states that are sensative to disturbances in adenine nucleoside metabolism.
1.2 Description of Related Art
1.2.1 Adenosine Deaminase Deficiency
Genetic defects in purine metabolism in humans result in serious metabolic disorders, often with pronounced tissue-specific phenotypes (Blaese, 1995a). A striking example of this is adenosine deaminase (ADA) deficiency, which results in impaired lymphoid development and a severe combined immunodeficiency disease (SCID) (Hershfield and Mitchell, 1995).
ADA deficient SCID was the first of the inherited immunodeficiencies for which the underlying molecular defect was identified (Giblett et al., 1972); however, despite over 20 years of subsequent research, a satisfactory explanation for the lymphoid specificity of this metabolic disease has not emerged. This is largely due to the inaccessibility of human tissue for detailed phenotypic and metabolic analysis and the absence of an animal model which retains features of ADA deficiency in humans.
Additional interest in ADA deficiency stems from recent attempts to use novel therapeutic strategies, including enzyme therapy (Hershfield et al., 1993) and gene therapy (Bordingon et al., 1995; Blaese et al., 1995), to treat the condition in humans. Although the results of these therapeutic approaches are encouraging, unexpected outcomes have raised numerous important questions regarding the efficacy of specific treatment protocols (Hershfield et al., 1993; Blaese, 1995b). The pace with which new enzyme and gene therapy protocols can be tested would be greatly increased by the availability of an animal model for ADA deficiency.
The availability of a genetic animal model for ADA deficiency would make possible a wide range of biochemical and immunological experiments that are not permissible with humans.
Attempts to generate ADA deficient mice were initially reported by two groups (Wakamiya et al., 1995; Migchielsen et al., 1995), resulting in animals with independent sites of Ada gene disruption. However, these attempts did not lead to the production of viable ADA deficient mice. In each case a similar phenotype was observed. ADA deficient fetuses died perinatally due to severe liver damage (Wakamiya et al., 1995; Migchielsen et al., 1995). This phenotype was accompanied by profound disturbances in purine metabolism, including marked increases in the ADA substrates adenosine and 2′-deoxyadenosine.
2′-Deoxyadenosine is a cytotoxic metabolite that can kill cells through mechanisms that include disturbances in deoxynucleotide metabolism (Ullmann et al., 1978; Cohen et al., 1978) and the inhibition of cellular transmethylation reactions (Hershfield, 1979; Hershfield et al., 1979). ADA deficient fetuses exhibited evidence for both of these mechanisms of 2′-deoxyadenosine cytotoxicity, in that levels of the 2′-deoxyadenosine metabolite, dATP, were markedly elevated, and the enzyme S-adenosylhomocysteine (AdoHcy) hydrolase was inhibited (Wakamiya et al., 1995; Migchielsen et al., 1995). These metabolic disturbances are thought to contribute to the liver damage and subsequent death of ADA deficient fetuses.
Previous attempts by the inventors to produce mice that expressed ADA in the fetus but not in the neonatal mouse had failed (Blackburn et al., 1995). The strategy was to introduce an ADA transgene that would only be expressed in the placenta into heterozygous ADA knockout mice. The heterozygous transgenic mice could then be mated to yield homozygous ADA knockout mice that were able to develop because of the transgene provided ADA expression in the placenta. Viable mice were obtained by this strategy; however, the promoter used to express the transgene was not specific to the placenta and ADA expression was detected in the gastrointestinal tract of these animals, predominantly in the forestomach. Therefore, although ADA was not expressed in the lungs of these animals, ADA expression was present in the gut.
Disclosed herein is description of a mouse lacking post-partum ADA expression. This was acheived by creating a placenta specific promoter (Shi et al., 1997) and using this promoter in a transgene construct to express ADA. Indeed, the ADA deficient mice have immunodeficiencies similar to that of humans with ADA deficiency (Blackburn, 1998). Surprisingly and unexpectedly, the ADA deficient mice showed lung abnormalities. Upon extensive examination, the inventors were able to determine that the lung abnormalities in ADA deficient mice were reminiscent of those seen in asthma.
1.2.2 Asthma
Asthma is an inflammatory disease of the airways. In the U.S., 13% of children and 6% of adults suffer from asthma and 1% of health care cost are devoted to asthma treatment (Vogel 1997, Cochrane et al., 1996; Weiss et al., 1992). The disease is typified by the infiltration and activation of immune cells in the lung, followed by airway inflammation and obstruction (Vogel 1997). Many factors can trigger asthma, however, the mechanisms by which these triggers lead to airway inflammation and damage are not well understood. There is increasing evidence that asthma has its roots in early life (Barker 1992; Busse et al., 1995), but the mechanisms involved are not understood. Nor is it known how genetic and environmental influences interact to manifest asthma. Therefore, there is a need in the art for a model system with a predetermined genetic background that would allow testing of the influence of environmental factor on asthma development.
1.2.3 Animal Models and Asthma
Because of the limitations on the availability of human tissues, a number of animal models have been developed to better define the structural and functional consequences produced by environmental agents within the respiratory tract (Larsen and Colasurdo, 1997; Abraham and Baugh, 1995). While an ideal animal model should exhibit all the features of human asthma, there is general agreement that a single animal model does not exhibit all the functional and biological changes that would mimic the disease process seen in humans (Larsen and Colasurdo, 1997).
1.2.4 Adenosine Signaling in Asthma
Adenosine is a regulatory nucleoside that has the potential to be produced from all cells as a product of ATP catabolism (Arch and Newsholme, 1978). In mammalian tissues there exist two metabolic pathways through which adenosine can be generated: One is through the enzyme 5′-nucleotidase, which generates adenosine by enzymatic dephosphorylation of 5′-AMP (Zimmerman 1992). The other involves the hydrolysis of S-adenosylhomocysteine to adenosine and homocysteine (Schutz et al., 1981). Once generated, adenosine is freely transported in and out of cells through an ubiquitous nucleoside transporter (Arch and Newsholme, 1978) and serves as an extracellular signal. Inside the cell it is either deaminated to inosine by ADA (Blackburn and Kellems 1996) or is phosphorylated to AMP by adenosine kinase (Arch and Newsholme, 1978). Extracellular adenosine can bind to cell surface receptors to elicit a wide variety of cellular responses (Stiles 1992).
The regulatory actions of adenosine are mediated by several distinct membrane receptors that are classified as P1 purinergic receptors (Olah and Stiles 1995). Distinct subtypes of adenosine receptors have been identified on the basis of cDNA sequence and pharmacological profiles and are termed A1, A2a, A2b, and A3 adenosine receptors (Libert et al., 1989; Stehle et al., 1992; Pierce et al., 1992; Rivkees and Reppert, 1992; Fink et al., 1992; Zhou et a., 1992; Salvatore et al., 1993). Adenosine receptors are tightly coupled to effector enzymes by guanine nucleotide (G-protein) regulatory proteins (Stiles 1992; Olah and Stiles 1995), and each receptor subtype has a distinct affinity for adenosine and its structural congeners. Both A2 receptor subtypes couple to adenylate cyclase by sti
Blackburn Michael R.
Datta Surjit K.
Kellems Rodney E.
Board of Regents , The University of Texas System
Fulbright & Jaworski LLP
Kaushal Sumesh
LeGuyader John L.
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