Major basic protein deficient animals

Details Major basic protein deficient animals Major basic protein deficient animals

1998-12-11

2001-01-23

LeGuyader, John L. (Department: 1633)

Multicellular living organisms and unmodified parts thereof and

Nonhuman animal

Transgenic nonhuman animal

C800S008000, C800S009000, C800S013000, C800S014000, C800S018000, C435S320100, C435S325000, C536S023100, C536S023500

Reexamination Certificate

active

06177610

ABSTRACT:

TECHNICAL FIELD
The invention relates to major basic protein (MBP) deficient animals.
BACKGROUND OF THE INVENTION
The management of asthma has changed significantly over the past decade, reflecting the recognition of a coincident chronic pulmonary inflammation. Asthma appears to be pluricausal in origin, involving several genes and exacerbating factors, such as air pollution, allergies, cold sensitivity, viral infection, and tobacco/chemical exposure. See, for example, Daniels, S. E. et al.,
Nature,
383(6597):247-250, 1996; McBride, D. E. et al.,
Am. J. Resp. Crit. Care Med.,
149(5):1192-1197, 1988; and O'Byrne, P. M.,
J. Aller. Clin. Immunol.,
81(1):119-127, 1988. The wide variability among patients, both in terms of etiology and presentation of symptoms, is anchored by three common characteristic features: reversible variable airflow limitations, specific airway histopathologies, and airway hyperresponsiveness (AHR, i.e., the development of bronchoconstriction in response to nonspecific stimuli). Additional indicators of asthma include mucus overproduction, increased expression of IL-4 and IL-5, increased serum IgE levels, and often (70-90% of reported cases) the increased presence of eosinophils in the airway mucosa and lumen. The pathophysiological manifestations of asthma correlate with this eosinophilic airway infiltration. Indeed, eosinophil influx has been associated with the development of lung dysfunction even in mild cases of asthma. The concurrent appearance of pulmonary pathologies and eosinophil recruitment to the lung suggests that a causative relationship exists.
Eosinophil effector functions are mediated by several concurrent mechanisms including the secretion of small molecule mediators of inflammation (e.g., PAF, leukotrienes, proinflammatory cytokines such as interleukins 4, 5, and 8), antigen presentation and the release of granule components (i.e., degranulation). These mechanisms contribute to several inflammatory pathways. For example, secretion of inflammatory signals by eosinophils results in vasodilation and tissue edema and the recruitment/activation of other effector cells. Moreover, the ability of eosinophils to act as antigen presentation cells suggests that their recruitment to the lung during allergic inflammation may result in presentation of aeroallergens to resident T cells and thus initiate and/or amplify pulmonary immune responses.
A growing literature exists indicating that the release of eosinophil secondary granule proteins (ESGPs) is a critical effector function. ESGPs effector functions include not only cytotoxic activities leading to airway damage and lung dysfunction (e.g., AHR), but also agonist activities on several other cell types. Gleich, G. J. and C. Adolphson,
Agents Actions Suppl.,
43:223-230, 1993. For example, in vitro studies of lung fibroblasts have shown that ESGPs are capable of modulating gene expression from these cells. Rochester, C. L. et al.,
J. Immunol.,
156(11):4449-4456, 1996. Furthermore, ESGPs also modulate the activation state and genes expressed by other leukocyte effector cells including neutrophils and mast cells. Agonist activities also extend to autocrine effects on eosinophils leading to the expression of inflammatory mediators and eosinophil degranulation. Patella, V. et al.,
J. Immunol.,
157(3):1219-1225, 1996. These studies suggest that the interplay of ESGP effector functions have pleiotropic effects on the lung resulting in both tissue destruction and the modulation of inflammatory responses associated with allergic pulmonary inflammation. The four prominent murine secondary granule constituents include eosinophil peroxidase (EPO), major basic protein (MBP), and eosinophil associated ribonucleases (EAR-1 and EAR-2). EPO, MBP, eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN) are the prominent secondary granule constituents in humans.
SUMMARY OF THE INVENTION
As described herein, MBP deficient animals fail to exhibit airway hyperresponsiveness. Thus, the release of MBP is linked to a lung disfunction in asthmatic patients. MBP deficient animals can be used in conjunction with wild-type animals to screen for anti-MBP therapies.
In one aspect, the invention features a DNA construct that includes an inactivated MBP gene, wherein the inactivation is due to disruption of a sequence in the gene such that expression of functional MBP is prevented. The inactivated MBP gene may lack exons 2, 3 and 4.
The invention also features a non-human mammal, and progeny thereof, that include an inactivated MBP gene, wherein the inactivation results in reduced levels of endogenous MBP in the non-human mammal. The non-human mammal can be heterozygous or homozygous for the inactivated MBP gene. In one embodiment, the non-human mammal lacks MBP. The non-human mammal can be, for example, a rodent, and in particular, a mouse. Non-human mammals of the invention do not exhibit airway hyperresponsiveness. The non-human mammal also can include a human MBP gene.
Non-human mammal eosinophil cells lacking MBP also are featured. The cells can be murine eosinophil cells. Murine eosinophil cells can be isolated from a mouse homozygous for an inactivated MBP gene, wherein the inactivation results in a mouse lacking MBP.
The invention also relates to a method for identifying inhibitors of MBP effector function that includes comparing airway hyperresponsiveness of a first non-human mammal in the presence of a compound with a second non-human mammal in the absence of the compound. The first and second non-human mammals each include a human MBP gene and an inactivated endogenous MBP gene, such that the first and second non-human mammals each have detectable levels of human MBP and reduced levels of endogenous MBP. Compounds are identified as inhibitors of MBP effector function if airway hyperresponsiveness is reduced in the first non-human mammal. The first and second non-human mammals can be rodents, and in particular, can be mice.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.


REFERENCES:
Mullins et al., J. Clin. Invest., vol. 98, pp. S37-S41, 1996.
Moreadith et al., J. Mol. Med., vol. 75, pp. 208-216, 1997.
Aszodi et al., J. Molecular Med., vol. 76, pp. 238-252, 1998.
Wall, Theriogenology, vol. 45, pp. 57-68, 1996.
Daniels et al., “A genome-wide search for quantitative trait loci underlying asthma”,Nature, 1996, 383:247-250.
McBride et al., “Inflammatory Effects of Ozone in the Upper Airways of Subjects with Asthma”,Am. J. Resp. Crit. Care Med., 1994, 149(5):1192-1197.
O'Byrne et al., “Allergen-induced airway hyperresponsiveness”,J. Allergy. Clin. Immunol., 1988, 81(1):119-127.
Gleich et al., “Bronchial Hyperreactivity and Eosinophil Granule Proteins”,Agents Actions Suppl., 1993, 43:223-230.
Rochester et al., “Eosinophil-Fibroblast Interactions —Granule Major Basic Protein Interacts with IL-1 and Transforming Growth Factor-&bgr; in the Stimulation of Lung Fibroblast IL-6-Type Cytokine Production”,J. Immunol., 1996, 156(11):4449-4456.
Patella et al., “Eosinophil Granule Proteins Activate Human Heart Mast Cells”,J. Immunol., 1996, 157(3):1219-1225.
Abu-Ghazaleh et al., “Interaction of Eosinophil Granule Major Basic Protein with Synthetic Lipid Bilayers: A Mechanism for Toxicity”,J. Membrane Biol., 1992, 128(2):153-164.
O'Donnell et al.,

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