Multicellular living organisms and unmodified parts thereof and – Method of using a transgenic nonhuman animal in an in vivo...
Reexamination Certificate
1999-01-08
2003-01-28
Priebe, Scott D. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of using a transgenic nonhuman animal in an in vivo...
C800S014000, C800S009000, C800S013000, C800S022000, C800S023000, C800S024000, C800S025000
Reexamination Certificate
active
06512161
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to models of human diseases and to methods of using these models for identifying compounds effective for the treatment of these diseases. In particular, the present invention represents a new experimental model for the in vivo analysis of human lipoprotein (a) [Lp(a)] and the development of therapeutic strategies to reduce the health risks associated with high levels of this lipoprotein.
Dyslipoproteinaemias are disorders in the metabolism of the lipoproteins which are responsible for transporting lipids such as cholesterol and triglycerides in the blood and the peripheral fluids. Dyslipoproteinaemias consequently are associated with life-threatening diseases linked to hypercholesterolaemia, hypocholesterolaemia or hypertriglyceridaemia, such as atherosclerosis.
Atherosclerosis is a complex, polygenic disease which is defined, on the histological plane, by deposits (lipid or fibro-lipid plaques) of lipids and other blood derivatives in the wall of the large arteries (aorta, coronary arteries and carotid). The plaques, which are more or less calcified according to the progress of the disease, can be associated with lesions and are linked to the accumulation, in the arteries, of fatty deposits which essentially consist of cholesterol esters. The plaques are accompanied by a thickening of the arterial wall together with hypertrophy of the smooth muscle, the appearance of foam cells and the accumulation of fibrous tissue. The plaques are raised on the arterial wall resulting in a stenosis, that is, a narrowing or stricture of the artery. In the worst-affected patients, this stenosis is responsible for the vascular occlusions, such as atheroma, thrombosis and embolisms. Accordingly, excess accumulation of cholesterol (hypercholesterolaemias) can lead to very serious cardiovascular diseases such as infarction, sudden death, cardiac decompensation, cerebrovascular diseases, and the like.
It is particularly important, therefore, to have immediately available treatments which diminish, in some disease situations, the levels of plasma cholesterol or stimulate the efflux of cholesterol (reverse transport of cholesterol) from the peripheral tissues in order to unload the cells which have accumulated the cholesterol in the context of forming an atheroma plaque. Cholesterol is transported in the blood by a variety of lipoproteins including the low density lipoproteins (LDL) and the high density lipoproteins (HDL). The LDLs are synthesized in the liver and are responsible for supplying the peripheral tissues with cholesterol. By contrast, the HDLs pick up cholesterol in the peripheral tissues and transport it to the liver where it is stored and/or broken down.
Numerous studies have correlated elevated plasma levels of lipoprotein (a) [Lp(a)] with increased incidence of cardiovascular disease and stroke (reviewed in Utermann, G.,
Science
(1989) 246, 904-910; Maher, V. M. G., & Brown, B. G., Curr. Opin. Lipidol. (1995) 6, 229-235). Lipoprotein(a) is a complex particle composed of a lipid moiety and two disulfide-linked subunits: apolipoprotein B-100 (apoB-100) and apolipoprotein(a) [apo(a)]. The presence of apo(a), a hydrophilic glycoprotein structurally related to plasminogen, distinguishes Lp(a) from low density lipoprotein (LDL) and confers its characteristic biological and physical properties.
Apolipoprotein B-100 (apoB) is the major protein constituent of very low density lipoproteins (VLDL), low density lipoproteins (LDL) and lipoprotein Lp(a). This protein is the physiological ligand of the LDL receptor, and its plasma concentration is positively correlated with the development of atherosclerosis (Brunzell et al. 1984 Arteriosclerosis 4, 79-93). ApoB-100 is one of the largest known proteins, with a mass of 550 kDa and containing 4536 amino acids (Chen et al. 1986 J. Biol. Chem, 261, 12919-21). This apolipoprotein is only synthesized in the liver. Its plasma concentration is 1.0-1.2 g/l. ApoB-100 plays the major role in transporting cholesterol which is synthesized in the liver through the plasma to the other cells of the organism. Another version of apoB, i.e. apoB-48, is present in the chylomicrons. In humans, apoB-48 is synthesized in the intestine. ApoB-48 has a mass of 260 kDa and contains 2152 amino acids which linearly correspond to 48% of the N-terminal end of apoB-100 (Powell et al., 1987, Cell 50, 831-40). Since the C-terminal moiety of apoB-100 contains the zone for binding the apoB-100 to the LDL receptor, apoB-48 does not attach to this latter receptor and behaves in a different manner metabolically.
To study the disease states relating to dysliproproteinaemias, it is advantageous to have available an animal model which expresses a protein or a protein complex which is associated with a risk for a disease which is linked to dyslipoproteinaemias. Such an animal model would be particularly advantageous for understanding these diseases and, more specifically, the regulatory mechanisms which these proteins or protein complexes initiate. This would make it possible to test, rapidly and in vivo, a considerable number of therapeutic agents or compounds for the purpose of detecting a potential activity associated with the expression of the proteins. Furthermore, such a model would be of interest for developing novel therapeutic methods for treating these types of diseases, such as methods which are based on gene therapy. The in vivo analysis of Lp(a), an independent atherosclerosis risk factor in humans, has been limited in part by its restricted distribution among mammals. Apo(a) is naturally present exclusively in old world monkeys, humans, and one non-primate species, the European hedgehog. Such limited distribution of apo(a) among mammals has limited studies of its in vivo properties.
Accordingly, the present invention relates to animal models of disease states involving Lp(a), including atherosclerosis, to enable screening and identification of compounds for the treatment of these diseases, in particular, atherosclerosis.
Reported Developments
Generally speaking, the murines, namely mice, rats and guinea pigs, are the most widely used animal models. They are easy to manipulate and inexpensive. Unfortunately, these small mammals are not always compatible with the intended application because they are not always representative of humans and their metabolism. Chimpanzees are used for testing therapeutic agents and vaccines directed against various diseases, including AIDS and cancer. However, the very substantial cost incurred in using chimpanzees as a model system constitutes a major and compelling handicap with regard to its use.
Despite the drawbacks of a system using mice, the development of transgenic mice expressing human apo(a) cDNA provided a means to test hypotheses accounting for the effect of Lp(a) on the vasculature. Specifically, these mice have been used to examine the ability of apo(a) to promote atherogenesis by inhibition of plasmin formation and associated consequences (Lawn, et al.,
Nature
(1992) 360, 670-672; Grainger, et al.,
Nature
(1994) 370, 460-462). Studies of apo(a) transgenic mice have also led to several important insights into Lp(a) assembly, including the observation that apo(a) was unable to form a covalent association with LDL containing murine apoB (Chiesa, et al.,
J Biol Chem
(1992) 267, 24369-24374). This result, coupled with evidence for Lp(a) formation when the mice were infused with human LDL or expressed a human apoB transgene (Linton, et al.,
J Clin Invest
(1993) 92, 3029-3037; Callow, et al.,
Proc Nat'l Acad Sci, USA
(1994) 91, 2130-2136) suggested that murine apoB lacked structural requirements necessary for Lp(a) assembly. This was not a completely unexpected finding in light of the absence of the apo(a) gene in mice and the sequence specific interactions between apo(a) and apoB believed to mediate Lp(a) assembly in humans. Two studies using site specific mutagenesis of human apoB transgenes in mice have reported localization of a single
Denefle Patrice
Duverger Nicolas
Emmanuel Florence
Houdebine Louis-Marie
Hughes Steven D.
Aventis Pharmaceuticals Inc.
Chen Shin-Lin
Coppola William C.
Priebe Scott D.
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