The invention provides gene-targeted non-human animals comprising a genetically modified apoE gene encodes a recombinant apoE polypeptide displaying domain interaction. The invention further provides cells isolated from the gene-targeted animals, which cells produce a recombinant apoE polypeptide displaying domain interaction. The invention further provides methods of identifying agents that reduce apoE4 domain interaction, and which are useful to treat apoE4-related neurological and cardiovascular disorders.

SUMMARY OF THE INVENTION

The present invention provides non-human gene-targeted animal models for the study of apolipoprotein E4-associated pathologies, wherein the endogenous apoE gene of the gene-targeted animal is genetically altered such that the encoded recombinant apoE polypeptide exhibits domain interaction. Since domain interaction is a hallmark of human apoE4, the non-human gene-targeted animals of the instant invention serve as models for human apoE4 domain interaction. The invention further provides cells isolated from a non-human gene-targeted animal of the invention, in particular, cells that produce recombinant apoE exhibiting domain interaction. The invention further provides isolated recombinant apoE protein produced by a cell of the invention. The non-human gene-targeted animals of the invention, as well as cells isolated from such animals and recombinant apoE protein produced by such cells, are useful for screening candidate agents for their ability to reduce apoE domain interaction in the recombinant apoE polypeptide. Such agents are useful for reducing apoE4 domain interaction in human cells producing apoE4, and are therefore useful for treating apoE4-related neurological and cardiovascular disorders.

The invention further provides methods of identifying agents that reduce apoE4 domain interaction. The methods generally comprise contacting a non-human gene-targeted animal (or a cell or a recombinant apoE protein) of the invention with a test agent, and determining the effect, if any, of the test agent on domain interaction, or a phenomenon associated with apoE4 domain interaction. In some embodiments, phenomena associated with apoE4 domain interaction are associated with neurological disorders. The phenomena measured include behavioral phenomena and physiological phenomena. Behavioral phenomena may include cognitive function, such as spatial learning. Physiological phenomena may include the presence of neurofibrillary tangles and A.beta. deposits. Alternatively, or in conjunction, the phenomena examined may be examining phenotypes such as neurodegeneration, including neurodegeneration that is age-dependent. In other embodiments, phenomena and/or disorders associated with apoE4 domain interaction are cardiovascular disorders. In these embodiments, phenomena include hyperlipidemia and associated disorders such as coronary artery disease and atherosclerosis.

A primary object of the invention is to provide a method of using a gene-targeted animal model for identifying candidate agents (e.g., a small molecule drug or an endogenous factor) that reduce apoE4 domain interaction and, in doing so, treat disorders related to the presence of apoE4, e.g., neurodegenerative disorders and cardiovascular disorders. Such methods are useful for screening candidate agents for use in treating or relieving the symptoms of apoE4-related pathologies. The cells derived therefrom are also useful for screening biologically active agents that reduce apoE4 domain interaction.

A feature of the invention is that a gene-targeted mouse of the invention expresses a form of apoE that differs from the endogenous apoE in that it exhibits domain interaction. The genetically modified apoE gene of gene-targeted non-human animals of the invention is in its normal genomic environment, and therefore is under control and regulation of the same enhancer and tissue-specific elements that direct expression of the wild-type apoE gene. Thus, an advantage of a gene-targeted non-human animal of the instant invention is that the genetically modified apoE gene is under control of the same elements that control the apoE gene in a wild-type animal of the same species, such that the genetically altered endogenous apoE exhibits wild-type expression patterns. Thus, gene-targeted animals of the invention provide animal models that are representative of human apoE4 in its native environment.

FEATURES OF THE INVENTION

In one aspect, the invention provides a gene-targeted non-human animal comprising a modified endogenous apoE allele, wherein said modified endogenous apoE allele is under transcriptional control of endogenous regulatory elements, and wherein the modified apoE allele encodes a recombinant apoE polypeptide that exhibits domain interaction characteristic of human apoE4.

In another aspect, the invention provides a gene-targeted non-human animal comprising a modified endogenous apoE allele, wherein said modified endogenous apoE allele is under transcriptional control of endogenous regulatory elements, and wherein the modified apoE allele comprises a Thr.fwdarw.Arg substitution at a position equivalent to amino acid 61 of human apoE4.

In another aspect, the invention provides a gene-targeted mouse comprising a modified endogenous mouse apoE allele, wherein said modified endogenous mouse apoE allele is under transcriptional control of endogenous regulatory elements, and wherein the modified mouse apoE allele comprises a Thr.fwdarw.Arg substitution at a position equivalent to amino acid 61 of human apoE4. In some aspects, the gene-targeted mouse is homozygous for the modified endogenous apoE allele.

In another aspect, the invention provides an isolated non-human cell comprising a modified endogenous apoE allele, wherein said modified endogenous apoE allele is under transcriptional control of endogenous regulatory elements, and wherein the modified endogenous apoE allele encodes a recombinant apoE polypeptide that exhibits domain interaction characteristic of human apoE4.

In another aspect, the invention provides an isolated non-human cell comprising a modified endogenous apoE allele, wherein said modified endogenous apoE allele is under transcriptional control of endogenous regulatory elements, and wherein the modified endogenous apoE allele comprises a Thr.fwdarw.Arg substitution at a position equivalent to amino acid 61 of human apoE4.

In another aspect, the invention provides an isolated mouse cell comprising a modified endogenous mouse apoE nucleic acid molecule, wherein said modified endogenous mouse apoE nucleic acid molecule is under transcriptional control of endogenous regulatory elements, and wherein the modified mouse apoE nucleic acid comprises a Thr.fwdarw.Arg substitution at a position equivalent to amino acid 61 of human apoE4.

In another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence derived from a non-human apoE gene, which nucleotide sequence is modified such that it encodes an apoE protein that exhibits domain interaction characteristic of human apoE4. In another aspect, the invention provides a recombinant vector comprising such a nucleic acid molecule. In another aspect, the invention provides a recombinant host cell comprising such a recombinant vector.

In another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence derived from a non-human apoE gene, which nucleotide sequence is modified such that it comprises a Thr.fwdarw.Arg substitution at a position equivalent to amino acid 61 of human apoE4, and encodes an apoE protein that exhibits domain interaction characteristic of human apoE4. In another aspect, the invention provides a recombinant vector comprising such a nucleic acid molecule. In another aspect, the invention provides a recombinant host cell comprising such a recombinant vector.

In another aspect, the invention provides a recombinant apoE protein encoded by a nucleic acid molecule comprising a nucleotide sequence derived from a non-human apoE gene, which nucleotide sequence is modified such that it encodes an apoE protein that exhibits domain interaction characteristic of human apoE4.

In another aspect, the invention provides a method of identifying an agent for treating an apoE-associated neurological disorder, comprising: contacting the gene-targeted mouse of the invention with a test agent; and b) determining the effect of the test agent on apoE4 activity. In some aspects, the apoE4 activity tested is apoE4 domain interaction.

In another aspect, the invention provides a method of identifying an agent that reduces a phenomenon associated with Alzheimer's disease (AD), comprising: contacting the gene-targeted mouse of the invention with a test agent; and b) determining the effect of the test agent on a phenomenon associated with AD. In some of these aspects, the phenomenon associated with AD is selected from the group consisting of amyloid deposits, neuronal cell loss, and neurofibrillary tangles.

In another aspect, the invention provides a method of identifying an agent that reduces serum cholesterol levels in an individual, comprising: contacting the gene-targeted mouse of the invention with a test agent; and b) determining the effect of the test agent on a serum cholesterol level in the mouse.

In another aspect, the invention provides a method of identifying an agent for reducing the risk of coronary artery disease, comprising: contacting the gene-targeted mouse of the invention with a test agent; and determining the effect, if any, on plaque deposition on a wall of a coronary artery.

In another aspect, the invention provides method of identifying an agent that reduces apoE4 domain interaction, comprising: contacting a recombinant apoE protein of the invention with a test agent; and determining the effect, if any, on an apoE4-associated activity. In some of these aspects, the determination is by an emulsion binding assay.
 

Claim 1 of 6 Claims

1. A gene-targeted mouse whose genome comprises a modified endogenous apolipoprotein E (apoE) allele, wherein said modified allele comprises an apoE-encoding nucleic acid under transcriptional control of endogenous ApoE regulatory sequences, wherein the modified allele encodes a modified apoE polypeptide that exhibits domain interaction characteristic of human apolipoprotein E4 (apoE4), wherein the modified apoE polypeptide comprises a Thr.fwdarw.Arg substitution at a position equivalent to amino acid 61 of human apoE4, wherein the gene targeted mouse is homozygous for the modified apoE allele, and wherein the modified apoE polypeptide exhibits preferential binding to lower density lipoproteins when compared to unmodified, wild-type mouse apoE, and wherein the mouse exhibits apoE4-related neurodegeneration.

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