Gene-targeted animal model of apolipoprotein E4 domain interaction and uses thereof

Abstract

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.

1 . A gene-targeted non-human animal comprising a modified endogenous apolipoprotein E (apoE) allele, wherein said modified allele comprises an apoE-encoding nucleic acid under transcriptional control of endogenous regulatory sequences, and wherein the modified allele encodes a modified apoE that exhibits domain interaction characteristic of human apolipoprotein E4 (apoE4).  
   
   
       2 . The non-human animal of  claim 1 , wherein the modified apoE comprises a Thr→Arg substitution at a position equivalent to amino acid 61 of human apoE4.  
   
   
       3 . The non-human animal of  claim 1 , wherein the gene-targeted non-human animal is homozygous for the modified apoE allele.  
   
   
       4 . The non-human animal of  claim 1 , wherein the gene-targeted animal is a mouse.  
   
   
       5 . An isolated non-human cell comprising a modified endogenous apolipoprotein E (apoE) allele, wherein said modified endogenous allele is under transcriptional control of endogenous regulatory sequences, and wherein the modified allele encodes a modified apoE that exhibits domain interaction characteristic of human apolipoprotein E4 (apoE4).  
   
   
       6 . The non-human cell of  claim 5 , wherein the modified apoE comprises a Thr→Arg substitution at a position equivalent to amino acid 61 of human apoE4.  
   
   
       7 . The non-human cell of  claim 5 , wherein the cell is homozygous for the modified apoE allele.  
   
   
       8 . The non-human cell of  claim 5 , wherein the cell is a mouse cell.  
   
   
       9 . An isolated nucleic acid molecule comprising a nucleotide sequence derived from a non-human apolipoprotein E (apoE) gene, which nucleotide sequence is modified such that it encodes a protein comprising a Thr→Arg substitution at a position equivalent to amino acid 61 of human apoE4.  
   
   
       10 . A recombinant vector comprising the nucleic acid of  claim 9 .  
   
   
       11 . A recombinant host cell comprising the vector of  claim 10 .  
   
   
       12 . A recombinant apolipoprotein E (apoE) protein encoded by a nucleic acid comprising a nucleotide sequence derived from a non-human apoE gene, which nucleotide sequence is modified such that it encodes a protein that exhibits domain interaction characteristic of human apolipoprotein E4 (apoE4).  
   
   
       13 . The recombinant protein of  claim 12 , wherein the recombinant protein comprises a Thr→Arg substitution at a position equivalent to amino acid 61 of human apoE4.  
   
   
       14 . A method of identifying an agent that reduces a phenomenon associated with Alzheimer's disease (AD), the method comprising: 
 a) contacting the gene-targeted non-human animal of  claim 1  with a test agent; and    b) determining the effect of the test agent on a phenomenon associated with AD.    
   
   
       15 . The method of  claim 15 , wherein the phenomenon associated with AD is selected from the group consisting of amyloid deposits, neuronal cell loss, and neurofibrillary tangles.  
   
   
       16 . A method for identifying an agent that reduces apolipoprotein E4 domain interaction, the method comprising: 
 a) contacting the recombinant protein of  claim 12  with a test agent; and    b) determining the effect of the test agent on domain interaction.    
   
   
       17 . The method of  claim 16 , wherein said determining comprises determining binding of the recombinant apoE to tau.  
   
   
       18 . The method of  claim 16 , wherein said determining comprises determining the effect of the agent on binding to VLDL.  
   
   
       19 . A method of identifying an agent that reduces the risk of heart disease, comprising: 
 a) contacting the non-human animal of  claim 1  with a test agent; and    b) determining the effect, if any, on apoE activity.