Models of prion disease

Abstract

The present invention provides a novel PrP protein, and nucleic acids encoding this protein, where the PrP protein is characterized in vivo by 1) incomplete glycosylation relative to glycosylation of wild-type PrP C and 2) proper cellular localization, i.e. an ability to be transported to the cell surface. This novel, under-glycosylated PrP, unlike its normal cellular counterpart, can easily be converted into a protease-resistant isoform by incubation with infectious prions. The invention further provides systems for the study of prion disorders and methods of using these systems, e.g. the study of the mechanical processes in progression of prion-mediated disease or the identification of new therapeutic agents for treatment of prion-mediated disorders. In such systems, protease-resistant under-glycosqvated PrP is generated de novo and can be detected by standard immunoblot techniques.

1 - 22 . (Canceled)  
     
     
         23 . An isolated PrP protein characterized by: 
 an amino acid sequence different from wild-type PrP;    a reduction in glycosylation relative to wild-type PrP; and    cellular localization sufficient to allow prion infection.    
     
     
         24 . The PrP protein of  claim 23 , wherein the amino acid sequence is different at one or more positions and wherein an asparagine in at least one glycosylation site is substituted with a different amino acid.  
     
     
         25 . The PrP protein of  claim 24 , wherein the asparagine is substituted by a glutamine.  
     
     
         26 . An isolated nucleic acid sequence or complement thereof encoding a PrP protein characterized by a reduction in glycosylation relative to wild-type PrP and cellular localization sufficient to allow prion infection.  
     
     
         27 . An assay, comprising the steps of: 
 contacting a sample putatively comprising prions with a recombinant cell line, wherein the cell line has operatively inserted therein an exogenous nucleic acid sequence encoding a protein characterized by an amino acid sequence different from wild-type PrP, a reduction in glycosylation relative to wild type PrP, and cellular localization sufficient to allow prion infection; and determining the presence of prions in the sample after contacting the cell line.    
     
     
         28 . The assay of  claim 27  wherein the determining is carried out with an antibody.  
     
     
         29 . The assay of  claim 27  wherein the determining is carried out with a transgenic mouse.  
     
     
         30 . A non-human transgenic animal, comprising: 
 a genome artificially altered to comprise an exogenous nucleic acid encoding a protein of  claim 24  and an inducer sequence which affects expression thereof.    
     
     
         31 . The transgenic animal of  claim 30 , wherein the animal is selected from the group consisting of a mouse, a rat and a hamster and the genetically diverse animal is selected from the group consisting of a human, cow, sheep, horse, pig, chicken, dog or cat.  
     
     
         32 . The transgenic animal of  claim 30 , wherein the animal is a mouse and the inducer system comprises an inducible transactivator sequence and an operator sequence wherein both are operably linked to the exogenous nucleic acid in a manner so as to control expression of the exogenous PrP gene.  
     
     
         33 . A method for detecting prion infectivity of a sample, the method comprising the steps of: 
 obtaining sample from an animal to be tested;    inoculating the transgenic animal of  claim 32  with the sample; and    observing the transgenic animal for symptoms of prion disease.    
     
     
         34 . The method of  claim 33 , wherein the animal to be tested is selected from the group consisting of human, cow, sheep, pig, horse, cat, dog, turkey or chicken, and the transgenic animal is selected from the group consisting of: mice, rats, rabbits, hamsters and guinea pigs.  
     
     
         35 . A method for identifying a biologically active agent that modulates infection or progression of a prion-mediated disorder, the method comprising: 
 combining a candidate agent with a detection system selected from the group consisting of:    (a) a mammalian PrP polypeptide characterized by a reduction in glycosylation relative to the wild-type PrP polypeptide and proper cellular localization;    (b) a cell comprising a nucleic acid encoding a mammalian PrP polypeptide characterized by a reduction in glycosylation relative to the wild-type PrP polypeptide and proper cellular localization;    (c) a non-human transgenic animal model having a genome comprising a mammalian PrP polypeptide characterized by a reduction in glycosylation relative to the wild-type PrP polypeptide and proper cellular localization;    inoculating the detection system with a prion preparation; and    determining the amount of protease resistant prions;    wherein the level of protease-resistant prions is indicative of the effect of the candidate agent on prion-mediated disorders.    
     
     
         36 . A process for diagnosing a patient suspected of having a prion-mediated disorder comprising the steps of: 
 a) obtaining cells or body fluids from the patient;    b) transfecting said cells with a PrP nucleic acid of  claim 26;  and    c) detecting prions in the cells.    
     
     
         37 . A recombinant neuroblastoma cell (N2a cell), wherein the N2a cell comprises an operative pSPOX expression vector comprising an exogenous PrP gene mutagenesized to contain a double mutation where two glycosylation sites are substituted from asparagene to glutamine (N180Q/N196Q) to allow prion infection.