Npp1 fusion proteins

Abstract

The present invention provides a novel fusion polypeptide containing a catalytic domain of NPP1 fused to a targeting moiety, nucleic acids encoding the fusion polypeptide, a vector containing the nucleic acid integrated thereinto, a host cell transformed with the vector and pharmaceutical compositions comprising the fusion polypeptide.

What is claimed is: 
     
         1 . An isolated polypeptide comprising a targeting moiety and a C-terminus catalytic domain of NPP1. 
     
     
         2 . The polypeptide of  claim 1 , wherein said catalytic domain is a pyrophosphatase or phosphodiesterase domain of NPP1. 
     
     
         3 . The polypeptide of  claim 1 , wherein said polypeptide is a recombinant protein. 
     
     
         4 . The polypeptide of  claim 1 , wherein said targeting moiety is chemically linked to said catalytic domain of NPP1. 
     
     
         5 . The polypeptide of  claim 1 , wherein said catalytic domain of NPP1 comprises A205 through L591 of SEQ ID NO:1. 
     
     
         6 . The polypeptide of  claim 5 , wherein said catalytic domain of NPP1 comprises P99 through L591 of SEQ ID NO:1. 
     
     
         7 . The polypeptide of  claim 5 , wherein said catalytic domain of NPP1 comprises A205 through D925 of SEQ ID NO: 1. 
     
     
         8 . The polypeptide of  claim 5 , wherein said catalytic domain of NPP1 comprises P99 through D925 of SEQ ID NO: 1. 
     
     
         9 . The polypeptide of  claim 8 , wherein said polypeptide is a consecutive fragment of the polypeptide having the amino acid sequence of P99 through D925 of SEQ ID NO:1 and containing at least the amino acid resides A205 through L591 of SEQ ID NO:1. 
     
     
         10 . The polypeptide of  claim 5 , wherein said targeting moiety is a peptide comprising at least four negatively charged amino acid residues. 
     
     
         11 . The polypeptide of  claim 5 , wherein said targeting moiety is a peptide comprising between about five and about fifteen negatively charged amino acid residues. 
     
     
         12 . The polypeptide of  claim 5 , wherein said negatively charged amino acid residues comprise at least one aspartic acid or glutamic acid. 
     
     
         13 . The polypeptide of  claim 5 , wherein said negatively charged amino acid residues comprise at least four aspartic acid residues. 
     
     
         14 . The polypeptide of  claim 5 , wherein said negatively charged amino acid residues comprise at least four glutamic acid residues. 
     
     
         15 . The polypeptide of  claim 5 , wherein said negatively charged amino acid residues comprise eight consecutive aspartic acid residues. 
     
     
         16 . The polypeptide of  claim 5 , wherein said targeting moiety is fused to either N-terminus or C-terminus of said catalytic domain of NPP1. 
     
     
         17 . The polypeptide of  claim 5 , wherein said targeting moiety is fused to N-terminus and C-terminus of said catalytic domain of NPP1. 
     
     
         18 . The polypeptide of  claim 12 , wherein said polypeptide further comprises a polypeptide linker between said targeting moiety and said catalytic domain of NPP1. 
     
     
         19 . The polypeptide of  claim 1 , wherein said polypeptide further comprises an Fc region of an immunoglobulin. 
     
     
         20 . The polypeptide of  claim 1 , wherein said polypeptide further comprises a signal peptide. 
     
     
         21 . The polypeptide of  claim 1 , wherein said polypeptide forms a homodimer. 
     
     
         22 . The polypeptide of  claim 1 , wherein said polypeptide protein is a monomer. 
     
     
         23 . An isolated nucleic acid encoding the polypeptide of  claim 1 . 
     
     
         24 . A replication or expression vector carrying the isolated nucleic acid of  claim 23 . 
     
     
         25 . A host cell transformed with the replication or expression vector according to  claim 23 . 
     
     
         26 . The host cell of  claim 25 , wherein said host cell is selected from the group consisting of CHO cell, HEK293 cell, or COS cell. 
     
     
         27 . The host cell of  claim 20 , wherein said host cell is an avian tumor cell. 
     
     
         28 . A transgenic animal producing the polypeptide of  claim 1 . 
     
     
         29 . The transgenic animal of  claim 28 , wherein said transgenic animal is mammalian or avian. 
     
     
         30 . A method producing said polypeptide of  claim 1  by an avian cell comprising culturing an avian cell transfected with at least one expression vector comprising a transcription unit having a nucleotide sequence encoding said polypeptide of  claim 1  operably linked to a promoter and a transcriptional terminator, and wherein the cultured avian cell produces said polypeptide. 
     
     
         31 . A method for producing said polypeptide of  claim 1  in an egg of a chicken, the method comprising:
 (a) providing an avian leukosis viral vector comprising a nucleic acid sequence encoding said polypeptide, and a promoter operably linked to said sequence, wherein said promoter drives expression of the encoding sequence in the chicken oviduct; 
 (b) introducing said vector into chicken stage X embryonic cells; 
 (c) incubating said embryonic cells under conditions conducive to hatching live chicks; 
 (d) nurturing growth of a mature chimeric chicken from said chicks; 
 (e) mating said chimeric chicken, either naturally or via artificial insemination, with a non-transgenic chicken; 
 (f) identifying a transgenic chicken by screening the progeny of step (e) for germ line incorporation of the protein encoding sequence; and 
 (g) mating the transgenic progeny with non-transgenic chickens to produce eggs containing the exogenous protein. 
 
     
     
         32 . A method of treating a disorder in a subject in need thereof comprising administering an effective amount of a pharmaceutical composition comprising the polypeptide of  claim 1 . 
     
     
         33 . The method of  claim 32 , wherein said subject is a mammal. 
     
     
         34 . The method of  claim 33 , wherein said mammal is a human. 
     
     
         35 . The method of  claim 32 , wherein said disorder is arterial calcification, insulin resistance, hypophosphatemic rickets, or ossification of posterior longitudinal ligament of spine. 
     
     
         36 . The method of  claim 35 , wherein said disorder is arterial calcification. 
     
     
         37 . The method of  claim 36 , wherein the treatment reduces calcification in the artery. 
     
     
         38 . The method of  claim 36 , wherein the arterial calcification is generalized arterial calcification of infancy.