NPP1 FUSION PROTEINS
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.
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.