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 an NPP1 component fused to a targeting moiety wherein the NPP1 component is a truncated NPP1 comprising the cysteine rich region and a catalytic domain having pyrophosphatase and/or phosphodiesterase activity but having the N-terminal cytosolic and the transmembrane domains removed, wherein the targeting moiety is capable of enhancing the targeting of the polypeptide to a site of calcification, and wherein said targeting moiety is fused to the NPP1 component.
2 . (canceled)
3 . (canceled)
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 NPP1 component comprises
a) A205 through L591 of SEQ ID NO:1,
b) P99 through L591 of SEQ ID NO:1,
c) A205 through D925 of SEQ ID NO: 1, or
d) P99 through D925 of SEQ ID NO: 1.
6 - 8 . (canceled)
9 . The polypeptide of claim 1 , 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
a) at least four negatively charged amino acid residues, or
b) between about five and about fifteen negatively charged amino acid residues.
11 . (canceled)
12 . The polypeptide of claim 10 , wherein said negatively charged amino acid residues comprise
a) at least one aspartic acid or glutamic acid,
b) at least four aspartic acid residues,
c) at least four glutamic acid residues, or
d) eight consecutive aspartic acid residues.
13 - 15 . (canceled)
16 . The polypeptide of claim 5 , wherein said targeting moiety is fused to either N-terminus or C-terminus of said NPP1 component or is fused to N-terminus and C-terminus of said NPP1 component.
17 . (canceled)
18 . The polypeptide of claim 12 , wherein said polypeptide further comprises
a) a polypeptide linker between said targeting moiety and said NPP1 component,
b) an Fc region of an immunoglobulin, or
c) a signal peptide.
19 . (canceled)
20 . (canceled)
21 . The polypeptide of claim 1 , wherein said polypeptide forms a homodimer.
22 . The polypeptide of claim 1 , wherein said polypeptide 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, avian tumor cell or COS cell.
27 . (canceled)
28 . A transgenic animal producing the polypeptide of claim 1 .
29 . (canceled)
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 . (canceled)
34 . (canceled)
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 . (canceled)
37 . (canceled)
38 . The method of claim 35 , wherein the arterial calcification is generalized arterial calcification of infancy.