Bone delivery conjugates and method of using same to target proteins to bone
View Patent ↗A bone delivery conjugate having a structure selected from the group consisting of: A) X-D n -Y-protein-Z; and B) Z-protein-Y-D n -X, wherein X is absent or is an amino acid sequence of at least one amino acid; Y is absent or is an amino acid sequence of at least one amino acid; Z is absent or is an amino acid sequence of at least one amino acid; and D n is a poly aspartate wherein n=10 to 16. Compositions comprising same and methods of use thereof.
1. An isolated nucleic acid molecule encoding a protein comprising a structure selected from the group consisting of:
(i) X-D n -Y-sALP-Z; and
(ii) Z-sALP-Y-D n -X,
wherein X is absent or is an amino acid sequence of at least one amino acid;
Y is absent or is an amino acid sequence of at least one amino acid;
Z is absent or is an amino acid sequence of at least one amino acid;
D n is a poly aspartate wherein n=10 to 16;
said sALP is a soluble alkaline phosphatase; and
said protein is catalytically competent to allow formation of hydroxyapatite crystals in bone.
2. The nucleic acid molecule of claim 1 , wherein said structure is: Z-sALP-Y-D n -X.
3. The nucleic acid molecule of claim 2 , wherein n=10.
4. The nucleic acid molecule of claim 3 , wherein said sALP is a secreted soluble form of an alkaline phosphatase comprising an amino acid sequence encoded by the sequence of SEQ ID NO: 5.
5. The nucleic acid molecule of claim 4 , wherein Y is an amino acid sequence of at least one amino acid and X and Z are absent from said protein.
6. The nucleic acid molecule of claim 1 , wherein said sALP is capable of catalyzing the cleavage of inorganic pyrophosphate (PPi).
7. A recombinant vector comprising the nucleic acid molecule of claim 1 .
8. A recombinant host cell comprising the nucleic acid molecule of claim 1 .
9. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of:
(a) the nucleic acid sequence of SEQ ID NO: 7;
(b) a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 8;
(c) a nucleic acid sequence completely complementary to the nucleic acid sequence of (a) or (b); and
(d) a nucleic acid sequence which hybridizes under high stringency conditions to any of the nucleic acid sequences of (a), (b) or (c), wherein said high stringency conditions comprise pre-hybridization and hybridization in 6×SSC, 5×Denhardt's reagent, 0.5% SDS and 100 mg/ml of denatured fragmented salmon sperm DNA at 68° C.; and washes in 2×SSC and 0.5% SDS at room temperature for 10 minutes; in 2×SSC and 0.1% SDS at room temperature for 10 minutes; and in 0.1×SSC and 0.5% SDS at 65° C. three times for five minutes;
wherein said nucleic acid molecule or its complement encodes a protein that is catalytically competent to allow formation of hydroxyapatite crystals in bone.
10. The nucleic acid molecule of claim 9 , said nucleic acid molecule comprising a nucleic acid sequence which hybridizes under high stringency conditions to the complement of the nucleic acid sequence of SEQ ID NO: 7 or to the complement of a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 8, wherein said high stringency conditions comprise pre-hybridization and hybridization in 6×SSC, 5×Denhardt's reagent, 0.5% SDS and 100 mg/ml of denatured fragmented salmon sperm DNA at 68° C.; and washes in 2×SSC and 0.5% SDS at room temperature for 10 minutes; in 2×SSC and 0.1% SDS at room temperature for 10 minutes; and in 0.1×SSC and 0.5% SDS at 65° C. three times for five minutes.
11. The nucleic acid molecule of claim 9 , wherein said nucleic acid molecule encodes a protein comprising a structure selected from the group consisting of:
(i) X-D n -Y-sALP-Z; and
(ii) Z-sALP-Y-D n -X,
wherein X is absent or is an amino acid sequence of at least one amino acid;
Y is absent or is an amino acid sequence of at least one amino acid;
Z is absent or is an amino acid sequence of at least one amino acid;
D n is a poly aspartate wherein n=10 to 16; and
said sALP is a soluble alkaline phosphatase.
12. The nucleic acid molecule of claim 11 , wherein said structure is: Z-sALP-Y-D n -X.
13. The nucleic acid molecule of claim 12 , wherein n=10.
14. The nucleic acid molecule of claim 13 , wherein Y is an amino acid sequence of at least one amino acid and X and Z are absent from said protein.
15. The nucleic acid molecule of claim 11 , wherein said sALP is capable of catalyzing the cleavage of inorganic pyrophosphate (PPi).
16. A recombinant vector comprising the nucleic acid molecule of claim 11 .
17. A recombinant host cell comprising the nucleic acid molecule of claim 11 .