Bone delivery conjugates and method of using same to target proteins to bone

Abstract

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 - 11 . (canceled) 
     
     
         12 . A bone delivery conjugate comprising sALP-Y-D n , wherein
 Y is an amino acid sequence of at least one amino acid;   D n  is a poly aspartate wherein n=10; and   sALP is a secreted soluble tissue non-specific alkaline phosphatase of SEQ ID NO: 6;   wherein said bone delivery conjugate is catalytically competent to allow formation of hydroxyapatite crystals in bone.   
     
     
         13 . The bone delivery conjugate of  claim 12 , wherein said sALP is capable of catalyzing the cleavage of inorganic pyrophosphate (PPi). 
     
     
         14 . The bone delivery conjugate of  claim 13 , wherein said bone delivery conjugate is encoded by a nucleic acid molecule which hybridizes under high stringency conditions to a nucleic acid molecule having a nucleic acid sequence complementary to at least one of SEQ ID NO: 5 and SEQ ID NO: 7. 
     
     
         15 . The bone delivery conjugate of  claim 14 , 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. 
     
     
         16 . A composition comprising the bone delivery conjugate of  claim 12  and at least one pharmaceutically acceptable carrier. 
     
     
         17 . The composition of  claim 16 , wherein said sALP is capable of catalyzing the cleavage of inorganic pyrophosphate (PPi). 
     
     
         18 . A method of delivering a protein to bone tissue of a mammal to treat a bone related undesired condition in said mammal, comprising administering to said mammal an effective amount of a bone delivery conjugate comprising sALP-Y-D n , wherein
 Y is an amino acid sequence of at least one amino acid;   D n  is a poly aspartate wherein n=10;   sALP is a secreted soluble tissue non-specific alkaline phosphatase of SEQ ID NO: 6; and   said bone delivery conjugate is catalytically competent to allow formation of hydroxyapatite crystals in bone.   
     
     
         19 . The method of  claim 18 ,
 wherein said bone delivery conjugate is encoded by a nucleic acid molecule which hybridizes under high stringency conditions to a nucleic acid molecule having a nucleic acid sequence complementary at least one of SEQ ID NO: 5 and SEQ ID NO: 7;   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.   
     
     
         20 . The method of  claim 18 , wherein said mammal is a human, and the bone delivery conjugate further comprises at least one pharmaceutically acceptable carrier. 
     
     
         21 . The method of  claim 20 , wherein said effective amount is administered to said human more than once at an interval ranging from daily to weekly. 
     
     
         22 . The method of  claim 21 , wherein said effective amount is from about 1 to about 50 milligrams per kilogram of body weight. 
     
     
         23 . The method of  claim 22 , wherein said effective amount is from about 1 to about 10 milligrams per kilogram of body weight. 
     
     
         24 . The method of  claim 23 , wherein said effective amount is about 1 milligram per kilogram of body weight. 
     
     
         25 . The method of  claim 20 , wherein said bone related undesired condition is at least one of hypophosphatasia (HPP), hypophosphatemic rickets, osteomalacia, a lack or insufficient amount of functional alkaline phosphatase, impaired skeletal mineralization, growth retardation, lower extremity deformity, hyperparathyroidism, parathyroidectomy, hypercalciuria, hypercalcemia, and nephrocalcinosis. 
     
     
         26 . The method of  claim 25 , wherein the HPP is perinatal HPP, infantile HPP, childhood HPP, or adult HPP. 
     
     
         27 . The method of  claim 20 , wherein the pharmaceutically acceptable carrier is saline. 
     
     
         28 . The method of  claim 18 , wherein said sALP is capable of catalyzing the cleavage of inorganic pyrophosphate (PPi). 
     
     
         29 . The composition of  claim 16 , wherein the pharmaceutically acceptable carrier is saline.