Delivery of therapeutic agents to the bone

Abstract

This invention relates to compositions and methods of delivering therapeutic agents to bone. More specifically, the invention relates to endowing a large molecule vectors i.e., adeno virus, retrovirus, liposomes, micelles, natural and synthetic polymers, or combinations thereof, with the ability to target bone tissue in vivo and with improved stability in the blood, by attaching multiple copies of acid amino acid peptides. One preferred embodiment of the invention relates to endowing an adeno-associated virus (AAV) vector with the ability to target bone-tissue in vivo and improve its stability, by the addition of multiple acidic amino acid peptides attached to the capsid of the viral vector.

1 . An insolated adeno-associated virus-2 comprising:
 a) a capsid encoded by adeno-associated virus-2 DNA modified by insertion of acid amino acid polypeptide encoding DNA immediately after the adeno-associated virus-2 initial codon of capsid protein VP2;   b) wherein the acid amino acid polypeptide consist of 4-15 acid amino acids; and   c) wherein the adeno-associated virus-2 targets bone in vivo and is capable of transfecting single stranded DNA into a mammalian cell.   
     
     
         2 . The insolated adeno-associated virus-2 of  claim 1 , wherein the acid amino acid polypeptide consist of 6-10 acid amino acids. 
     
     
         3 . The insolated adeno-associated virus-2 of  claim 1 , wherein the acid amino acid polypeptide consist of 8 acid amino acids. 
     
     
         4 . The insolated adeno-associated virus-2 of  claim 1 , wherein the acid amino acid polypeptide consist of aspartic acid. 
     
     
         5 . The insolated adeno-associated virus-2 of  claim 1 , wherein the acid amino acid polypeptide consist of glutamic acid. 
     
     
         6 . The insolated adeno-associated virus-2 of  claim 1 , wherein the DNA sequence encoding for the acid amino acid polypeptide consists of SEQ ID NO:3. 
     
     
         7 . The insolated adeno-associated virus-2 of  claim 1 , wherein the adeno-associated virus-2 comprises a single stranded DNA encoding an N-acetylgalactosamine-6-sulfate-sulfatase, and wherein the single stranded DNA expresses a physiologically active N-acetylgalactosamine-6-sulfate-sulfatase after transfection into a mammalian cell. 
     
     
         8 . The insolated adeno-associated virus-2 of  claim 1 , wherein the adeno-associated virus-2 comprises a single stranded DNA encoding a tissue non-specific alkaline phosphatase, and wherein the single stranded DNA expresses a physiologically active tissue non-specific alkaline phosphatase after transfection into a mammalian cell. 
     
     
         9 . The insolated adeno-associated virus-2 of  claim 1 , wherein the adeno-associated virus-2 comprises a single stranded DNA encoding a β-glucuronidase, and wherein the single stranded DNA expresses a physiologically active β-glucuronidase after transfection into a mammalian cell. 
     
     
         10 . The insolated adeno-associated virus-2 of  claim 1 , further comprising a pharmaceutical composition. 
     
     
         11 . An insolated adeno-associated virus-2 comprising:
 a) a capsid encoded by adeno-associated virus-2 DNA modified by insertion of SEQ ID NO:3 immediately after the adeno-associated virus-2 initial codon of capsid protein VP2;   b) wherein the adeno-associated virus-2 targets bone in vivo and is capable of transfecting single stranded DNA into a mammalian cell.   
     
     
         12 . The insolated adeno-associated virus-2 of  claim 11 , wherein the adeno-associated virus-2 comprises a single stranded DNA encoding a N-acetylgalactosamine-6-sulfate-sulfatase, and wherein the single stranded DNA expresses a physiologically active N-acetylgalactosamine-6-sulfate-sulfatase after transfection into a mammalian cell. 
     
     
         13 . The insolated adeno-associated virus-2 of  claim 11 , wherein the adeno-associated virus-2 comprises a single stranded DNA encoding a tissue non-specific alkaline phosphatase, and wherein the single stranded DNA expresses a physiologically active tissue non-specific alkaline phosphatase alter transfection into a mammalian cell. 
     
     
         14 . The insolated adeno-associated virus-2 of  claim 11 , wherein the adeno-associated virus-2 comprises a single stranded DNA encoding a β-glucuronidase, and wherein the single stranded DNA expresses a physiologically active β-glucuronidase after transfection into a mammalian cell. 
     
     
         15 . The insolated adeno-associated virus-2 of  claim 11 , further comprising a pharmaceutical composition. 
     
     
         16 . A method of transfecting a gene into a mammalian cell, comprising:
 a) incorporating the gene into the adeno-associated virus-2 of  claim 1 ;   b) contacting the mammalian cell with the composition in a);   c) wherein the gene is expressed in the mammalian cell.   
     
     
         17 . The method of  claim 16 , wherein the mammalian cell is in vitro. 
     
     
         18 . The method of  claim 16 , wherein the mammalian cell is in an animal and the adeno-associated virus-2 is administered intravenously. 
     
     
         19 . The method of  claim 16 , wherein the gene encodes for N-acetylgalactosamine-6-sulfate-sulfatase. 
     
     
         20 . The method of  claim 16 , wherein the adeno-associated virus-2 further comprises a pharmaceutical composition.