Methods and compositions for liver specific delivery of therapeutic molecules using recombinant AAV vectors

Abstract

Provided are methods for selectively expressing therapeutic molecules, such as secretory proteins, antisense molecules and ribozymes, in the liver. The methods find use in treating hepatic diseases or conditions. The methods also find use in treating any disease or condition in which systemic administration of the therapeutic substance, for example, a secretory protein, is desired. The methods involve administering to a mammalian patient having a need for liver expression of a therapeutic molecule an AAV vector containing a therapeutically effective amount of the therapeutic molecule. Also provided are novel vectors employable in these methods.

What is claimed is:  
     
         1 . A method for realizing liver specific delivery of a therapeutic molecule in a mammalian patient comprising administering to said mammalian patient a therapeutically effective amount of an AAV vector containing said therapeutic molecule.  
     
     
         2 . A method according to  claim 1  wherein said therapeutic molecule is a nucleic acid sequence encoding a secretory protein, an antisense molecule or a ribozyme.  
     
     
         3 . A method according to  claim 1  wherein the AAV vector is selected from the group consisting of pD-5, pD-10, pD-15, pD-20.  
     
     
         4 . A method according to  claim 1  wherein said AAV vector is administered by intravenous or intraportal injection.  
     
     
         5 . A method according to  claim 1  wherein said AAV vector is administered ex vivo.  
     
     
         6 . A method according to  claim 1  wherein said AAV vector is administered by direct injection.  
     
     
         7 . A method according to  claim 1  wherein said therapeutic molecule is a nucleic acid encoding the LDL receptor.  
     
     
         8 . A method according to  claim 1  wherein said AAV vector additionally contains a liver specific promoter.  
     
     
         9 . A method according to  claim 8  wherein said AAV vector additionally contains a liver specific enhancer.  
     
     
         10 . A method according  claim 8  wherein said promoter is selected from the group consisting of the hepatitis B virus X gene promoter, the hepatitis B virus core protein promoter, the AFP gene promoter, the albumin gene promoter, the α-1 antitrypsin gene promoter, the fibrinogen gene promoter, the APO-A1 gene promoter and the promoter genes for liver transference enzymes.  
     
     
         11 . A method according to  claim 10  wherein said AAV vector additionally contains a liver specific enhancer.  
     
     
         12 . A hybrid helper AAV vector comprising a DNA sequence encoding hepatitis B virus surface antigen or a functional fragment thereof linked to a DNA sequence encoding the AAV VP-1 protein to form a chimeric DNA sequence.  
     
     
         13 . A recombinant AAV vector comprising two AAV ITRs (inverted terminal repeats) in which the native D-sequences of each of said ITRs are modified by the substitution of nucleotides such that at least 5 native nucleotides and up to 18 native nucleotides are retained and the remaining nucleotides of the D-sequence are deleted or replaced with non-native nucleotides.  
     
     
         14 . A recombinant AAV vector according to  claim 13  wherein said at least 5 native nucleotides are 5′ CTCCA 3′.  
     
     
         15 . A recombinant AAV vector comprising two AAV ITRs (inverted terminal repeats) in which the native D-sequences of each of said ITRs are modified by the substitution of nucleotides such that at least 10 native nucleotides up to 18 native nucleotides are retained and the remaining nucleotides of the D-sequence are deleted or replaced with non-native nucleotides.  
     
     
         16 . A recombinant AAV vector comprising two AAV ITRs (inverted terminal repeats) in which the native D-sequences of each of said ITRs are modified by the substitution of nucleotides such that 10 native nucleotides are retained and the remaining nucleotides of the D-sequence are deleted or replaced with non-native nucleotides.  
     
     
         17 . A recombinant AAV vector according to  claim 16  wherein said 10 native nucleotides comprise nucleotides 5′ CTCCA 3′ and five other native nucleotides of said D-sequence.  
     
     
         18 . A recombinant AAV vector selected from the group consisting of pD-5, pD-10, pD-15 and pD-20.