Nucleic acid constructs and gene therapy vectors for use in the treatment of wilson disease

Abstract

The invention relates to nucleic acid constructs and gene therapy vectors that comprise an ATP7B variant for use in the treatment of conditions associated with a deficiency or dysfunction of Copper-transporting ATPase 2, and particularly of Wilson's disease. An AAV vector devised according to the invention significantly reduced urine Cu excretion, and liver Cu content in Wilson's disease mice treated with the vector, while ceruloplasmin activity was significantly restored. On the other hand, the administration of the vector resulted in the normalization of serum transaminases' levels and of liver histology, together with a marked reduction of the inflammatory infiltrate.

1 . A method for the treatment of a condition caused by a deficiency or dysfunction of Copper-transporting ATPase 2 in a patient that comprises administering to the patient a therapeutically effective amount of an expression vector comprising a nucleic acid construct, and a pharmaceutically acceptable carrier, wherein the nucleic acid construct comprises:
 a) a nucleotide sequence of a eukaryotic promoter;   b) a nucleotide sequence encoding a truncated Copper-transporting ATPase 2 in which the N-terminal Heavy-Metal-Associated sites HMA 1, HMA 2, HMA 3, and HMA 4 are not present and wherein HMA 5 and HMA 6 are present; and   c) a polyadenylation signal sequence.   
     
     
         2 . The method according to  claim 1 , wherein the condition caused by a deficiency or dysfunction of Copper-transporting ATPase 2 is Wilson disease. 
     
     
         3 . The method according to  claim 1 , wherein the N-terminal Heavy-Metal-Associated sites HMA 1, HMA 2, HMA 3, and HMA 4 comprise amino acids 57 to 486 of SEQ ID NO:2. 
     
     
         4 . The method according to  claim 1 , wherein the truncated Copper-transporting ATPase2 comprises the amino acid sequence of SEQ ID NO:7. 
     
     
         5 . The method according to  claim 1 , wherein the nucleic acid construct further comprises 5′ITR and 3′ITR sequences of a virus. 
     
     
         6 . The method according to  claim 5 , wherein the 5′ITR and 3′ITR sequences are of an adeno-associated virus (AAV). 
     
     
         7 . The method according to  claim 6 , wherein the 5′ITR and 3′ITR sequences of an AAV are of a serotype selected from the group consisting of AAV1, AAV2, and AAV4. 
     
     
         8 . The method according to  claim 1 , wherein the expression vector is comprised within a recombinant AAV virion (rAAV). 
     
     
         9 . The method according to  claim 1 , wherein the nucleotide sequence encoding the truncated Copper-transporting ATPase 2 is selected from the group consisting of:
 a) nucleotides 473 through 3580 of the nucleotide sequence of SEQ ID NO:6;   b) the nucleotide sequence of SEQ ID NO:8;   c) a nucleotide sequence wherein at least 827 of the codons encoding the truncated Copper-transporting ATPase 2 are identical to the codons of SEQ ID NO:8; and   d) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:7.   
     
     
         10 . The method according to  claim 1 , wherein the nucleotide sequence of the eukaryotic promoter is a nucleotide sequence of the α1-antitrypsin gene promoter, or a chimeric promoter sequence that comprises an al-antitrypsin gene promoter sequence combined with an albumin gene enhancer element. 
     
     
         11 . The method according to  claim 1 , wherein the nucleotide sequence of the eukaryotic promoter consists of nucleotides 156 through 460 of SEQ ID NO:1 (AAT), or consists of the nucleotide sequence of SEQ ID NO:5 (EalbPa1AT). 
     
     
         12 . The method according to  claim 1 , wherein the expression vector comprises a nucleic acid construct comprising:
 a) a nucleotide sequence of a eukaryotic promoter, consisting of:
 nucleotides 156 through 460 of SEQ ID NO:1 (AAT), or 
 the nucleotide sequence of SEQ ID NO:5 (EalbPa1AT); 
   b) a nucleotide sequence encoding a truncated Copper-transporting ATPase 2 in which the N-terminal Heavy-Metal-Associated sites HMA 1, HMA 2, HMA 3, and HMA 4 are not present and wherein HMA 5 and HMA 6 are present;   c) a polyadenylation signal sequence; and   d) a 5′ ITR sequence and a 3′ ITR sequence of an adeno-associated virus (AAV).   
     
     
         13 . The method according to  claim 1 , wherein the expression vector comprises a nucleic acid construct comprising:
 a) a nucleotide sequence of a eukaryotic promoter;   b) a nucleotide sequence encoding a truncated Copper-transporting ATPase 2 in which the N-terminal Heavy-Metal-Associated sites HMA 1, HMA 2, HMA 3, and HMA 4 are not present and wherein HMA 5 and HMA 6 are present, wherein the nucleotide sequence encoding the Copper-transporting ATPase 2 is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:7;   c) a polyadenylation signal sequence; and   d) a 5′ ITR sequence and a 3′ ITR sequence of an adeno-associated virus (AAV).   
     
     
         14 . The method according to  claim 1 , wherein the therapeutically effective amount of the expression vector is administrated intravenously or by intrahepatic injection. 
     
     
         15 . The method according to  claim 8 , wherein the rAAV virion is administered to the patient in an amount of or at a dose of 5×10 11  to 1×10 14  viral genomes/kg. 
     
     
         16 . The method according to  claim 1 , wherein the expression vector is comprised within a recombinant AAV virion (rAAV) comprising a capsid protein of an AAV. 
     
     
         17 . The method according to  claim 16 , wherein the capsid protein of the AAV is of a serotype selected from the group consisting of AAV1, AAV3, AAVS, AAV7, AAV8, AAV9 and AAV10. 
     
     
         18 . The method according to  claim 16 , wherein the rAAV virion comprises a capsid protein of an AAV8 or AAV3 serotype. 
     
     
         19 . The method according to  claim 16 , wherein the rAAV virion comprises a capsid protein of an AAV3 serotype. 
     
     
         20 . The method according to  claim 16 , wherein the rAAV virion comprises a capsid protein of an AAV3B serotype. 
     
     
         21 . The method according to  claim 16 , wherein the 5′ ITR and 3′ ITR sequences of the nucleic acid construct are of an AAV2 serotype and the capsid protein is of an AAV3 serotype. 
     
     
         22 . The method according to  claim 16 , wherein the rAAV virion comprises:
 a nucleic acid construct comprising:
 a) a nucleotide sequence of an al-antitrypsin gene promoter consisting of nucleotides 156 through 460 of SEQ ID NO:1; 
 b) a nucleotide sequence encoding a truncated Copper-transporting ATPase 2 in which the N-terminal Heavy-Metal-Associated sites HMA 1, HMA 2, HMA 3, and HMA 4 are not present and HMA 5 and HMA 6 are present, and wherein the N-terminal Heavy-Metal-Associated sites HMA 1, HMA 2, HMA 3, and HMA 4 comprise amino acids 57 to 486 of SEQ ID NO:2; 
 c) a polyadenylation signal sequence; and 
 d) a 5′ ITR sequence and a 3′ ITR sequence of AAV2; and 
   a capsid protein of AAV3B.   
     
     
         23 . The method according to  claim 16 , wherein the rAAV virion comprises:
 a nucleic acid construct comprising:
 a) a nucleotide sequence of an α1-antitrypsin gene promoter consisting of nucleotides 156 through 460 of SEQ ID NO:1; 
 b) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:7; 
 c) a polyadenylation signal sequence; and 
 d) a 5′ ITR sequence and a 3′ ITR sequence of AAV2; and 
   a capsid protein of AAV3B.   
     
     
         24 . The method according to  claim 23 , wherein the rAAV virion is administered to the patient in an amount of or at a dose of 5×10 11  to 1×10 14  viral genomes/kg.