Treatment of glycogen storage disease iii

Abstract

The present invention relates to vectors and compositions for the treatment of glycogen storage disease III.

We claim: 
     
         1 . A dual AAV vector system comprising two AAV vectors, wherein:
 a first AAV vector comprises, between 5′ and 3′ AAV ITRs, a first nucleic acid sequence that encodes a N-terminal part of a glycogen debranching enzyme (GDE), and   a second AAV vector comprises, between 5′ and 3′ AAV ITRs, a second nucleic acid sequence that encodes a C-terminal part of said GDE, and   wherein the first and second nucleic acid sequences encoding said GDE comprise a polynucleotide region that permits the production of a full-length GDE protein.   
     
     
         2 . The dual AAV vector system according to  claim 1 , wherein said polynucleotide region is a GDE polynucleotide sequence that overlaps between said first and second nucleic acid sequences. 
     
     
         3 . The dual AAV vector system according to  claim 2 , wherein said polynucleotide sequence that overlaps between said first and second nucleic acid sequences is between about 100 and about 4500 nucleotides. 
     
     
         4 . The dual AAV vector system according to  claim 1 , wherein:
 said first nucleic acid sequence comprises a sequence encoding said N-terminal part of said GDE followed by a splice donor site; and   said second nucleic acid sequence comprises a splice acceptor site followed by a sequence encoding said C-terminal part of said GDE.   
     
     
         5 . The dual AAV vector system according to  claim 4 , wherein:
 the splice donor site in said first nucleic acid sequence is followed by a recombinogenic sequence and the splice acceptor site in said second nucleic acid sequence is preceded by said recombinogenic sequence.   
     
     
         6 . The dual AAV vector system according to  claim 5 , wherein said recombinogenic sequence is:
 an alkaline phosphatase (AP1) fragment; or   an AK, F1 phage recombinogenic sequence.   
     
     
         7 . The dual AAV vector system according to  claim 5 , wherein said recombinogenic sequence is:
 an alkaline phosphatase (AP1) fragment, wherein said AP1 fragment is selected from the sequences shown in SEQ ID NO:1 to 7; or   an AK, F1 phage recombinogenic sequence as shown in SEQ ID NO:8.   
     
     
         8 . The dual AAV vector system according to  claim 5 , wherein said recombinogenic sequence is:
 an alkaline phosphatase (AP1) fragment, wherein said AP1 fragment is as shown in SEQ ID NO:7; or   an AK, F1 phage recombinogenic sequence as shown in SEQ ID NO:8.   
     
     
         9 . The dual AAV vector system according to  claim 1 , wherein said first and second nucleic acid sequences are optimized sequences. 
     
     
         10 . The dual AAV vector system according to  claim 1 , wherein said first nucleic acid sequence is preceded by a promoter optionally followed by an intron, and said second nucleic acid sequence is followed by a polyadenylation signal. 
     
     
         11 . The dual AAV vector system according to  claim 1 , wherein the polynucleotide region is a GDE polynucleotide sequence that overlaps between said first and second nucleic acid sequences, and wherein the nucleic acid sequence encoding the N-terminal part of GDE and the nucleic acid sequence encoding the C-terminal part of GDE are selected from the combinations shown in table 2. 
     
     
         12 . The dual AAV vector system according to  claim 11 , wherein:
 a) the first AAV vector comprises a genome comprising, in the 5′ to 3′ orientation:
 a 5′ ITR; 
 a promoter optionally preceded by an enhancer; 
 optionally, an intron; 
 a nucleic acid sequence encoding a N-terminal part of GDE selected in the group consisting of the nucleic acid sequences encoding a N-terminal part of GDE shown in table 2; and 
 a 3′-ITR; and 
   b) the second AAV vector comprises a genome comprising, in the 5′ to 3′ orientation: 
 - a 5′ ITR; 
 a nucleic acid sequence encoding a C-terminal part of GDE selected in the group consisting of the nucleic acid sequences encoding a C-terminal part of GDE shown in table 2; 
 a polyadenylation signal; and 
 a 3′-ITR. 
   
     
     
         13 . The dual AAV vector system according to  claim 11 , wherein:
 the nucleic acid sequence encoding the N-terminal part of GDE is the nucleotide sequence comprised between nucleotides 1 and 2688 of SEQ ID NO:13 or a corresponding optimized sequence; and   the nucleic acid sequence encoding the C-terminal part of GDE is the nucleotide sequence comprised between nucleotides 1693 and 4599 of SEQ ID NO:13 or a corresponding optimized sequence.   
     
     
         14 . The dual AAV vector system according to  claim 11 , wherein:
 the nucleic acid sequence encoding the N-terminal part of GDE is the nucleotide sequence comprised between nucleotides 1 and 2688 of SEQ ID NO:13 or a corresponding optimized sequence selected from SEQ ID NO:27 or SEQ ID NO:28; and   the nucleic acid sequence encoding the C-terminal part of GDE is the nucleotide sequence comprised between nucleotides 1693 and 4599 of SEQ ID NO:13 or a corresponding optimized sequence selected from SEQ ID NO:29 or SEQ ID NO:30.   
     
     
         15 . The dual AAV vector system according to  claim 11 , wherein:
 the nucleic acid sequence encoding the N-terminal part of GDE is the nucleotide sequence comprised between nucleotides 1 and 1809 of SEQ ID NO:13, or a corresponding optimized sequence selected from nucleotides 1-1809 of SEQ ID NO:25, 27 or 28; and   the nucleic acid sequence encoding the C-terminal part of GDE is the nucleotide sequence comprised between nucleotides 2641 and 4599 of SEQ ID NO:13, or a corresponding optimized sequence selected from nucleotides 949-2907 of SEQ ID NO:26, 29 or 30.   
     
     
         16 . The dual AAV vector system according to  claim 10 , wherein:
 said promoter is a muscle-specific promoter, an ubiquitous promoter or a promoter directing expression in muscle and in liver cells; and/or   said intron is selected in the group consisting of a human beta globin b2 intron, a FIX intron and a chicken beta-globin intron, wherein said intron is optionally a modified intron selected from a modified HBB2 intron of SEQ ID NO:9, a modified FIX intron of SEQ ID NO:10, or a modified chicken beta-globin intron of SEQ ID NO:11; and/or   said polyadenylation signal is selected from the human beta globin polyadenylation signal, the bovine growth hormone polyadenylation signal, the SV40 polyadenylation signal, or another naturally occurring or artificial polyadenylation signal.   
     
     
         17 . The dual AAV system according to  claim 1 , wherein each of said first and second AAV vectors is an AAV vector with an AAV-derived capsid. 
     
     
         18 . A cell transduced with the dual AAV system according to  claim 1 . 
     
     
         19 . A composition comprising, in a pharmaceutically acceptable carrier, the dual AAV system according to  claim 1  or a cell comprising said dual AAV system. 
     
     
         20 . A method of treating glycogen storage disease III comprising administering the dual AAV system according to  claim 1  or a cell comprising said dual AAV system to a subject having glycogen storage disease III. 
     
     
         21 . An AAV vector corresponding to the first or second AAV vector of the dual AAV vector system according to  claim 1 .