Compositions and methods for treating mpsi

Abstract

A vector have an expression cassette containing have a hIDUA gene has a sequence of SEQ ID NO: 1 (FIG. 1 ) or a sequence at least about 95% identical thereto which encodes a functional human alpha-L-iduronidase is provided. The vector may be a production vector or a rAAV8. Also provided are compositions containing these vectors and methods of treating MPSI and the symptoms associated with Hurler, Hurler-Scheie and Scheie syndromes.

1 . A method for producing recombinant adeno-associated virus (rAAV) particles in host cells comprising: culturing host cells which contain: at least (a) an expression cassette comprising a human alpha-L-iduronidase (hIDUA) gene under the control of regulatory sequences which control expression thereof, wherein the expression cassette is flanked by AAV inverted terminal repeat (ITR) sequences at its 5′ end and its 3′ ends and wherein said hIDUA gene has a sequence of SEQ ID NO: 1, or a sequence at least about 95% identical to SEQ ID NO: 1, which encodes a functional human alpha-L-iduronidase, (b) rep sequences operably linked to regulatory sequences which direct expression of rep proteins thereof in the host cells; and (c) nucleic acid sequences encoding an AAV capsid protein operably linked to regulatory sequences which direct expression of the AAV capsid in the host cells, and sufficient helper sequences to package the expression cassette into the AAV capsid, wherein the host cells express the rep and capsid proteins and package the expression cassette into the assembled capsid to produce the rAAV particles. 
     
     
         2 . The method according to  claim 1 , further comprising purifying the rAAV particles. 
     
     
         3 . The method according to  claim 1 , wherein the host cell is selected from mammalian cell culture and prokaryotic cell culture. 
     
     
         4 . The method according to  claim 3 , wherein the host cell is an HEK293 cell. 
     
     
         5 . The method according to  claim 1 , wherein the functional human alpha-L-iduronidase is selected from the group consisting of:
 (a) amino acids 1 to about 653 of SEQ ID NO: 2;   (b) a synthetic human enzyme comprising a heterologous leader sequence fused to amino acids 27 to 653 of SEQ ID NO: 2; and   (c) a variant of amino acid sequence of SEQ ID NO:2 having one or more of the modifications comprising: an H to Q reduction at amino acid position 82 (SEQ ID NO: 8); a change from R to Q at position 105 (SEQ ID NO: 9); a change from G to R at position 116 (SEQ ID NO: 10); a change from V to A at position 279 (SEQ ID NO: 11); a change from L to R at position 346 (SEQ ID NO: 12); a change from A to T at position 361 (SEQ ID NO: 13); a change from H to N at position 449 (SEQ ID NO: 14); a change from V to I at position 454 (SEQ ID NO: 15); a change from A to T at position 591 (SEQ ID NO: 16); and a change from A to T at position 622 (SEQ ID NO: 17).   
     
     
         6 . The method according to  claim 1 , wherein the regulatory sequences comprise a liver-specific promoter. 
     
     
         7 . The method according to  claim 6 , wherein the promoter is a TBG promoter. 
     
     
         8 . The method according to  claim 1 , wherein the AAV capsid is selected from AAV8 and AAV9. 
     
     
         9 . The method according to  claim 1 , wherein the ITR sequences are full-length ITRs or self-complementary ITRs. 
     
     
         10 . An rAAV produced according to the method of  claim 1 . 
     
     
         11 . A pharmaceutical composition useful for treating mucopolysaccharidosis type I (MPS I), wherein the composition comprises an effective amount of the rAAV virus particles produced according to the method of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         12 . The pharmaceutical composition according to  claim 11 , wherein the rAAV is formulated at a dosage comprising 3×10 9  to 3×10 13  genome copies of the rAAV virus particle per kg.