Production of lysosomal enzymes in plants by transient expression

Abstract

The invention relates to α-galactosidase truncated at the carboxy terminus and the production of enzymatically active recombinant human and animal lysosomal enzymes involving construction and expression of recombinant expression constructs comprising coding sequences of human or animal lysosomal enzymes in a plant expression system. The plant expression system provides for post-translational modification and processing to produce a recombinant gene product exhibiting enzymatic activity. The invention is demonstrated by working examples in which transgenic tobacco plants express recombinant expression constructs comprising human glucocerebrosidase nucleotide sequences. The invention is also demonstrated by working examples in which transfected tobacco plants express recombinant viral expression constructs comprising human a galactosidase nucleotide sequences. The recombinant lysosomal enzymes produced in accordance with the invention may be used for a variety of purposes, including but not limited to enzyme replacement therapy for the therapeutic treatment of human and animal lysosomal storage diseases.

We claim:  
     
         1 . A pharmeceutic composition comprising a polypeptide represented by the amino acid sequence of SEQ ID NO: 18.  
     
     
         2 . A pharmeceutical composition as set forth in  claim 1 , wherein the C-terminus of said polypeptide is the amino acid sequence depicted in SEQ ID NO: 30.  
     
     
         3 . A pharmeceutical composition as set forth in  claim 1 , wherein said polypeptide is expressed in a plant.  
     
     
         4 . A pharmeceutical composition as set forth in  claim 1 , wherein said polypeptide includes plant glycosylations.  
     
     
         5 . A pharmaceutical composition as set forth in  claim 1 , wherein said polypeptide is glycosylated at least at one amino acid position selected from the group consisting of amino acid positions 108, 161, 184, and 337.  
     
     
         6 . A pharmeceutical composition as set forth in  claim 1 , wherein said polypeptide is glycosylated at least at one amino acid position selected from the group consisting of amino acid positions 108, 161, and 184.  
     
     
         7 . A pharmeceutical composition as set forth in  claim 1 , wherein said polypeptide has a plant glycosylation pattern at amino acid positions 108, 161, and 184, wherein an alpha 1, 3 fucose is present on the proximal N-acetylglucosamine (GlcNac).  
     
     
         8 . A pharmeceutical composition as set forth in  claim 1 , wherein said polypeptide has a plant glycosylation pattern at amino acid positions 108, 161, and 184, wherein an beta 1, 2 xylose is present on a beta-linked mannose of the core.  
     
     
         9 . A pharmeceutical composition as set forth in  claim 1 , wherein said polypeptide is for usage in enzyme replacement therapy.  
     
     
         10 . A pharmaceutical composition as set forth in  claim 9 , wherein said polypeptide is for treatment of Fabry disease.