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 α 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 polynucleotide comprising the nucleotide sequence depicted in SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 31,or 32.  
     
     
         2 . A viral vector comprising the polynucleotide according to  claim 1 .  
     
     
         3 . The viral vector according to  claim 2 , wherein said viral vector is an RNA viral vector.  
     
     
         4 . A virus particle comprising the viral vector according to  claim 3 .  
     
     
         5 . A plant cell or a plant comprising the virus particle according to  claim 4 .  
     
     
         6 . A plant cell or a plant comprising the polynucleotide according to  claim 1 .  
     
     
         7 . A polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 4, 6, 8, 10, 12, 14 ,16, 18, or 20.  
     
     
         8 . A polynucleotide comprising a nucleotide sequence encoding the polypeptide according to  claim 7 .  
     
     
         9 . A plant cell or a plant expressing the polypeptide according to  claim 7 .  
     
     
         10 . A polypeptide comprising (a) the complete, or a fragment of, the amino acid sequence of α-galactosidase and (b) the amino acid depicted in SEQ ID NO: 37, wherein the amino acid sequence depicted in SEQ ID NO: 37 is at the carboxy end of the complete, or a fragment of, the amino acid sequence of α-galactosidase, wherein said fragment of the amino acid sequence of α-galactosidase comprises a deletion of at the carbozy end of α-galactosidase, wherein said deletion is one to twenty-five amino acids.  
     
     
         11 . The polypeptide according to  claim 10 , wherein said deletion is one to twelve amino acids.  
     
     
         12 . The polypeptide according to  claim 11 , wherein said deletion is four to twelve amino acids.  
     
     
         13 . A polynucleotide comprising a nucleotide sequence encoding the polypeptide according to  claim 10 .  
     
     
         14 . A polynucleotide comprising a nucleotide sequence encoding the polypeptide according to  claim 11 .  
     
     
         15 . A polynucleotide comprising a nucleotide sequence encoding the polypeptide according to  claim 12 .  
     
     
         16 . A plant cell or a plant expressing the polypeptide according to  claim 10 .  
     
     
         17 . A plant cell or a plant expressing the polypeptide according to  claim 11 .  
     
     
         18 . A plant cell or a plant expressing the polypeptide according to  claim 12.