Production of lysosomal enzymes in plants by transient expression

#This invention is in the field of therapeutic peptides. Specifically this invention relates to the production of pharmaceutical peptides and proteins encoded on a recombinant plant virus or and produced by an infected plant or produced by a recombinant plant. The present invention relates especially to the production of human and animal lysosomal enzymes in plants comprising expressing the genetic coding sequence of a human or animal lysosomal enzyme in a plant expression system. The plant expression system provides for post-translational modification and processing to produce recombinant protein having enzymatic activity. The invention is demonstrated herein by working examples in which transgenic or transfected tobacco plants produce a modified human α galactosidase and a glucocerebrosidase, both of which are enzymatically active. 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 lysosomal storage diseases, research for development of new approaches to medical treatment of lysosomal storage diseases, and industrial processes involving enzymatic substrate hydrolysis.

#Lysosomes, which are present in all animal cells, are acidic cytoplasmic organelles that contain an assortment of hydrolytic enzymes. These enzymes function in the degradation of internalized and endogenous macromolecular substrates. When there is a lysosomal enzyme deficiency, the deficient enzyme's undegraded substrates gradually accumulate within the lysosomes causing a progressive increase in the size and number of these organelles within the cell. This accumulation within the cell eventually leads to malfunction of the organ and to the gross pathology of a lysosomal storage disease, with the particular disease depending on the particular enzyme deficiency. More than thirty distinct, inherited lysosomal storage diseases have been characterized in humans.

#Enzyme Replacement Therapy

#FIG. 1 shows a Tobamovirus expression vectors.

#FIG. 2 shows a Tobamovirus expression vector containing the human α galactosidase gene or a variant of the gene. The amino acid sequence of αASP and Native SP depicted in FIG. 2 are depicted in SEQ ID. NO: 1 and 2, respectively. The entire amino acid sequence of WT rGAL-A is depicted in SEQ ID NO: 4.

#FIG. 3A shows accumulation by Western Analysis of total plant soluble extract anti human GAL-A sera.

#The present invention provides a polynucleotide comprising the nucleotide sequence depicted in SEQ ID NO:3, 5, 7, 9, 11, 13, 15, 17, 19, 31, or 32. The present invention also provides a polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 4, 6, 8, 10, 12, 14,16, 18, or 20.

#The present invention further provides for a polypeptide comprising (a) the complete, or a fragment of, the amino acid sequence of α-galactosidase and (b) the amino acid SEKEL (SEQ ID NO:37), wherein SEKEL 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. Preferably, the deletion is one to twelve amino acids. More preferably, the deletion is four to twelve amino acids. The present invention further provides for a polynucleotide comprising a nucleotide sequence encoding the aforementioned polypeptide(s).

#The present invention further provides for a polynucleotide encoding the aforementioned polypeptide(s).