SBIR-STTR Award

New methods are needed to produce pharmaceutical quality proteins for therapeutic use in humans on a large scale. Many heritable metabolic storage disorders of humans are potentially treatable by supplementation with exogenously produced enzymes. For example, Gaucher's disease is caused by genetic deficiencies in the enzyme glucocerebrosidase and can be successfully treated by enzyme replacement therapy. However, cost and availability of the enzyme limit access to treatment. Plants are relatively unexplored production hosts for cost-effective biosynthesis of human proteins. Using recently developed transient expression vectors based on tobacco mosaic virus, we will examine the feasibility of producing custom-designed proteins in plants using glucocerebrosidase as a test enzyme. These vectors allow both high levels of expression and rapid cycles of testing and modification of the enzyme. If the enzyme produced in plants shows catalytic activity toward the accumulating glucocerebroside, we can rapidly test experimental alterations to facilitate purification, stability, and targeting of the enzyme to lipid storing cells. The long-term objectives are to develop methods for producing copious amounts of glucocerebrosidase and other human enzymes for routine therapeutic treatments. Using this method, custom peptide synthesis could be designed for individuals that may be intolerant to the wild type enzyme. PROPOSED COMMERCIAL APPLICATION: The inexpensive biomass and economy of scale of plant growth offer the potential to drastically reduce the cost of manufacture of human proteins. There is an ever increasing commercial demand for these proteins in therapeutic applications.

A considerable number of hereditary metabolic storage disorders of humans are potentially treatable by supplementation with exogenously produced enzymes. Glucocerebrosidase, either derived from human placental tissue or a recombinant form from Chinese hamster ovary cell, is presently used in an effective but costly treatment of Gaucher disease. We discovered that genetically modified plants have the capacity to synthesize this human lysosomal enzyme with high specific activity. We developed a novel bioprocessing method to extract the enzyme from tobacco leaves, providing at least a 30-fold enrichment for enzyme to levels that will enable commercial production. Through development of a full-scale pilot process for the extraction and purification of multigram quantities of glucocerebrosidase, we will establish that plants are an important low- cost source for the large quantities of complex recombinant proteins required to advance modern medical therapies. Our goals are to reduce the costs of enzyme replacement therapy for Gaucher patients; validate the concept of using plants as a source of recombinant proteins for clinical use in humans; and provide an accurate economic production model to evaluate a wide variety of future medical applications.Thesaurus termsbiological product, enzyme biosynthesis, glucosylceramidase, protein engineering drug design /synthesis /production, drug screening /evaluation, gene targeting, glycosylation, method development protein purification, tobacco, tobacco mosaic virus, transfection, transfection vectorNational Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)