Date of Award
1-17-2013
Document Type
Dissertation
Degree Name
Molecular Biosciences, Ph.D.
First Advisor
Carole Cramer
Committee Members
Fabricio Medina-Bolivar; Malathi Srivatsan; Maureen Dolan; Shiguang Yu
Call Number
LD 251 .A566d 2012 A32
Abstract
One of the main challenges for successful drug development is the creation of molecules that are able to cross the cell membrane to deliver therapeutics into specific subcellular compartments where they can serve their purpose. Among the therapeutic applications burdened with cell uptake limitations are enzyme replacement therapeutics (ERTs) for both lysosomal storage disorders (LSD) and endoplasmic reticulum storage diseases (ERSD). LSD and ERSD are rare disorders caused by defects in specific proteins within these cellular compartments, and current treatments rely on the regular provision of active enzyme to replace the missing/defective protein in patients. Plant lectin RTB, naturally present in castor bean seeds, mediates endocytotic uptake into mammalian cells, transcytosis, and trafficking to lysosomes or endoplasmic reticulum (ER) of associated proteins, or "payloads". In order to test the potential of RTB to deliver lysosomal and ER ERTs, RTB was genetically fused to four different human enzymes: α-L-iduronidase (IDUA), the lysosomal enzyme deficient in Mucopolysaccharidosis type I patients; β-glucocerebrosidase (GBA), the lysosomal enzyme deficient in Gaucher Disease; UDP-glucuronosyltransferase 1A1 (UGT1A1), an ER enzyme defective in hyperbilirubinemia disorders, Criggler-Najjar, and Gilbert Syndrome; and UDP-glucuronosyltransferase 1A9 (UGT1A9), an ER-localized enzyme involved in the detoxification of small molecules. A plant-based transient expression platform was used to produce RTB-human enzyme fusion proteins for these studies. This bioproduction platform provides supply, cost, safety, and delivery advantages over mammalian cell-based production platforms for enzyme replacement therapies. Gene constructs encoding RTB-IDUA, RTB-GBA, UGT1A1-RTB and UGT1A9-RTB were developed and expressed in leaves of Nicotiana benthamiana plants using an Agrobacterium-mediated transient expression system. Recombinant fusion products were purified and characterized for RTB lectin activity, human enzyme activity, and, where applicable, the ability to direct uptake into human cells. Results presented in this work demonstrate that RTB:IDUA is very effective at delivering molecules across the human cell membrane through a completely novel mechanism which is independent of the glycan receptor mechanisms currently used by the commercially available recombinant ERT proteins. The novel enzyme delivery technology described in this dissertation has the potential of delivering replacement enzymes to a greater spectrum of cells and tissues than the current treatment, enhancing treatment efficacy. The present study also shows that RTB does not work with all partners, and that properties of the payload play an important role in RTB bioactivity. Both GBA and UGT negatively affected the sugar binding activity of this lectin. Finally, for the particular case of UDP-glucuronosyltransferases (UGTs) studies, we have the first reported evidence that plants are capable of expressing active human UGT1A1 and UGT1A9 enzymes. Results demonstrate that it is possible to solubilize UGT1A9 from a plant expression system in an active presentation. These finding have the potential to provide a cost-effective and abundant source of soluble UGT1A9 for therapeutic implications.
Rights Management
This work is licensed under a Creative Commons Attribution 4.0 International License.
Recommended Citation
Acosta Gamboa, Walter, "Development Of Plant Lectin RTB for Delivery of Therapeutic Proteins" (2013). Student Theses and Dissertations. 853.
https://arch.astate.edu/all-etd/853
