Date of Award
9-27-2016
Document Type
Thesis
Degree Name
Biology, MS
First Advisor
Carole Cramer
Committee Members
Maureen Dolan; Susan Motts
Call Number
LD 251 .A566t 2016 M24
Abstract
Therapeutic proteins are becoming one of the fastest growing classes of drugs on the market. These large biologically-synthesized macromolecules are typically administered intravenously and are dependent upon cellular receptors and other active uptake mechanisms for delivery into tissues and cells. There is significant interest in developing cell-based assays to assess kinetics and mechanisms of endocytosis and transcytosis of large therapeutic proteins across various cell layers. The goal of this research was to develop a transwell “disease tissue on a chip” model to compare rates and efficiencies of different delivery systems for moving bioactive lysosomal enzymes across a human epithelial cell layer to correct disease fibroblasts. The experimental model was designed to mimic a disease-specific assay to differentiate transcellular delivery of lysosomal enzyme replacement therapeutics (ERTs) across epithelial cell layers. Epithelial cells, which line all internal and external surfaces of the human body create the first obstacle for protein delivery because these cells will not spontaneously endocytose large molecules. Current ERTs for lysosomal storage diseases (LSDs) exemplify receptor-dependent therapeutic proteins that are dependent upon the mannose 6-phosphate receptor (M6PR) for delivery into cells and lysosomes. While these therapies effectively treat many of the visceral organs associated with these diseases, the low abundance of M6PR in certain cells limits the ability of the ERT to provide corrective doses to the central nervous system, bone, eyes, ears and other “hard ttreat” tissues. A “disease on a chip” model will provide a useful system to compare different strategies to deliver large lysosomal enzymes across cell layers. The specific disease model developed in this research represents GM1 gangliosidosis, a rare neurodegenerative LSD caused by deficiencies of the acid β-galactosidase enzyme. The transwell system comprised a confluent polarized epithelial cell layer established within an insert with fibroblast cells grown in the basolateral chamber as the “target” tissue. Both cell lines were genetically modified by CRISPR/Cas9 genome-editing technology to knock-out the endogenous β-galactosidase enzyme creating a GM1 disease-specific transwell model. This system was used to differentiate and monitor transcellular delivery of recombinant β-galactosidase comparing receptor-mediated (M6PR) and absorptivemediated mechanisms for delivery of corrective enzyme. Relative correction of the lysosomal disease phenotype was monitored by a high-content screening system (HCS). The system clearly delineated differences between the enzyme delivery systems and provides a broadly applicable platform that can be modified to model specific organs or specific diseases.
Rights Management
This work is licensed under a Creative Commons Attribution 4.0 International License.
Recommended Citation
Martin, Reid Allan, "Developing High-Content Screening to Assay Trans-Epithelial Transport of Complex Proteins" (2016). Student Theses and Dissertations. 662.
https://arch.astate.edu/all-etd/662
