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Title:Design of folic acid, transferrin, and integrin receptor specific polyamidoamine dendrimers for gene delivery
Author(s):Hwang, Mark
Director of Research:Pack, Daniel W.
Doctoral Committee Chair(s):Pack, Daniel W.
Doctoral Committee Member(s):Zhao, Huimin; Cheng, Jianjun; Wang, Yingxiao; Kong, Hyun Joon
Department / Program:Bioengineering
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Non-viral gene delivery
polyamidoamine dendrimers
receptor-targeted vectors
Abstract:Gene therapy holds promise for the treatment of many medical ailments. Understanding the genetic basis of disease, coupled with advances in modern medicine, fuels the search for disease treatment at the genetic level. However, gene therapy is far from the standard of care after thirty years of research and development. Progress is limited by the need for a gene delivery platform that is both safe and highly efficient. Viral vectors have evolved to deliver genetic material efficiently to target cells, but suffer from a host of potential safety and immunogenic concerns. Conversely, comparatively less efficient non-viral delivery methods result from poor understanding of rational design required for a successful synthetic, ground-up approach to gene delivery. Barriers to gene delivery must thus first be identified and overcome before rational design can take place. The plasma membrane represents one important barrier in the gene delivery process. The method by which vectors are internalized and subsequently trafficked plays a key role in determining their intracellular fate. Recent research has shown, for example, that vector interaction with different regions of the plasma membrane can result in endocytosis directed to endolysosomes, the Golgi apparatus, or the endoplasmic reticulum. Each of these destinations affects whether the ultimate goal of vector delivery to the nucleus for expression is achieved. Moreover, the milieu of intracellular sorting vesicles points to the complexity of the trafficking process – one in which endocytic pathways are not clearly defined, and may involve pathway interplay that has been hitherto unexplored. Our primary aim was therefore to understand the interplay of common endocytic mechanisms in non-viral gene delivery. Vector-targeting to cell surface receptors is one method of enhancing gene delivery to specific cells. However, receptor-specific internalization has a direct effect on the endocytic trafficking process that must be taken into account in vector design. While many ligands are used successfully to target vectors to different tissues that overexpress their receptor, their precise mechanisms of endocytosis and processing en route to the nuclear region is undefined. We thus also seek to elucidate the relationships between receptor-specific targeting and endocytosis as they relate to synthetic gene delivery. The research presented in this thesis describes the issue of different methods of vector internalization and how they affect subsequent gene delivery. Chapter 2 discusses the roles of clathrin and caveolar processing in polyethylenimine and polyamidoamine polymer gene delivery. Our results show that these processes are not necessarily independent of each other. Intracellular sorting exists by which synthetic vectors internalized by one mechanism may still be dependent on another for successful gene delivery. We further developed this study approach in Chapter 3 by examining the impact of targeting polyplexes to clathrin and caveolae, using transferrin and folic acid ligands, respectively. Once again, our findings suggest that pathways are not delineated simply by their method of endocytosis because of the existence of complex sorting mechanisms that take effect following vector uptake into the cell. In the final chapter, we explored the trafficking mechanisms of the integrin receptor, one that is highly implicated in cancer, metastasis, and angiogenesis, but of which the precise method of trafficking as it relates to gene delivery is currently unexplored. This logical approach to understanding gene delivery started with identifying significant pathways involved in the delivery process, characterizing these pathways by studying the processing of ligands known to traverse them, and finally exploring the arginine-glycine-aspartic acid (RGD) ligand targeted to the integrin receptor in the context of our previous discoveries. Lysosomal avoidance and trafficking via the caveolar pathway were recurring motifs for successful gene delivery in our experiments. Incorporating these principles to design of polymeric gene delivery vehicles will likely enhance gene delivery efficiency and the development of the synthetic vector field for clinical application.
Issue Date:2012-09-18
Rights Information:Copyright 2012 Mark Hwang
Date Available in IDEALS:2012-09-18
Date Deposited:2012-08

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