Author ORCID Identifier

Year of Publication


Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation




Chemical and Materials Engineering

First Advisor

Dr. Daniel W. Pack


With the promise to treat a multi-faceted list of serious inherited and acquired diseases, such as cancer, neurodegenerative and infectious diseases, and inherited genetic indications, gene therapy has continued to push the boundaries of traditional medicine since its earliest implementation. While much progress has been made, clinical success has largely remained elusive. Immunogenicity, difficulty producing commercially relevant quantities, and having a limited genetic payload still limits the ability of viruses to act as directed delivery agents for genetic material. As such, researchers have turned to cationic synthetic materials as a means of delivering nucleic acids, which can circumvent the immune response but suffer decreased delivery efficiency relative to viral vectors. To advance non-viral vectors towards clinical relevance, special consideration of the various barriers associated with nucleic acid delivery must be applied to the design of the synthetic agents. How does the material interact with the cell surface, are the vectors stable in circulation, can the structure escape the endocytic environment of cells, and will the structure release the genetic payload? All of these questions, and many others, need to be explored and implemented in a set of design criteria that optimizes the ability of a certain vector to consistently induce adequate expression for the desired application.

In chapter 3, we apply the aforementioned considerations to polyethylenimine (PEI), the “gold standard” material for polymeric gene delivery vectors (polyplexes). Through simple modifications of PEI via succinylation, we generated zwitterion-like polymers (zPEI) that increased transfection efficiency by up to 50 fold over that of traditional PEI vectors when in the presence of serum proteins. These vectors also show remarkable resilience when lyophilized and stored for long periods, maintaining transfection efficacies similar to freshly prepared polyplexes over the course of 8 weeks. Since vector development is quick, efficient, consistent, and can be stored for long periods of time, zPEI could serve as a platform for testing applications of non-viral gene therapies or for explicit expression of specific proteins.

In chapter 4, we explore this notion by investigating applications where non-viral particles have shown success and elucidate situations where certain vectors may be preferred over others. Finally, in chapters 5 and 6 we deployed our own application of non-viral genetic engineering to induce production of the plant-derived biomolecule, curcumin, in a mammalian cell. This is the first time an entire plant enzyme cluster has been expressed in a mammalian cell, as well as the first time a plant biomolecule has been synthesized in a mammalian cell. We optimized the transient expression of the gene cluster and maximized the production of bisdemethoxycurcumin, demethoxycurcumin, and curcumin. Bioactivity of curcuminoids produced in cells were assessed via metabolic and migration assays. When cultured in the presence of curcuminoid-producing HEK293 cells, metabolic activity of MDA-MB-231 and MCF7 cancer cells was reduced and cell migration was inhibited. We believe this work may represent the first step towards a drastic shift in how diseases are treated, focusing not on the external delivery of drugs, but instead engineering patients’ bodies to produce their own supply of therapeutic compounds.

Digital Object Identifier (DOI)

Funding Information

The material is based upon work supported by NASA Kentucky under NASA award No: NNX15AR69H. (2018-2020)