Date of Graduation

8-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Engineering (PhD)

Degree Level

Graduate

Department

Biomedical Engineering

Advisor/Mentor

Nelson, Christopher E.

Committee Member

Wolchok, Jeffrey C.

Second Committee Member

Greene, Nicholas P.

Third Committee Member

Rao, Raj R.

Keywords

Engineering; Biomedical engineering; Gene deliver; Transcriptional control; Macrophages; Inflammatory response

Abstract

Macrophages are an important cell type in the body responsible for orchestrating the inflammatory response and return to homeostasis. Macrophages are often implicated in a multitude of disease pathologies including delayed wound healing, excessive fibrosis, autoimmune disease, and immunosuppression within the tumor microenvironment. Although macrophages have been targeted for therapeutic applications, success in utilizing them has remained elusive due to their relative ease of phenotype switching between inflammatory and anti-inflammatory. The challenge that remains is developing clinically relevant delivery vehicles that can target macrophages without causing a phenotypic switch counter-productive to the desired response. This is especially important when targeting regenerative processes. Alongside determining an appropriate delivery vehicle, it is necessary to determine the effects and utility of transcriptional modifiers in macrophages and the ability to target therapeutically relevant genes. This dissertation develops a high-throughput screening technique to determine inflammatory response experienced by macrophages in response to different clinically relevant delivery vehicles and establishes the ability to transcriptionally modify macrophages. In Chapter 2, we developed the inflammatory reporter and characterized the response experienced by RAW264.7 cells, a mouse macrophage cell line. Our results showed that the reporter expressed under NFκB response elements correlated with inflammatory markers and could be used as a sensitive and high-throughput screening technique for inflammatory response in macrophages. In Chapter 3, we used the inflammatory reporter to determine the inflammatory response to different viral and non-viral delivery vehicles and determined the inflammatory response and delivery efficiency of Adeno-associated virus (AAV) 1, 2, 5, 8, and 9, Adenovirus (Ad) 5/35, and non-viral delivery methods Lipofectamine 2000® and TransIT-X2®. We found that relatively, only AAV 1 and Ad5/35, which was designed for macrophage delivery, showed any increase in vehicle uptake with minimal inflammatory response while both non-viral delivery methods showed increased delivery and inflammatory response. In Chapter 4, we determined the ability to use catalytically dead CRISPR associated protein 9 (dCas9) fused with a proven transcriptional activator, VP64, to increase levels of interleukin 10 (IL-10) within RAW264.7 cells. This work in its entirety has developed a platform for quick and efficient screening of inflammatory responses experienced in macrophages and determined the ability to transcriptionally modify macrophages for therapeutic applications.

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