Thesis Format
Integrated Article
Degree
Master of Science
Program
Medical Biophysics
Collaborative Specialization
Molecular Imaging
Supervisor
Ronald, John A.
2nd Supervisor
Scholl, Timothy J.
Co-Supervisor
Abstract
Base editing is a powerful genome editing tool with the potential to treat thousands of genetic diseases caused by single-base changes in DNA called point mutations. Adenine base editors (ABEs) specifically convert adenine (A) to guanine (G) and can theoretically correct almost half of all disease-causing point mutations. Currently, there is a need for a safe and efficient method of delivering ABEs into tissues. In Chapter 2, we developed DNA minicircles as a novel non-viral delivery method for ABEs. To assess delivery efficiency, we also developed activatable reporter imaging systems for the visualization of ABE activity using fluorescence or bioluminescence imaging. Using our imaging reporters, we show for the first time that minicircles are superior to plasmids for the delivery of ABE into a variety of cancer cell types. Chapter 3 summarizes the main findings and discusses limitations and future work.
Summary for Lay Audience
The human genome is composed of over three billion DNA letters known as bases, with the four possible bases being: adenine (A), cytosine (C), guanine (G), and thymine (T). An error in which a single base is changed to another base is known as a point mutation, and is the cause for thousands of genetic diseases including sickle cell disease, Duchenne muscular dystrophy, and many types of cancer. Base editing is a recently developed genome editing technology that enables precise targeting and modification of DNA bases. Base editors consist of an RNA component that targets a specific location in the DNA and a protein component that makes the base change. Adenine base editors (ABEs) specifically change an A to a G and can theoretically correct almost half of all disease-causing point mutations. One of the major challenges with base editing is delivery: we need a way to efficiently get the base editors into cells. Minicircles are small circular pieces of DNA that can be used to express a gene of interest in cells. Minicircles are more efficient and safer at delivering genes than plasmids, which are circular pieces of DNA but also contain bacterial components. In this study, we developed and tested minicircles for the delivery of ABEs for the first time. Additionally, to track the DNA edits being made, we developed imaging tools that are turned on when a base editor is working in a cell. These tools allowed us to evaluate the efficiency of minicircle delivery using two imaging techniques called fluorescence imaging and bioluminescence imaging – the latter of which might be useful one day for imaging base editor activity inside the body. We found that minicircles are better than plasmids at delivering ABE DNA into multiple cancer cell types. Our results demonstrate that minicircles are a promising method for efficient and safe delivery of base editors into cells. Future work will test these minicircles for base editing in animal models of cancer. With these tools, we will be able to develop better base editors and delivery methods for the treatment of cancer and other genetic diseases.
Recommended Citation
Liu, Shirley, "Evaluation of DNA Minicircles for Delivery of Adenine Base Editors Using Activatable Reporter Imaging Systems" (2023). Electronic Thesis and Dissertation Repository. 9317.
https://ir.lib.uwo.ca/etd/9317