Date of Award

Spring 2022

Document Type

Open Access Thesis

Department

Biomedical Science

First Advisor

Sujit Pujhari

Abstract

Severe Acute Respiratory Syndrome Corona Virus-2, also known as SARS-CoV-2, is a newly emerged respiratory viral pathogen. The outbreak of SARS-CoV-2 started in Wuhan, China, in late 2019 and rapidly transformed into a pandemic. Several vaccines have been developed and are available to the public on an unprecedented timeline, protecting individuals from a severe infection and saving precious lives. However, none of the vaccines can provide a sterile immunity, and the SARS-CoV-2 transmission cycle continues. Additionally, because of the mutation-prone RNA genome and immunological selection pressure, SARS-CoV-2 is rapidly mutating, especially the Spike(S) protein, and new viral strains are arising with varying transmissibility rates severity. S-protein is an essential structural component of the SARS-CoV-2, which interacts with the host cell receptors and facilitates the entry of the virus into the host cells. The progeny of infectious viruses produced either infect healthy cells or are released to the environment infecting new hosts. Knock-down of S-protein in an infected cell severely impairs the production of infectious viral particles and could be a promising approach in blocking the transmission cycle of SARS-CoV-2. Chapter 1 presents a proof of concept to deliver highly effective shRNA against SARS-CoV-2 Sprotein using the Adeno Associated Virus delivery system. Multiple shRNAs targeting spike protein were screened, and the candidate shRNA with significant knock-down efficiency was selected. The selected shRNA was packaged into a respiratory tissue-specific AAV6 viral vector. The transduction of AAV6 encoding S-protein shRNA or luciferase shRNA into HEK293T cells demonstrated a robust suppression of S-protein and paved the path for in vivo studies.

Recently AAV viral vectors have become popular candidates for gene therapy. However, an eight-day-long viral production procedure challenges keeping the production cost low. Contrary to the time-consuming freeze-thaw (FT) water lysis method of virus-producing HEK293T cells, we developed a simple water-based hypotonic (WH) method for AAV production. Utilizing this novel method, AAV can be produced in 6 days. We explored a fundamental biophysical property of water to release cell associated AAV particles. Distilled water has the highest water potential due to the near absence of solutes allowing it to passively move into the cell. A shear force applied through pipetting ruptures the hypotonic cells and releases the virus into the supernatant. The quality of virus preparation through FT-lysis and WH-lysis evaluated by Coomassie Brilliant Blue stain is comparable. The GFP expression was measured as a function of the transduction efficiency of virus preparation, wherein the WH-lysis method performed better than the FT-lysis method.

In conclusion, the implications of this thesis provide a proof of concept to explore an AAV-based shRNA-mediated S-protein knock-down as a new therapeutic option to block the transmission chain of SARS-CoV-2, which can be extended to other respiratory viruses. In addition, we demonstrated that the WH-lysis method of AAV purification is a fast, convenient, and simple method of producing a high-quality viral vector for various applications.

Rights

© 2022, Ronald Anderson Smithwick

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