Selectively Replicating Trojan Virus Vectors as Programmable CRISPR-Based Antiviral Therapies

PROJECT SUMMARY The COVID-19 pandemic has highlighted our long-standing vulnerability to new viral infectious diseases with which there is no acquired immunity. While vaccines and antiviral therapies can eventually be developed to treat many viral infectious diseases, these interventions require significant time and resources to acquire. This results in a critical period of time where there are no therapeutic options to slow the spread of the virus, besides physical countermeasures that have dramatic economic and social consequences. The truly alarming insight is that all of the vaccines and therapies we develop now against SARS-CoV-2 will be useless against the next cycle of viral outbreaks (e.g., influenza, ebola, etc.). There is a paramount need to fundamentally transform our approach to combating emerging viral diseases by developing antiviral strategies that can be rapidly deployed at the onset of a new viral outbreak. We propose a revolutionary new viral-antiviral technology that has the potential to target emerging viral pathogens at all stages of the disease outbreak cycle. This includes targeting viral pathogens in animal hosts prior to human transmission, preventing viral infections in healthy individuals, and treating ongoing viral infections. This new "Trojan virus" technology uses engineered viral vectors that imitate viral pathogens, yet contain potent CRISPR antiviral machinery that degrades pathogenic viral particles. These Trojan virus vectors have an incomplete viral genome that can selectively replicate only in previously infected cells by hijacking viral derived proteins, which it uses to multiply and spread throughout the infected areas of the body. The spread of the Trojan virus acts to prevent viral infection in healthy tissue by targeting invading viral particles, while at the same time suppressing active sites of viral infection. The integration of CRISPR antiviral technology into Trojan virus vectors allows the system to be reprogrammed to target new viral strains without extensive protein engineering or clinical testing, facilitating the rapid mobilization of the technology during viral disease outbreaks. The proposed research will focus on developing SARS-CoV-2 Trojan virus technology as a therapeutic option for active viral infections. The research will use engineered non-infectious cellular model systems to evaluate SARS-CoV-2 Trojan virus genome designs that can selectively replicate only in previously infected cells. We will determine the optimal strategy for incorporating CRISPR antiviral technology into the SARS-CoV-2 Trojan virus vectors, while identifying key CRISPR vulnerabilities in the SARS-CoV-2 virus. To validate that the genome engineering principles developed for the SARS-CoV-2 Trojan virus therapy can mitigate active viral infections, we will duplicate the approach to target the mouse hepatitis coronavirus (MHV-A59), tracking the effectiveness of the Trojan virus in murine model systems. If successful, our research will generate a functional SARS-CoV-2 Trojan virus therapy, validating the genome design in preclinical model systems, to establish the foundation for commercial development of the technology for use in the current and future SARS- CoV outbreaks.