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

项目总结 新冠肺炎大流行突显了我们长期以来对新的病毒传染病的脆弱性 没有后天的豁免权。虽然疫苗和抗病毒疗法最终可以被开发出来治疗 许多病毒性传染病，这些干预措施需要大量的时间和资源才能获得。这将导致 在一个关键时期，除了物理治疗外，没有任何治疗方法可以减缓病毒的传播 具有重大经济和社会后果的对策。真正令人震惊的洞察是，所有 我们现在开发的针对SARS-CoV-2的疫苗和疗法对下一个周期的 病毒爆发(如流感、埃博拉等)。迫切需要从根本上改变我们的方法 通过开发可在一开始就快速部署的抗病毒策略来抗击新出现的病毒疾病 一场新的病毒爆发。我们提出了一种革命性的新病毒-抗病毒技术，它有可能针对 在疾病暴发周期的所有阶段出现新的病毒病原体。这包括以病毒病原体为目标 动物宿主在人类传播之前，预防健康个体的病毒感染，并正在进行的治疗 病毒感染。这种新的“特洛伊病毒”技术使用的是模拟病毒病原体的工程病毒载体， 但含有有效的CRISPR抗病毒机制，可降解致病病毒颗粒。这些特洛伊木马病毒 载体具有不完整的病毒基因组，只能通过以下方式选择性地在先前感染的细胞中复制 劫持病毒衍生的蛋白质，利用这些蛋白质在体内受感染的区域繁殖和传播。 特洛伊木马病毒的传播通过瞄准入侵的病毒颗粒来防止健康组织中的病毒感染， 同时抑制病毒感染的活性部位。CRISPR抗病毒技术的集成 特洛伊木马病毒载体允许系统重新编程以针对新的病毒株，而不需要大量的 蛋白质工程或临床测试，促进病毒疾病期间技术的快速动员 疫情爆发。拟议的研究将集中于开发SARS-CoV-2特洛伊病毒技术作为 活动性病毒感染的治疗选择。这项研究将使用工程化的非传染性细胞模型 用于评估SARS-CoV-2特洛伊木马病毒基因组设计的系统，该设计只能在以前的 被感染的细胞。我们将确定将CRISPR抗病毒技术纳入 SARS-CoV-2特洛伊木马病毒媒介，同时识别SARS-CoV-2病毒中的关键CRISPR漏洞。至 验证为SARS-CoV-2特洛伊病毒治疗开发的基因组工程原理可以缓解 对于活动性病毒感染，我们将复制针对小鼠肝炎冠状病毒(MHV-A59)的方法， 在小鼠模型系统中跟踪特洛伊病毒的有效性。如果成功，我们的研究将产生 一种功能性SARS-CoV-2特洛伊病毒疗法，在临床前模型系统中验证基因组设计，以 为用于当前和未来SARS的技术的商业开发奠定基础- 冠状病毒暴发。

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.