International Laboratory of Molecular Biology for Tropical Disease Agents, School of Veterinary Medicine, University of California, Davis, CA 95616.
Proc Natl Acad Sci U S A. 2013 Sep 17;110(38):15407-12. doi: 10.1073/pnas.1314483110. Epub 2013 Aug 29.
Replication-competent viruses, such as Vaccinia virus (VACV), are powerful tools for the development of oncolytic viral therapies and elicit superior immune responses when used as vaccine and immunotherapeutic vectors. However, severe complications from uncontrolled viral replication can occur, particularly in immunocompromised individuals or in those with other predisposing conditions. VACVs constitutively expressing interferon-γ (IFN-γ) replicate in cell culture indistinguishably from control viruses; however, they replicate in vivo to low or undetectable levels, and are rapidly cleared even in immunodeficient animals. In an effort to develop safe and highly effective replication-competent VACV vectors, we established a system to inducibly express IFN-γ. Our SMART (safety mechanism assisted by the repressor of tetracycline) vectors are designed to express the tetracycline repressor under a constitutive VACV promoter and IFN-γ under engineered tetracycline-inducible promoters. Immunodeficient SCID mice inoculated with VACVs not expressing IFN-γ demonstrated severe weight loss, whereas those given VACVs expressing IFN-γ under constitutive VACV promoters showed no signs of infection. Most importantly, mice inoculated with a VACV expressing the IFN-γ gene under an inducible promoter remained healthy in the presence of doxycycline, but exhibited severe weight loss in the absence of doxycycline. In this study, we developed a safety mechanism for VACV based on the conditional expression of IFN-γ under a tightly controlled tetracycline-inducible VACV promoter for use in vaccines and oncolytic cancer therapies.
复制型病毒，如牛痘病毒（VACV），是开发溶瘤病毒疗法的有力工具，用作疫苗和免疫治疗载体时可引起更强的免疫反应。然而，当病毒复制不受控制时，可能会发生严重的并发症，尤其是在免疫功能低下的个体或有其他易感条件的个体中。持续表达干扰素-γ（IFN-γ）的 VACV 在细胞培养中与对照病毒无法区分地复制；然而，它们在体内以低水平或无法检测到的水平复制，并在免疫缺陷动物中甚至迅速清除。为了开发安全且高效的复制型 VACV 载体，我们建立了诱导表达 IFN-γ的系统。我们的 SMART（受四环素抑制剂辅助的安全机制）载体旨在在组成型 VACV 启动子下表达四环素抑制剂，并在工程化的四环素诱导启动子下表达 IFN-γ。接种不表达 IFN-γ 的 VACV 的免疫缺陷 SCID 小鼠表现出严重的体重减轻，而接种在组成型 VACV 启动子下表达 IFN-γ 的 VACV 的小鼠则没有感染迹象。最重要的是，在给予强力霉素的情况下，表达 IFN-γ 基因的 VACV 接种小鼠保持健康，但在不给予强力霉素的情况下则表现出严重的体重减轻。在这项研究中，我们开发了一种基于 IFN-γ 条件表达的 VACV 安全机制，该机制基于在严格控制的四环素诱导型 VACV 启动子下表达，可用于疫苗和溶瘤癌症治疗。
