SMART Virus Vectors with a Built-in Safety Mechanism

描述（由申请方提供）：病毒是开发疫苗和基因疗法的有力工具。然而，活重组载体的安全性始终是一个问题，因为不受控制的复制或并发症并非无关紧要，特别是在免疫抑制的个体中。在努力开发更安全，但仍然有效的活病毒载体，我们建议构建具有安全机制的完全复制的病毒载体，该安全机制设计用于当载体发生并发症或需要停止治疗时使用。我们的SMART（四环素阻遏物辅助的安全机制）病毒载体将使用来自泰特操纵子的元件来调节“安全”基因的表达。牛痘病毒（Vaccinia virus，VV）是一种理想的载体系统。泰特系统已经成功地适应VV，允许表达受到抗生素四环素的严格调控。此外，我们已经表明，干扰素-γ（IFN-γ）作为一个安全的基因在体内表达时，VV，减毒超过百万倍的病毒在免疫缺陷小鼠。我们的假设是，表达安全基因的活SMART VV载体将显著更安全：任何不良反应的治疗将与抗生素治疗一样简单，因为它将允许安全基因的表达并显著增强病毒清除。更重要的是，四环素治疗将只需要当并发症发生或怀疑，或当治疗应该停止。本R21申请将涉及两个具体目标：（1）开发仅在诱导剂存在下表达安全基因的SMART VV载体，以及（2）评估新载体的安全性和有效性。首先，将产生在组成型VV启动子下表达四环素阻遏物和在工程化诱导型启动子下表达报告基因绿色荧光蛋白（GFP）的SMART VV载体，并将检查GFP的诱导以优化系统。然后，将开发可诱导表达鼠IFN-γ的SMART载体作为模型安全基因。将给予正常和免疫缺陷小鼠诱导性表达IFN-γ的VV载体，并在存在和不存在诱导剂的情况下评估存活、痘病变消退、疾病恢复、体重减轻和病毒复制。此外，将评估对VV的免疫应答，以确保新载体的功效不受表达四环素阻遏物表达或四环素处理的影响。

DESCRIPTION (provided by applicant): Viruses are powerful tools for the development of vaccines and gene therapies. However, the safety of live recombinant vectors is always a concern, as uncontrolled replication or complications are not inconsequential, particularly in immunosuppressed individuals. In an effort to develop safer, yet still effective live viral vectors, we propose to construct fully replicating virus vectors with a safety mechanism designed to be used when complications with the vector occur or therapy needs to be stopped. Our SMART (Safety Mechanism Assisted by the Repressor of Tetracycline) virus vectors will use elements from the tet operon to regulate the expression of a "safety" gene. Vaccinia virus (VV) is an ideal vector system to test this strategy. The tet system has been successfully adapted to VV, allowing expression to be tightly regulated by the antibiotic tetracycline. In addition, we have shown that interferon-gamma (IFN-gamma) acts as a safety gene in vivo when expressed by VV, attenuating the virus by more than million-fold in immunodeficient mice. Our hypothesis is that live SMART VV vectors expressing a safety gene would be significantly safer: treatment of any adverse reactions would be as simple as antibiotic therapy, since it would allow the expression of the safety gene and significantly enhance virus clearance. More importantly, tetracycline treatment would only be needed when complications occur or are suspected, or when treatment should be stopped. Two specific aims will be addressed under this R21 application: (1) to develop SMART VV vectors expressing a safety gene only in the presence of inducer, and (2) to assess the safety and efficacy of the new vectors. First, SMART VV vectors expressing the tetracycline repressor under a constitutive VV promoter and the reporter gene green fluorescent protein (GFP) under an engineered inducible promoter will be generated and the induction of GFP will be examined in an effort to optimize the system. Then, a SMART vector inducibly expressing murine IFN-gamma, as a model safety gene will be developed. Normal and immunodeficient mice will be given the VV vector expressing IFN-gamma inducibly and survival, pock lesion resolution, disease recovery, weight loss, and virus replication will be assessed in the presence and absence of inducer. In addition, immune responses to VV will be assessed to ensure that the efficacy of the new vectors is not compromised by expression tetracycline repressor expression or tetracycline treatment.