SMART Virus Vectors with a Built-in Safety Mechanism

描述(由申请人提供)：病毒是开发疫苗和基因疗法的强大工具。然而，活的重组载体的安全性一直是一个令人担忧的问题，因为不受控制的复制或并发症并不是无关紧要的，特别是在免疫抑制的个体中。为了开发更安全但仍然有效的活病毒载体，我们建议构建完全复制的病毒载体，并设计一种安全机制，以便在载体发生并发症或治疗需要停止时使用。我们的SMART(由四环素抑制子辅助的安全机制)病毒载体将使用tet操纵子中的元件来调节“安全”基因的表达。痘苗病毒(VV)是测试这一策略的理想载体系统。TET系统已经成功地适应了VV，允许表达受到抗生素四环素的严格调控。此外，我们已经证明，当干扰素-伽马(干扰素-伽马)通过VV表达时，在体内起到安全基因的作用，在免疫缺陷小鼠中使病毒减弱100多万倍。我们的假设是，表达安全基因的活的智能VV载体将显著更安全：任何不良反应的治疗都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，只有在出现或怀疑出现并发症，或者应该停止治疗时，才需要四环素治疗。在R21的应用下，将解决两个具体目标：(1)开发仅在诱导剂存在的情况下表达安全基因的智能VV载体；(2)评估新载体的安全性和有效性。首先，构建在构成VV启动子下表达四环素抑制子的SMART VV载体和在工程可诱导启动子下表达报告基因绿色荧光蛋白(GFP)的SMART VV载体，并检测GFP的诱导作用以优化系统。然后，将开发一种可诱导表达小鼠干扰素-γ的SMART载体，作为模型安全基因。正常和免疫缺陷小鼠将被给予诱导性表达干扰素-γ的VV载体，在存在和不存在诱导剂的情况下，将评估存活率、斑点病变消退、疾病恢复、体重减轻和病毒复制。此外，还将评估对VV的免疫反应，以确保新载体的效力不会因四环素抑制物的表达或四环素治疗而受到影响。

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.