Transgene Regulation Using the Tetracycline-Inducible TetR-KRAB System after AAV-Mediated Gene Transfer in Rodents and Nonhuman Primates

Numerous studies have demonstrated the efficacy of the Adeno-Associated Virus (AAV)-based gene delivery platform in vivo. The control of transgene expression in many protocols is highly desirable for therapeutic applications and/or safety reasons. To date, the tetracycline and the rapamycin dependent regulatory systems have been the most widely evaluated. While the long-term regulation of the transgene has been obtained in rodent models, the translation of these studies to larger animals, especially to nonhuman primates (NHP), has often resulted in an immune response against the recombinant regulator protein involved in transgene expression regulation. These immune responses were dependent on the target tissue and vector delivery route. Here, using AAV vectors, we evaluated a doxycyclin-inducible system in rodents and macaques in which the TetR protein is fused to the human Krüppel associated box (KRAB) protein. We demonstrated long term gene regulation efficiency in rodents after subretinal and intramuscular administration of AAV5 and AAV1 vectors, respectively. However, as previously described for other chimeric transactivators, the TetR-KRAB-based system failed to achieve long term regulation in the macaque after intramuscular vector delivery because of the development of an immune response. Thus, immunity against the chimeric transactivator TetR-KRAB emerged as the primary limitation for the clinical translation of the system when targeting the skeletal muscle, as previously described for other regulatory proteins. New developments in the field of chimeric drug-sensitive transactivators with the potential to not trigger the host immune system are still needed.多项研究已证实，基于腺相关病毒(AAV)的基因递送平台在体内应用中具备优异有效性。出于治疗应用或安全性考量，对转基因表达实现精准调控是多数实验方案的核心诉求。截至目前，四环素依赖型与雷帕霉素依赖型调控系统是受评估最为广泛的两类基因表达调控方案。尽管在啮齿动物模型中已成功实现转基因的长期调控，但将此类研究成果推广至大型动物——尤其是非人类灵长类动物(NHP)时，往往会引发针对转基因表达调控所需重组调控蛋白的免疫应答。此类免疫应答的发生情况与靶组织及载体递送途径密切相关。本研究依托AAV载体，在啮齿类动物与猕猴中评估了一款将四环素阻遏蛋白(TetR)与人Krüppel相关盒(KRAB)蛋白融合的多西环素诱导调控系统。研究结果表明，分别通过视网膜下途径递送AAV5载体、肌内途径递送AAV1载体后，该系统可在啮齿类动物中实现长效基因调控。然而，正如此前针对其他嵌合反式激活因子的研究结论，基于TetR-KRAB的系统在猕猴体内经肌内载体递送后，因免疫应答的产生而未能实现长效基因调控。由此可见，当靶向骨骼肌时，针对嵌合反式激活因子TetR-KRAB的免疫应答是制约该系统临床转化的核心限制因素，这与此前其他调控蛋白的相关研究结果一致。目前，研发可避免触发宿主免疫系统的嵌合药物敏感型反式激活因子仍是该领域亟待突破的方向。