Zerby Dennis, Sakhuja Kiran, Reddy P Seshidhar, Zimmerman Heather, Kayda Dawn, Ganesh Shanthi, Pattison Scott, Brann Terrence, Kadan Michael J, Kaleko Michael, Connelly Sheila
Genetic Therapy, Inc. (A Novartis Company), Gaithersburg, MD 20878, USA.
Hum Gene Ther. 2003 May 20;14(8):749-61. doi: 10.1089/104303403765255147.
Transcriptional regulation that is rapid, reversible, and repeatedly inducible would greatly enhance the safety and efficacy of many gene therapy strategies. We developed a chimeric ligand-inducible regulation system based on the human estrogen receptor. This system has two components, the responsive promoter driving expression of the transgene of interest, and the ligand-inducible chimeric transcription factor. The transcription factor is composed of a novel DNA binding domain and a modified estrogen receptor ligand-binding domain. A point mutation in the ligand-binding domain significantly reduces estrogen binding while allowing binding of the estrogen antagonist, tamoxifen. We used a gutless adenoviral vector system and incorporated both components into two separate vectors. A single gutless vector encoding both system components was also generated. The tamoxifen-mediated induciblity of transgene expression of the gutless vector system was compared in vitro and in vivo with the analogous components incorporated into early generation, E1/E2a/E3-deficient adenoviral vectors. In normal mice, both the gutless vector and early generation systems displayed inducibility in the presence of tamoxifen. Importantly, the gutless vector system was inducible to extremely high levels, at least four times over a 2-month period. In contrast, the early generation vector system was inducible only once. Furthermore, the early generation system displayed significant toxicity, as evidenced by extremely high liver enzyme levels, abnormal liver pathology, and rapid loss of vector DNA from the liver, while the gutless vector system displayed minimal toxicity. These data directly demonstrate the improved in vivo function of the tamoxifen-inducible transcriptional regulation system in the context of the gutless adenoviral vectors.
快速、可逆且可反复诱导的转录调控将极大提高许多基因治疗策略的安全性和有效性。我们基于人雌激素受体开发了一种嵌合配体诱导调控系统。该系统有两个组成部分，即驱动目的转基因表达的响应启动子和配体诱导嵌合转录因子。转录因子由一个新型DNA结合结构域和一个修饰的雌激素受体配体结合结构域组成。配体结合结构域中的一个点突变显著降低了雌激素结合能力，同时允许雌激素拮抗剂他莫昔芬结合。我们使用了无病毒腺病毒载体系统，并将这两个组成部分分别整合到两个载体中。还构建了一个编码这两个系统组成部分的单一无病毒载体。将无病毒载体系统中他莫昔芬介导的转基因表达诱导性在体外和体内与整合到早期一代E1/E2a/E3缺陷型腺病毒载体中的类似组成部分进行了比较。在正常小鼠中，无病毒载体和早期一代系统在存在他莫昔芬的情况下均表现出诱导性。重要的是，无病毒载体系统可诱导到极高水平，在2个月内至少诱导四倍。相比之下，早期一代载体系统仅能诱导一次。此外，早期一代系统表现出显著毒性，表现为肝酶水平极高、肝脏病理异常以及肝脏中载体DNA迅速丢失，而无病毒载体系统表现出最小毒性。这些数据直接证明了在无病毒腺病毒载体背景下，他莫昔芬诱导转录调控系统在体内功能的改善。
