Xu L, Zerby D, Huang Y, Ji H, Nyanguile O F, de los Angeles J E, Kadan M J
Genetic Therapy, Inc. (A Novartis Company), 9 West Watkins Mill Road, Gaithersburg, Maryland, 20878, USA.
Mol Ther. 2001 Feb;3(2):262-73. doi: 10.1006/mthe.2000.0254.
The ability to regulate transgene expression will be essential for the safety and efficacy of many gene therapies. Various ligand-dependent transcription factors, including steroid hormone receptors, have been modified to enable transgene-specific regulation. To minimize effects on cellular gene expression, chimeric steroid receptors have been constructed by replacing their native DNA binding domain (DBD) with a heterologous DBD, like that from the yeast transcription factor GAL4. This approach has limitations for human gene therapy, including the potential immunogenicity of the GAL4 domain and the inability to discriminate between different GAL4-linked transgenes in the same cell. To address this, we have constructed chimeric regulators containing the human estrogen receptor (ER) ligand binding domain (LBD) and a Cys(2)-His(2)-type zinc finger DBD. Cys(2)-His(2) zinc finger domains are common among human DNA binding proteins and can be engineered to selectively bind different DNA sequences. We demonstrate over 500-fold drug-dependent transgene induction with these chimeric regulators in vitro and the ability to regulate an adenovirus-delivered transgene in mice. Two chimeras containing different Cys(2)-His(2) domains displayed highly sequence-specific binding and regulation. Incorporating a point mutation in the ER LBD that ablates estrogen binding enables selective in vivo regulation with the clinically useful anti-estrogen tamoxifen. These Cys(2)-His(2)-ER LBD chimeras represent a versatile framework for creating transgene-specific regulators potentially useful for human gene therapy applications.
对许多基因治疗的安全性和有效性而言，调控转基因表达的能力至关重要。包括类固醇激素受体在内的多种配体依赖性转录因子已被改造，以实现转基因特异性调控。为了尽量减少对细胞基因表达的影响，人们构建了嵌合类固醇受体，方法是用异源DNA结合结构域（DBD）取代其天然DBD，比如来自酵母转录因子GAL4的DBD。这种方法在人类基因治疗中存在局限性，包括GAL4结构域的潜在免疫原性以及无法在同一细胞中区分不同的与GAL4相连的转基因。为了解决这个问题，我们构建了包含人雌激素受体（ER）配体结合结构域（LBD）和Cys(2)-His(2)型锌指DBD的嵌合调节因子。Cys(2)-His(2)锌指结构域在人类DNA结合蛋白中很常见，可以通过工程设计使其选择性结合不同的DNA序列。我们在体外证明了这些嵌合调节因子能实现超过500倍的药物依赖性转基因诱导，以及在小鼠中调控腺病毒递送的转基因的能力。两个包含不同Cys(2)-His(2)结构域的嵌合体表现出高度序列特异性的结合和调控。在ER LBD中引入一个消除雌激素结合的点突变，能够用临床上有用的抗雌激素他莫昔芬进行选择性体内调控。这些Cys(2)-His(2)-ER LBD嵌合体代表了一个通用框架，可用于创建可能对人类基因治疗应用有用的转基因特异性调节因子。
