de Leeuw Charles N, Dyka Frank M, Boye Sanford L, Laprise Stéphanie, Zhou Michelle, Chou Alice Y, Borretta Lisa, McInerny Simone C, Banks Kathleen G, Portales-Casamar Elodie, Swanson Magdalena I, D'Souza Cletus A, Boye Shannon E, Jones Steven J M, Holt Robert A, Goldowitz Daniel, Hauswirth William W, Wasserman Wyeth W, Simpson Elizabeth M
Centre for Molecular Medicine and Therapeutics at the Child and Family Research Institute, University of British Columbia, Vancouver, BC, Canada V5Z 4H4 ; Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada V5Z 4H4.
Department of Ophthalmology, College of Medicine, University of Florida, Gainesville, FL, USA 32610.
Mol Ther Methods Clin Dev. 2014 Jan 8;1:5. doi: 10.1038/mtm.2013.5.
Critical for human gene therapy is the availability of small promoter tools to drive gene expression in a highly specific and reproducible manner. We tackled this challenge by developing human DNA MiniPromoters using computational biology and phylogenetic conservation. MiniPromoters were tested in mouse as single-copy knock-ins at the locus on the X Chromosome, and evaluated for lacZ reporter expression in CNS and non-CNS tissue. Eighteen novel MiniPromoters driving expression in mouse brain were identified, two MiniPromoters for driving pan-neuronal expression, and 17 MiniPromoters for the mouse eye. Key areas of therapeutic interest were represented in this set: the cerebral cortex, embryonic hypothalamus, spinal cord, bipolar and ganglion cells of the retina, and skeletal muscle. We also demonstrated that three retinal ganglion cell MiniPromoters exhibit similar cell-type specificity when delivered via adeno-associated virus (AAV) vectors intravitreally. We conclude that our methodology and characterization has resulted in desirable expression characteristics that are intrinsic to the MiniPromoter, not dictated by copy number effects or genomic location, and results in constructs predisposed to success in AAV. These MiniPromoters are immediately applicable for pre-clinical studies towards gene therapy in humans, and are publicly available to facilitate basic and clinical research, and human gene therapy.
对于人类基因治疗至关重要的是要有小型启动子工具，以便以高度特异性和可重复的方式驱动基因表达。我们通过利用计算生物学和系统发育保守性开发人类DNA微型启动子来应对这一挑战。微型启动子在小鼠中作为X染色体上该位点的单拷贝敲入进行测试，并评估其在中枢神经系统和非中枢神经系统组织中lacZ报告基因的表达。鉴定出18个在小鼠脑中驱动表达的新型微型启动子，2个用于驱动泛神经元表达的微型启动子，以及17个用于小鼠眼睛的微型启动子。这一组中涵盖了治疗关注的关键区域：大脑皮层、胚胎下丘脑、脊髓、视网膜的双极细胞和神经节细胞以及骨骼肌。我们还证明，当通过腺相关病毒（AAV）载体玻璃体内递送时，三个视网膜神经节细胞微型启动子表现出相似的细胞类型特异性。我们得出结论，我们的方法和表征产生了微型启动子固有的理想表达特征，不受拷贝数效应或基因组位置的影响，并导致构建体易于在AAV中取得成功。这些微型启动子可立即应用于人类基因治疗的临床前研究，并已公开提供，以促进基础和临床研究以及人类基因治疗。
