Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO 63110, USA.
Gene Ther. 2010 Nov;17(11):1390-9. doi: 10.1038/gt.2010.77. Epub 2010 May 13.
Given the remarkable recent progress in gene therapy-based treatments for retinal disease, there is an urgent need for the development of new approaches to quantitative design and analysis of photoreceptor-specific promoters. In this study, we determined the relative binding affinity of all single-nucleotide variants of the consensus binding site of the mammalian photoreceptor transcription factor, Crx. We then showed that it is possible to use these data to accurately predict the relative binding affinity of Crx for all possible 8 bp sequences. By rationally adjusting the binding affinity of three Crx sites, we were able to fine-tune the expression of the rod-specific Rhodopsin promoter over a 225-fold range in living retinas. In addition, we showed that it is possible to fine-tune the activity of the rod-specific Gnat1 promoter over ∼275-fold range by modulating the affinity of a single Crx-binding site. We found that the action of individual binding sites depends on the precise promoter context of the site and that increasing binding affinity does not always equate with increased promoter output. Despite these caveats, this tuning approach permits quantitative engineering of photoreceptor-specific cis-regulatory elements, which can be used as drivers in gene therapy vectors for the treatment of blindness.
鉴于基因治疗在视网膜疾病治疗方面的显著进展，迫切需要开发新的方法来定量设计和分析光感受器特异性启动子。在这项研究中，我们确定了哺乳动物光感受器转录因子 Crx 的共识结合位点的所有单核苷酸变异的相对结合亲和力。然后我们表明，使用这些数据可以准确预测 Crx 与所有可能的 8 个碱基对序列的相对结合亲和力。通过合理调整三个 Crx 结合位点的结合亲和力，我们能够在活体视网膜中精细调节 Rod 特异性视紫红质启动子的表达，范围为 225 倍。此外，我们还表明，通过调节单个 Crx 结合位点的亲和力，可以在约 275 倍的范围内精细调节 Rod 特异性 Gnat1 启动子的活性。我们发现，单个结合位点的作用取决于该位点的精确启动子背景，并且增加结合亲和力并不总是等同于增加启动子输出。尽管存在这些注意事项，但这种调谐方法允许对光感受器特异性顺式调控元件进行定量工程设计，可作为基因治疗载体中治疗失明的驱动子。
