Directed Evolution of Homing Endonucleases for Human Gene Therapy

描述（由申请人提供）：归巢核酸内切酶是催化DNA序列特异性双链断裂的酶，并可显著刺激细胞中这些断裂处的同源重组。这些酶具有巨大的应用潜力，例如基因治疗中的基因校正或系统生物学、功能基因组学、干细胞工程和代谢工程中的基因改变。然而，归巢核酸内切酶具有有限的靶序列的天然库，这严重阻碍了它们的应用。本研究的广泛和长期目标是设计归巢核酸内切酶，识别和切割与人类遗传疾病相关的新DNA序列，并研究归巢核酸内切酶的精致DNA序列特异性的分子决定因素。使用一个很好的特点归巢核酸内切酶I-SceI作为模型系统，我们将使用组合蛋白质工程策略，以产生I-SceI变异体，切割新的靶DNA序列中发现的突变血红蛋白β基因与镰状细胞贫血症。这种工程I-SceI突变体作为镰状细胞性贫血的新型基因治疗剂具有巨大的潜力。此外，我们将使用各种生物化学和生物物理方法来表征选择工程I-SceI变体在体外和哺乳动物细胞中。这些研究将为归巢核酸内切酶DNA序列特异性的分子基础提供新的见解。同样重要的是，我们预计这个探索性/开发性（R21）项目将建立一个技术平台，用于生物医学研究和人类基因治疗的各种DNA修饰酶的工程。

DESCRIPTION (provided by applicant): Homing endonucleases are enzymes that catalyze DNA sequence specific double-strand breaks and can significantly stimulate homologous recombination at these breaks in cells. These enzymes have great potential for applications such as gene correction in gene therapy or gene alteration in systems biology, functional genomics, stem cell engineering, and metabolic engineering. However, homing endonucleases have a limited natural repertoire of target sequences, which severely hampers their applications. The broad and long-term goal of this research is to engineer homing endonucleases that recognize and cleave novel DNA sequences that are linked with human genetic disorders and to investigate the molecular determinants of the exquisite DNA sequence specificity of homing endonucleases. Using a well-characterized homing endonuclease I-SceI as a model system, we will use combinatorial protein engineering strategies to generate I-SceI variants that cleave new target DNA sequences found in a mutant hemoglobin beta gene associated with sickle cell anemia. Such engineered I-SceI mutants hold tremendous potential as novel gene therapy agents for sickle cell anemia. In addition, we will use a variety of biochemical and biophysical methods to characterize select engineered I-SceI variants both in vitro and in mammalian cells. Such studies will provide novel insights into the molecular basis of the homing endonuclease DNA sequence specificity. Equally important, we anticipate that this exploratory/developmental (R21) project will establish a technology platform for engineering a wide variety of DNA-modifying enzymes for biomedical research and human gene therapy.