Using Zinc Finger Nucleases to Stimulate Gene Targeting in HSC

描述(申请人提供)：遗传性单基因疾病渗透医学。在基因组时代的黎明，我们对遗传基因差异对疾病易感性的贡献的理解只会越来越多。镰状细胞病是第一种发现氨基酸和核酸突变的单基因疾病，它警告说，了解一种疾病的遗传原因并不容易导致治疗。有几种潜在的方法可以在基因组水平上治疗遗传病。一种特别耐人寻味的方法是通过纠正导致疾病的突变来治愈这些疾病。对于镰状细胞病，这需要在造血干细胞中将突变的胸腺嘧啶重新转化为p-珠蛋白基因第6密码子中的腺嘌呤，然后将校正后的干细胞取回给患者，就像自体干细胞移植一样。在过去的几年里，两个主要的进展使得通过基因打靶进行基因校正的可能性变得更有希望。第一个是发现目标基因中的DNA双链断裂可以刺激基因靶向。我们已经发现，刺激可以达到50,000倍。第二个是我们的发现，模型锌指核酸酶可以通过在哺乳动物基因组中产生双链断裂来刺激基因打靶，我们的初步结果表明，锌指核酸酶可以被设计成刺激内源序列的基因打靶。双链断裂介导的基因打靶研究的下一步是在原代细胞而不是细胞系中研究这一过程。这项建议旨在研究基因打靶的过程和锌指核酸酶在造血祖细胞中的应用。这些研究的目的既是为了了解这些细胞中基因靶向的生物学，也是为了利用这种理解开发靶向作为治疗单基因疾病的工具，如镰状细胞疾病。

DESCRIPTION (provided by applicant): Inherited monogenic diseases permeate medicine. At the dawn of the genome era, our understanding of the contribution of inherited genetic differences to disease susceptibility is only increasing. Sickle cell disease, the first monogenic disease for which the amino acid and nucleic acid mutations were identified, cautions that knowing the genetic cause of a disease does not easily lead to therapies. There are several potential approaches to treating genetic diseases at the genome level. A particularly intriguing approach is to cure such diseases by correcting the mutation that causes the disease. For sickle cell disease this would entail converting the mutated thymine back to an adenine in codon 6 of the p-globin gene in hematopoietic stem cells and then retrieving the corrected stem cells to the patient as in an autologous stem cell transplant. In the last several years, two major advances have made the possibility of gene correction by gene targeting more promising. The first is the discovery that a DMA double-strand break in the target gene can stimulate gene targeting. We have found that the stimulation can be up to 50,000 fold. The second is our discovery that model zinc finger nucleases can stimulate gene targeting by creating double-strand breaks in the mammalian genome and our preliminary results demonstrating that zinc finger nucleases can be designed to stimulate gene targeting at endogenous sequences. The next step in the study of double-strand break mediated gene targeting is to study the process in primary cells rather than cell lines. This proposal aims to study the process of gene targeting and the use of zinc finger nucleases in hematopoietic progenitor cells. The goal of these studies is both to understand the biology of gene targeting in these cells and to use that understanding to develop targeting as a therapeutic tool to treat monogenic diseases such as sickle cell disease.