Using Zinc Finger Nucleases to Stimulate Gene Targeting in HSC

遗传性单基因疾病渗透到医学领域。在基因组时代的黎明， 理解遗传基因差异对疾病易感性的贡献， 增加。镰状细胞病，第一个单基因疾病的氨基酸和核酸 突变被确定，警告说，知道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疾病。 一种特别有趣的方法是通过纠正导致这种疾病的突变来治愈这种疾病。 疾病对于镰状细胞病，这需要将突变的胸腺嘧啶转化回腺嘌呤 在造血干细胞中的β-珠蛋白基因的密码子6中， 干细胞移植是一种自体干细胞移植。近年来，两大 这些进展使得通过基因靶向进行基因校正的可能性更有希望。一是 发现靶基因中的DMA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。第二个是我们发现模型锌 指状核酸酶可以通过在哺乳动物中产生双链断裂来刺激基因靶向 基因组和我们的初步结果表明，锌指核酸酶可以被设计成 刺激内源序列基因靶向。双链断裂研究的下一步 介导的基因靶向是在原代细胞而不是细胞系中研究该过程。这项建议 目的研究锌指核酸酶基因打靶的过程及其在造血干细胞移植中的应用， 祖细胞这些研究的目的是了解这些基因靶向的生物学， 细胞，并利用这种理解来开发靶向治疗工具，以治疗单基因 镰状细胞病等疾病。

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.