Engineering epigenetic therapy for sickle cell disease

描述(由申请人提供)：定制的DNA结合结构域融合到限制酶的出现改变了生物医学研究的格局，并预示着基因治疗的新纪元。在第一阶段临床试验中，锌指核酸酶被用来敲除艾滋病毒感染者CD34+T细胞中的CCR5受体，以产生抗艾滋病毒的T细胞，这一事实得到了突显(临床试验#NCT00842634)。除了有针对性的基因敲除外，“设计核酸酶”的其他生物医学应用还包括基因校正和基因激活。然而，基因纠正需要在DNA切割后进行同源重组--这是一个非常低效的过程--而目前稳定的基因激活策略需要异位蛋白的结构性表达，这些限制可能会阻碍治疗效果。与内源性基因编辑或基因激活不同，我建议开发一个平台，用于对任何感兴趣的基因座进行表观遗传重新编程。作为这项技术的概念验证，我建议设计一种带有DNA去甲基化酶的位点特异性DNA结合模块融合，用于表观遗传诱导胎儿血红蛋白(HBF)，用于治疗镰状细胞病(SCD)。SCD是由成人b-珠蛋白基因突变引起的，该基因迫使红细胞镰刀状并阻塞小血管，导致剧烈疼痛和广泛的医疗并发症。胎儿的血红蛋白通常在出生时被g-珠蛋白基因的DNA甲基化抑制，而成人的血红蛋白增加。由于HBF的小幅增加可以治愈这种疾病，SCD是开发表观遗传学重新编程技术的最佳应用。表观遗传工程的主要优点是成功地使CPGS在两条链上的DNA去甲基化将导致持久的HBF诱导，而不需要持续表达异位蛋白。这种高度创新的策略在SCD文献中是没有先例的。这项技术的发展将带来强大和持久的

DESCRIPTION (provided by applicant): The advent of custom built DNA binding domains fused to restriction enzymes changed the landscape of biomedical research and heralded a new age in gene therapy. This fact is highlighted by Phase I clinical trials in which zinc finger nucleases are being used to knockout the CCR5 receptor in CD34+ T cells of HIV infected individuals to generate HIV-resistant T cells (clinical trial #NCT00842634). In addition to targeted gene knockout, other biomedical applications of the "designer nucleases" are gene correction and gene activation. However, gene correction requires homologous recombination following a DNA cut - a very inefficient process - and current strategies for stable gene activation require constitutive expression of an ectopic protein, limitations that are likely to hinder therapeutic efficacy. In contrast to endogenous gene editing or genetic activation, I propose to develop a platform for epigenetic reprogramming of any locus of interest. As a proof-of-concept for this technology, I propose to engineer site-specific DNA binding module fusions with DNA demethylating enzymes for epigenetic induction of fetal hemoglobin (HbF) for therapy of sickle cell disease (SCD). SCD is caused by mutation of the adult b-globin gene which forces red blood cells to sickle and occlude small blood vessels leading to exquisite pain and a wide range of medical complications. Fetal hemoglobin is normally silenced at birth by DNA methylation of the g-globin locus as adult hemoglobin increases. Because small increases in HbF can cure this disease, SCD is an optimal application for developing epigenetic re-programming technologies. The major advantage of epigenetic engineering is that successful DNA demethylation of CpGs on both strands will lead to durable HbF-induction, not requiring continuous expression of ectopic proteins. This highly-innovative strategy has no precedent in the SCD literature. Development of this technology will lead to potent and durable