Engineering epigenetic therapy for sickle cell disease

描述（由申请人提供）：与限制性内切酶融合的定制DNA结合域的出现改变了生物医学研究的前景，并预示着基因治疗的新时代。I期临床试验强调了这一事实，其中锌指核酸酶用于敲除HIV感染个体的CD 34 + T细胞中的CCR 5受体以产生HIV抗性T细胞（临床试验#NCT00842634）。除了靶向基因敲除之外，“设计者核酸酶”的其他生物医学应用是基因校正和基因激活。然而，基因校正需要在DNA切割后进行同源重组-这是一个非常低效的过程-并且目前用于稳定基因活化的策略需要异位蛋白的组成型表达，这些限制可能阻碍治疗功效。与内源性基因编辑或遗传激活相反，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编程。 作为这项技术的概念验证，我建议工程师位点特异性DNA结合模块融合与DNA去甲基化酶的胎儿血红蛋白（HbF）的表观遗传诱导治疗镰状细胞病（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形并阻塞小血管，导致剧烈疼痛和广泛的医疗并发症。胎儿血红蛋白通常在出生时由于g-珠蛋白基因座的DNA甲基化而沉默，因为成人血红蛋白增加。由于HbF的小幅增加可以治愈这种疾病，SCD是开发表观遗传重编程技术的最佳应用。 表观遗传工程的主要优点是两条链上CpG的成功DNA去甲基化将导致持久的HbF诱导，而不需要异位蛋白的连续表达。这种高度创新的策略在SCD文献中没有先例。这项技术的发展将带来强大而持久的

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