Engineering epigenetic therapy for sickle cell disease

描述(由申请人提供)：融合到限制性内切酶的定制DNA结合域的出现改变了 生物医学研究的景观，并预示着基因治疗的新纪元。这一事实在以下方面得到了强调 锌指核酸酶用于敲除CCR5受体的I期临床试验 HIV感染者的CD34 T细胞产生对HIV有抵抗力的T细胞(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 基因修正和基因激活。然而，基因校正需要同源重组。 在DNA切割之后--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，这可能会阻碍治疗效果。在……里面 相对于内源性基因编辑或基因激活，我建议开发一个表观遗传平台 对任何感兴趣的轨迹进行重新编程。 作为这项技术的概念验证，我建议设计特定于位点的DNA结合模块 DNA脱甲基酶融合用于表观遗传诱导胎儿血红蛋白治疗 镰状细胞病(SCD)。SCD是由成人-珠蛋白基因突变引起的，这种基因迫使红色 血细胞镰刀状和闭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因-珠蛋白的DNA甲基化而沉默 随着成人血红蛋白的增加而增加。由于HBF的小幅增加可以治愈这种疾病，SCD是一种 开发表观遗传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将CPGS的DNA去甲基化 这两条链都将导致持久的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 将导致对SCD进行有效和持久的HBF诱导治疗，这将成为 研究性新药申请。此外，这项技术还可以适用于表观遗传学 其他生化修饰的重新编程，包括DNA甲基化和组蛋白乙酰化 和甲基化。这些修饰中的每一个都可能被用于治疗性基因沉默或 激活，这将是各种疾病的分子导向疗法的革命性变化。

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 -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 -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 HbF-induction therapy for SCD that will become the basis for an investigational new drug application. Moreover, this technology can be adapted to epigenetic reprogramming of other biochemical modifications including DNA methylation and histone acetylation and methylation. Each of these modifications could be targeted for therapeutic gene silencing or activation which will revolutionize molecularly-directed therapy for a variety of diseases.