APOE-targeted therapy for precision medicine in late onset Alzheimer's disease: A novel epigenome editing approach for downregulation of APOEe4 expression

摘要 阿尔茨海默病（AD）是老年痴呆症最常见的原因。随着人口老龄化的加剧， 在人口中，AD病例的数量正在快速增长，预计在未来三年内将大幅上升 几十年因此，AD给社会带来巨大的经济负担，给社会带来巨大的压力。 医疗保健系统。这些趋势将恶化，因为没有治疗方法来阻止或预防AD，预计 到2050年每年将花费超过1.1万亿美元。尽管所有的研究努力，金钱和承诺， 没有治愈AD的方法，也没有任何疾病修饰疗法（DMT）来减缓甚至延迟AD的进展。 疾病此外，许多临床试验，以确定疾病修饰疗法（DMT）的AD已经失败。 因此，AD仍然是一个关键的未满足的医疗需求，迫切需要重新关注其他目标， 将AD药物开发的范式转向精准医学。载脂蛋白E（APOE）是 是晚发性阿尔茨海默病（LOAD）最强和最可重复的遗传风险因素。最近 在细胞和小鼠模型中的研究表明，APOE水平降低50%具有有益效果。 总的来说，这些观察结果支持APOE作为一种新兴治疗药物的发展 目标为LOAD。CLAIRIGene和杜克大学的合作伙伴在STTR第一阶段提出开发 表观基因组编辑工具，以e4等位基因特异性方式精确下调APOE表达。的 技术原型基于CRISPR/失活（d）Cas技术与表观基因组修饰剂融合 其抑制基因表达并通过慢病毒（LV）载体递送。我们将开发这种技术原型 实现两个具体目标。目的1将开发精确减少APOE e4等位基因的系统 表达，并使用携带该基因的等基因hiPSC系评估该技术的功效和特异性。 e4/4、e3/4和e3/3基因型。我们预计APOE e4-mRNA和蛋白水平的特异性降低， 至≥ 50%。目标2将使用来自患者的hiPSC验证系统的有益影响 APOE e4等位基因的纯合子，其将分化为神经元、星形胶质细胞和小胶质细胞样细胞 模型我们希望这些实验将为系统的可行性提供概念验证， 有效地挽救LOAD特征性病理表型。预期成果与 NIA的使命是开发创新产品，推动预防和治疗疾病的进展。 AD和相关痴呆（ADRD）。在第1阶段完成后，CLAIRIGene将证明以下方法的可行性： 靶向表观基因组编辑，以特异性和有效地降低APOE e4水平，并提供体外证据- 这种策略在逆转分子和细胞病理表型方面具有有益作用 与负载有关。这将为II期研究奠定基础，II期研究将重点关注AD动物体内验证 模型，并最终将这种以APOE为靶点的表观基因组疗法推向临床研究， 医学在负载。

ABSTRACT Alzheimer's disease (AD) is the most common cause of dementia in aging. With a rapidly growing aging population, the number of AD cases is growing fast and projected to rise drastically over the next three decades. Therefore, AD poses a huge economic burden on society, placing overwhelming strain on the healthcare system. These trends will worsen because there are no therapies to halt or prevent AD, projected to cost more than $1.1 trillion annually by 2050. Despite all the research effort, money, and commitment, there is no cure for AD, nor any disease-modifying therapies (DMT) to slow down or even delay the progression of the disease. Moreover, numerous clinical trials to identify disease-modifying therapies (DMT) for AD have failed. Thus, AD remains a critical unmet medical need, and there is an urgent need to refocus on other targets and shifting the paradigm of AD drug development towards precision medicine. Apolipoprotein E (APOE) is the strongest and most reproducible genetic risk factor for late-onset Alzheimer's disease (LOAD). Recent studies in cellular and mouse models demonstrated that 50% reduction in APOE levels has beneficial effects. Collectively these observations lend support to the development of APOE as a new emerging therapeutic target for LOAD. CLAIRIgene, and partners at Duke University in this STTR Phase 1 propose to develop epigenome editing tools to downregulate APOE expression precisely and in e4 allele-specific manner. The technology prototype is based on CRISPR/deactivated(d)Cas technologies fused with epigenome modifiers that repress gene expression and delivered by lentiviral (LV) vehicle. We will develop this technology prototype by accomplishing two specific aims. Aim 1 will develop the system to precisely reduce APOE e4-allele expression and evaluate the efficacy and specificity of the technology using isogenic hiPSC lines carrying the e4/4, e3/4 and e3/3 genotypes. We expect specific reduction in APOE e4-mRNA and protein levels amounted to ≥50%. Aim 2 will validate the beneficial impact of the system using hiPSC derived from a patient homozygote for the APOE e4 allele that will be differentiated into neurons, astrocytes and microglia-like cellular models. We expect that these experiments will provide proof-of-concept for the feasibility of the system to effectively rescue pathological phenotypes characteristic of LOAD. The expected outcomes are relevant to the NIA's mission of the development of innovative products that may advance progress in preventing and treating AD and related dementias (ADRD). Upon completion of Phase 1, CLAIRIgene will have proven the feasibility of targeted epigenome editing to reduce APOE e4 levels specifically and efficiently and provide an in vitro proof- of-concept that this strategy has beneficial effects in reversing molecular and cellular pathological phenotypes related to LOAD. This will provide the foundation for Phase II which will focus on in vivo validation in AD animal models, and ultimately to advance this APOE-targeted epigenome therapy towards clinical studies for precision medicine in LOAD.