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。随着老龄化的快速增长 在人口方面，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给社会带来了巨大的经济负担，给人们带来了巨大的压力 医疗保健系统。这些趋势将会恶化，因为没有阻止或预防AD的治疗方法，预计 到2050年，每年的成本超过1.1万亿美元。尽管有所有的研究努力、资金和承诺，但仍有 没有治愈AD的方法，也没有任何疾病修改疗法(DMT)来减缓甚至延缓AD的进展 疾病。此外，许多确定AD疾病修正疗法(DMT)的临床试验都失败了。 因此，AD仍然是一个严重的未得到满足的医疗需求，迫切需要重新关注其他目标和 将AD药物开发的范式转向精准医学。载脂蛋白E(ApoE)是 迟发性阿尔茨海默病(LOAD)的最强和最具重复性的遗传风险因素。近期 在细胞和小鼠模型中的研究表明，APOE水平降低50%是有益的。 总而言之，这些观察结果支持APOE作为一种新的新兴疗法的发展 目标已装填。CLAIRIgene和杜克大学的合作伙伴在这个STTR阶段1提议开发 表观基因组编辑工具，精确地以e4等位基因特异的方式下调APOE的表达。这个 技术原型基于CRISPR/失活(D)CAS技术与表观基因组修饰物融合 这抑制了基因表达，并通过慢病毒(LV)载体传递。我们将开发这项技术原型 通过实现两个具体目标。Aim 1将开发该系统以精确减少apoE e4等位基因 表达并评价利用携带HIPSC基因的等基因hPSC系的技术的有效性和特异性 E4/4、E3/4和E3/3三种基因型。我们预计apoE e4-mrna和蛋白水平会有特定的降低。 至≥50%。目标2将使用来自患者的HiPSC来验证该系统的有益影响 APOE e4等位基因纯合，将分化为神经元、星形胶质细胞和小胶质细胞样细胞 模特们。我们预计这些实验将为该系统的可行性提供概念证明 有效挽救LOAD特有的病理表型。预期结果与 NIA的使命是开发可能推动预防和治疗进展的创新产品 AD及相关痴呆(ADRD)。在第一阶段完成后，CLAIRIgene将证明 有针对性的表观基因组编辑，以特异性和高效地降低载脂蛋白e4水平，并提供体外证据- 该策略在逆转分子和细胞病理表型方面具有有益效果的概念 与负载相关。这将为将重点放在AD动物体内验证的第二阶段奠定基础 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，以提高精确度 药装好了。

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.