Nonviral delivery of CRISPR-Cas9 into hepatocytes combined with APAP selection for treatment of Familial Hypercholesterolemia

项目总结 家族性高胆固醇血症(FH)每250人中就有1人患病，其特征是低密度脂蛋白受损 脂蛋白(LDL)代谢导致过早的心血管疾病。CRISPR-CAS9-导致的 编码血管生成素样3(Angptl3)的基因的功能突变被认为是一种治疗方法 永久降低低密度脂蛋白和甘油三酯水平并降低心血管疾病风险的战略。 然而，缺乏安全和有效的方法将CRISPR组分输送到肝细胞是一种 主要障碍。腺相关病毒(AAVs)是传递基因编辑试剂的首选平台 但却伴随着严重的局限性。此外，Cas9免疫在人类中高度流行。 人口数量进一步增加了AAV传播的复杂性。我们建议将基因编辑试剂引入肝细胞 使用非病毒体外传递方法，然后移植工程肝细胞来取代疾病 肝细胞治疗FH。我们的非病毒体外策略避免了体内方法的两个并发症，因为 CRISPR-Cas9编辑仅限于预期的目标单元格，并使有机会保持 细胞在培养中，直到它们不再具有免疫原性。但这种体外方法有一个潜在的缺点： 如何增加移植到肝脏的编辑肝细胞的数量。为了应对这一挑战，我们将使用 一种用发热药物对乙酰氨基酚(APAP)选择编辑肝细胞的新方法。我们 假设缺乏NADPH-细胞色素P450氧化还原酶(Cypor)的基因编辑的肝细胞 通过APAP给药体内浓缩，不会对肝脏造成永久性损害。在建议的研究中，我们会 使用CRISPR-Cas9干扰Cypor和AAVs直接比较LNPs、电穿孔和AAVs进行基因编辑 Angptl3.然后，我们将用基因编辑的肝细胞替换已建立的小鼠中的患病肝细胞 使用APAP选择的跳频模型(Ldlr-/-)。在目标1中，我们将比较多路传输的特异性和效率 电穿孔、LNP和AAV介导的CRISPR-Cas9基因在Cypor和Angptl3中的基因编辑 原代小鼠肝细胞。对于目标2，我们将评估和比较LNP、电穿孔和 腺相关病毒介导的Cypor-CRISPR-Cas9体外载体对肝细胞克隆扩增能力的影响 用APAP选择对野生型C57BL/6小鼠的肝脏进行再填充。在目标3中，我们将比较 非病毒和AAV介导的Cypor和Angptl3-CRISPR-Cas9复合递送对Cypor和Angptl3-CRISPR-Cas9能力的影响 肝细胞降低Ldlr-/-小鼠血浆胆固醇和甘油三酯水平及规避Cas9免疫 接受一过性APAP治疗。这个项目是第一个直接比较不同非病毒的 体外构建肝细胞的AAVs方法及对肝细胞编辑程度的评价 用APAP治疗FH进行体内克隆性扩增。此外，这项研究是第一次研究疫苗的免疫原性。 体外基因编辑的肝细胞。

PROJECT SUMMARY Familial hypercholesterolemia (FH) affects 1 in 250 people and is characterized by impaired low-density lipoprotein (LDL) metabolism resulting in premature cardiovascular disease. CRISPR-Cas9-induced loss of function mutations in the gene encoding Angiopoietin-like 3 (ANGPTL3) has been proposed as a therapeutic strategy to permanently reduce LDL cholesterol and triglyceride levels and lower cardiovascular disease risks. However, the absence of safe and effective methods for delivering CRISPR components into hepatocytes is a major barrier. Adeno-associated viruses (AAVs) are the platform of choice for delivering gene-editing reagents but are associated with severe limitations. In addition, Cas9 immunity is highly prevalent in the human population further complicating AAV delivery. We propose to introduce gene-editing reagents into hepatocytes using nonviral delivery approaches ex vivo, then transplanting the engineered hepatocytes to replace diseased hepatocytes to treat FH. Our nonviral ex vivo strategy avoids two complications of in vivo approaches since CRISPR-Cas9 editing is only restricted to the intended target cells and enables the opportunity to maintain cells in culture until they are no longer immunogenic. But this ex vivo approach has one potential drawback: How to enhance the number of edited hepatocytes engrafted in the liver. To address this challenge, we will use a novel approach for selecting edited hepatocytes using fever medicine acetaminophen (APAP). We hypothesize that gene edited hepatocytes lacking NADPH-cytochrome P450 oxidoreductase (Cypor) will be enriched in vivo by APAP administration without permanent liver damage. In the proposed studies, we will directly compare LNPs, electroporation and AAVs for gene editing using CRISPR-Cas9 to disrupt Cypor and Angptl3. We will then replace diseased hepatocytes with gene edited hepatocytes in an established mouse model of FH (Ldlr–/– ) using APAP selection. In Aim 1, we will compare specificity and efficiency of multiplex gene editing in Cypor and Angptl3 by electroporation, LNP, and AAV-mediated delivery of CRISPR-Cas9 in primary mouse hepatocytes. For Aim 2, we will evaluate and compare the effects of LNP, electroporation, and AAV-mediated ex vivo delivery of Cypor-CRISPR-Cas9 on the capacity of hepatocytes to clonally expand and repopulate the liver in wild type C57BL/6 mice using APAP selection. In Aim 3, we will compare the effects of nonviral and AAV-mediated multiplex delivery of Cypor and Angptl3-CRISPR-Cas9 on the capacity of hepatocytes to lower plasma cholesterol and triglyceride levels and circumvent Cas9 immunity in Ldlr–/– mice subjected to transient APAP treatment. This project is the first to directly compare different nonviral approaches to AAVs for engineering hepatocytes ex vivo and evaluate the extent that edited hepatocytes clonally expand in vivo using APAP to treat FH. In addition, this study is the first to study the immunogenicity of ex vivo gene edited hepatocytes.