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

项目摘要 家族性高胆固醇血症（FH）影响1/250人，其特征是低密度受损 脂蛋白（LDL）代谢导致过早的心血管疾病。CRISPR-Cas9诱导的细胞凋亡的丧失 已经提出在编码血管生成素样3（ANGPTL 3）的基因中的功能突变作为治疗剂 长期降低低密度脂蛋白胆固醇和甘油三酯水平，降低心血管疾病风险的策略。 然而，缺乏安全有效的方法将CRISPR组分递送到肝细胞中是一个挑战。 主要障碍。腺相关病毒（AAV）是递送基因编辑试剂的首选平台 但是与严重的局限性相关联。此外，Cas9免疫在人类中高度流行， 这进一步使AAV递送复杂化。我们建议将基因编辑试剂引入肝细胞 使用非病毒递送方法离体，然后移植工程化肝细胞以替代病变的肝细胞， 肝细胞治疗FH。我们的非病毒离体策略避免了体内方法的两个并发症， CRISPR-Cas9编辑仅限于预期的靶细胞，并使维持CRISPR-Cas9编辑的机会成为可能。 培养细胞，直到它们不再具有免疫原性。但这种离体方法有一个潜在的缺点： 如何增加移植到肝脏中的编辑肝细胞的数量。为了应对这一挑战，我们将使用 使用发热药物对乙酰氨基酚（APAP）选择编辑的肝细胞的新方法。我们 假设缺乏NADPH-细胞色素P450氧化还原酶（Cypor）的基因编辑肝细胞将 通过APAP施用在体内富集，而没有永久性肝损伤。在建议的研究中，我们会 使用CRISPR-Cas9直接比较LNP、电穿孔和AAV进行基因编辑以破坏Cypor， Angptl3.然后，我们将在建立的小鼠中用基因编辑的肝细胞替换患病的肝细胞 使用APAP选择的FH（Ldlr-/-）模型。在目标1中，我们将比较多重PCR的特异性和效率。 通过电穿孔、LNP和AAV介导的CRISPR-Cas9递送在Cypor和Angptl 3中进行基因编辑， 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 AAV介导的Cypor-CRISPR-Cas9的离体递送对肝细胞克隆扩增和增殖能力的影响 使用APAP选择在野生型C57 BL/6小鼠中重新填充肝脏。在目标3中，我们将比较 Cypor和Angptl 3-CRISPR-Cas9的非病毒和AAV介导的多重递送对免疫调节能力的影响 肝细胞降低血浆胆固醇和甘油三酯水平并规避Ldlr-/-小鼠中的Cas9免疫 进行短暂的APAP治疗。这个项目是第一个直接比较不同的非病毒 用于离体工程化肝细胞的AAV的方法，并评估编辑的肝细胞 使用APAP在体内克隆扩增以治疗FH。此外，本研究首次研究了 离体基因编辑的肝细胞。

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.