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

将 CRISPR-Cas9 非病毒递送至肝细胞结合 APAP 选择治疗家族性高胆固醇血症

基本信息

批准号：
10905153
负责人：
Renee Nicole Cottle
金额：
$ 56.58万
依托单位：
CLEMSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-11 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10905153
关键词：
ANGPTL3 gene Acetaminophen Address Affect Back C57BL/6 Mouse CRISPR/Cas technology Cardiovascular Diseases Cell Maintenance Cells Cholesterol Clinical Trials Clonal Expansion Clustered Regularly Interspaced Short Palindromic Repeats Cytochrome P450 Cytotoxic T-Lymphocytes Dependovirus Disease Electroporation Engineering Familial Hypercholesterolemia Fever Foundations Genes Genetic Engineering Hematopoietic stem cells Hepatocyte Human Immune response Immunity Impairment Inherited Insertional Mutagenesis LDL Cholesterol Lipoproteins Liver Liver diseases Low-Density Lipoproteins Major Histocompatibility Complex Malignant Neoplasms Mediating Medicine Membrane Proteins Messenger RNA Metabolic Metabolic Diseases Metabolism Methods Mus NADPH-Ferrihemoprotein Reductase Oxidoreductase Patients Peptides Persons Plasma Population Reagent Research Resected Resistance Ribonucleoproteins Risk Selection for Treatments Specificity T cell response T-Lymphocyte Testing Therapeutic Time Toxic effect Transplantation Triglycerides Viral Work cardiovascular disorder risk disease phenotype genome editing genotoxicity hepatocyte engraftment immunogenic immunogenicity in vivo lipid nanoparticle lipoprotein triglyceride liver injury liver transplantation loss of function mutation mouse model novel novel strategies premature programs

项目摘要

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.

项目概要家族性高胆固醇血症 (FH) 影响每 250 人中就有 1 人，其特点是低密度脂蛋白受损脂蛋白（LDL）代谢导致 CRISPR-Cas9 诱导的过早心血管疾病的丧失。编码血管生成素样 3 (ANGPTL3) 的基因的功能突变已被提议作为一种治疗方法永久降低低密度脂蛋白胆固醇和甘油三酯水平并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 成分导入肝细胞是一个问题腺相关病毒（AAV）是提供基因编辑试剂的首选平台。但存在严重的局限性。此外，Cas9 免疫在人类中非常普遍。我们建议将基因编辑试剂引入肝细胞中，使 AAV 递送进一步复杂化。使用非病毒体外递送方法，然后移植工程肝细胞来替代患病的肝细胞我们的非病毒体外策略避免了体内方法的两个并发症。 CRISPR-Cas9 编辑仅局限于预期的靶细胞，并有机会维持但这种离体方法有一个潜在的失败之处：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。一种使用退烧药对乙酰氨基酚（APAP）选择编辑肝细胞的新方法。开发出缺乏 NADPH-细胞色素 P450 氧化还原酶 (Cypor) 的基因编辑肝细胞将在拟议的研究中，我们将通过 APAP 给药在体内富集，而不会造成永久性肝损伤。使用 CRISPR-Cas9 破坏 Cypor 直接比较 LNP、电穿孔和 AAV 进行基因编辑然后，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 FH (Ldlr–/– ) 模型在目标 1 中，我们将比较多重分析的特异性和效率。通过电穿孔、LNP 和 AAV 介导的 CRISPR-Cas9 传递对 Cypor 和 Angptl3 进行基因编辑对于目标 2，我们将评估和比较 LNP、电穿孔和原代小鼠肝细胞的效果。 AAV 介导的 Cypor-CRISPR-Cas9 离体递送对肝细胞克隆扩增和能力的影响使用 APAP 选择在野生型 C57BL/6 小鼠中重新填充肝脏在目标 3 中，我们将比较不同方法的效果。 Cypor 和 Angptl3-CRISPR-Cas9 的非病毒和 AAV 介导的多重递送的能力肝细胞降低 Ldlr–/– 小鼠血浆胆固醇和甘油三酯水平并规避 Cas9 免疫该项目是第一个直接比较不同非病毒治疗的项目。用于离体工程肝细胞的 AAV 方法并评估编辑肝细胞的程度使用 APAP 进行体内克隆扩增来治疗 FH 此外，这项研究也是首次研究 FH 的免疫原性。离体基因编辑的肝细胞。