Therapeutic potential of CRISPR/Cas9 genome engineering in humanized mouse

项目摘要 肝脏定向基因编辑有可能纠正许多严重的单基因 紊乱将这种疗法转化为临床的两个主要障碍是有效性和安全性。 我们将使用一种创新的治疗性基因组编辑方法来解决这两个问题， 新型人源化小鼠模型。 CRISPR/Cas9基因组编辑技术已被证明非常有效地引入 双链制动器（DSB）。在哺乳动物中，DSB优先 通过易错非同源末端连接（NHEJ）修复，并在较小程度上通过 同源定向修复（HDR）。在这里，我们建议治疗难治性脂质紊乱， 从脂质基因中补偿性缺失整个外显子（外显子切除）， 血液中的胆固醇水平或有助于血液胆固醇清除较慢。删除将是 在两个侧翼中引入两个CRISPR/Cas9诱导的DSB后， 内含子这种修复机制在非增殖肝细胞中非常有效，并避免了肝细胞的损伤。 在编码序列中引入潜在有害的突变。 我们最近开发了一种小鼠品系， 肝细胞由于基因组编辑依赖于序列，因此必须在上下文中进行测试 所需靶组织的人类细胞。用正常人类重新繁殖的老鼠 肝细胞，我们将能够确定肝定向人 CRISPR/Cas9基因编辑然后，我们将在小鼠中测试他的方法， 来自家族性高胆固醇血症（FH）患者的人肝细胞。第一次异种移植 FH模型可用于评估治疗效果和潜在的代偿性 适应这种实验疗法。

PROJECT SUMMARY Liver-directed gene editing has the potential to correct numerous severe monogenic disorders. The two major hurdles of translating this therapy to the clinic are efficacy and safety. We will address both concerns using an innovative therapeutic genome editing approach in a novel humanized mouse model. CRISPR/Cas9 genome editing technology has proven extremely efficient in introducing double strand brakes (DSB) in many cellular systems. In mammals, DSB are preferentially repaired by the error prone non-homologous end joining (NHEJ) and to a lesser extent by homology-directed repair (HDR). Here, we propose to treat refractory lipid disorders with compensatory deletion of a whole exon (exon-excision) from lipid genes that increase the cholesterol levels in blood or contribute to slower clearance of blood cholesterol. Deletion will be mediated by NHEJ after introduction of two CRISPR/Cas9 induced DSBs in the two flanking introns. This repair mechanism is very efficient in non-proliferating hepatocytes and avoids the introduction of potentially harmful mutations in the coding sequence. We recently developed a mouse strain that can be repopulated with cadaveric human hepatocytes. Since genome editing is sequence dependent, it must be tested within the context of human cells of the desired target tissue. Using mice repopulated with normal human hepatocytes, we will be able to determine the safety and efficacy of liver-directed human genome editing by CRISPR/Cas9. We will then test his approach in mice repopulated with human hepatocytes from a patient with familial hypercholesterolemia (FH). This first xenograft model for FH can be used to evaluate the therapeutic efficacy and potential compensatory adaptation for this experimental therapy.