FANCC mutation correction using homology-independent targeted integration for gene therapy of Fanconi Anemia group C

项目总结 Fanconi贫血(FA)是一种与进行性骨髓衰竭相关的破坏性遗传性疾病 (BFM)、先天异常和癌症易感性。FA患者中任何一个都存在双等位基因突变 FA途径的基因成员，由22个基因组成。大多数突变发生在FA核心复合体中 包括FA互补C组(FANCC)基因。FANCC介导的FA(C组)患者显示 FA的典型临床症状。目前，治疗的重点是减轻BMF，这是早期的主要原因 儿科患者的死亡率，以及继发性恶性肿瘤。异基因干细胞移植是首选 治疗供者匹配的BMF患者的治疗。然而，移植患者表现出更高的风险。 移植物抗宿主病(GVHD)和继发性癌症。一种克服限制的替代方法 异基因干细胞移植涉及基因治疗以纠正患者干细胞的突变，然后 将矫正后的干细胞移植回患者体内。CRISPR/CAS9是最先进的技术，可以 无缝修改基因组。科学家已经使用这项技术精确地纠正了血液中的突变 干细胞可用于治疗遗传性血液疾病(血红蛋白病和 免疫缺陷性疾病)。这种方法依赖于一种称为同源定向修复(HDR)的途径 仅在分裂细胞时处于活动状态。然而，FA患者的血液干细胞在细胞生长方面存在缺陷，原因是 持续的DNA损伤。因此，HDR方法的效率可能达到FA的治疗阈值 基因疗法。在这里，我们提出了另一种方法，称为同源无关的靶向整合 将编码功能性FANCC基因的完整DNA序列导入内源性FANCC 启动子(控制FANCC基因表达的调控DNA序列)。该系统具有一定的实用价值 以纠正所有FA C组患者中发生的所有突变。Hiti方法依赖于DNA修复 途径称为非同源末端连接(NHEJ)。与HDR不同，NHEJ在所有细胞中都高度活跃，包括 慢/不分裂细胞。因此，我们期望HITI介导的基因校正在FA患者中是有效的 干细胞。我们已经开发了所有必要的系统来验证基因编辑的效率和功能 编辑体内和体外的细胞。我们还通过敲除FANCC生成了FA的替代模型 人CD34+细胞。这些细胞表现出典型的FA-HSPC表型，从而为 优化我们的基因编辑系统。值得注意的是，尽管效率很低，但HDR校正的小鼠HSPC的功能 在体外被部分解救出来。因此，使用HITI的高编辑效率将充分恢复校正后的词干的功能 细胞。我们的建议将提供一种改进的方法来精确纠正高FANCC突变患者 功效。我们的长期目标是开发一种全面的FA C组基因治疗的临床前模型。 该提案的结果将为开发治疗FA C组的基因疗法奠定坚实的基础 以及了解疾病的发病机制。

Project summary Fanconi Anemia (FA) is a devastating inherited disease associated with progressive bone marrow failure (BFM), congenital abnormalities, and cancer predisposition. FA patients harbor biallelic mutations in any one gene member of the FA pathway consisting of 22 genes. Most mutations happen in the FA core complex including the FA Complementation Group C (FANCC) gene. FANCC-mediated FA (group C) patients show typical clinical symptoms of FA. Currently, the treatment focuses on mitigating BMF, the leading cause of early mortality in pediatric patients, and secondary malignancies. Allogenic stem cell transplantation is the preferred therapy to treat BMF in patients with matched donors. However, transplanted patients show enhanced risk of graft-versus-host disease (GVHD) and secondary cancer. An alternative approach that overcomes the limitations of allogenic stem cell transplantation involve the gene therapy to correct mutations in patient stem cells, and then transplant back corrected stem cells into the patient. CRISPR/Cas9 is the state-of-the-art technology that allows modifying the genome seamlessly. Scientists have used this technology to precisely correct mutations in blood stem cells that can be applied for the treatment of genetic blood diseases (hemoglobinopathies and immunodeficient disorders). This approach depends on a pathway called homology-directed repair (HDR) that is only active in dividing cells. However, blood stem cells from FA patients are defective in cell growth due to sustained DNA damage. Thus, the efficiency of HDR approach might reach the therapeutic threshold for FA gene therapy. Here, we propose an alternative approach called homology-independent targeted integration (HITI) to introduce an intact DNA sequence encoding for functional FANCC gene into the endogenous FANCC promoter (a regulatory DNA sequence that controls expression of the FANCC gene). This system can be applied to correct all mutations occurring in all FA group C patients. The HITI approach is dependent on the DNA repair pathway called non-homologous end joining (NHEJ). Unlike HDR, NHEJ is highly active in all cells including slow/non-dividing cells. Thus, we expect that HITI-mediated gene correction will be efficient in FA patient derived stem cells. We have developed all necessary systems to validate the gene editing efficiency and functions of the edited cells both in vivo and in vitro. We also generated a surrogated model of FA by knockout of FANCC in human CD34+ cells. These cells show typical phenotypes of FA-HSPCs, thus providing a powerful model for optimizing our gene editing system. Of note, albeit low efficiency, the function of HDR-corrected mouse HSPCs was partially rescued in vitro. Thus, high editing efficiency using HITI will fully rescue functions of corrected stem cells. Our proposal will provide an improved approach to precisely correct patient FANCC mutations with high efficacy. Our long-term goal is to develop a comprehensive pre-clinical model for gene therapy of FA group C. Outcomes from this proposal will create a strong foundation for developing gene therapy to treat FA group C disease as well as understanding the disease mechanism.