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

使用同源无关的靶向整合校正 FANCC 突变，用于范可尼贫血 C 组的基因治疗

• 批准号: 10653342
• 负责人: Ngoc Tung Tran
• 金额: $ 19.81万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-15 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10653342
• 关键词: Alleles; Allogenic; Apoptosis; Autologous; Back; Biological Assay; Bone marrow failure; CD34 gene; CRISPR correction; CRISPR/Cas technology; Cell Cycle; Cells; Clinical; Code; Complement; Complementary DNA; Complex; Congenital Abnormality; DNA Damage; DNA Integration; DNA Repair; DNA Repair Pathway; DNA Sequence; DNA biosynthesis; Data; Defect; Development; Disease; Engraftment; Face; Fanconi Anemia pathway; Fanconi' s Anemia; Foundations; Future; Genes; Genetic; Genome; Goals; Hematological Disease; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Hemoglobinopathies; Hereditary Disease; Human; Human Cell Line; Immunofluorescence Immunologic; Immunologic Deficiency Syndromes; Impairment; In Vitro; Interphase Cell; Knock-out; Lentivirus; Mediating; Mendelian disorder; Mitomycin C; Modeling; Mus; Mutate; Mutation; Nonhomologous DNA End Joining; Outcome; Outcome Study; Pathway interactions; Patients; Phenotype; Pre-Clinical Model; Proliferating; Proteins; Protocols documentation; Regimen; Risk; Safety; Scientist; Second Primary Cancers; Stains; Stem cell transplant; Subgroup; Symptoms; System; Systems Integration; Technology; Testing; Therapeutic; Time; Transplant Recipients; Transplantation; Virus Integration; Western Blotting; Xenograft Model; arm; cancer predisposition; cell growth; cell type; clinical application; conditioning; congenital bone marrow failure; design; functional restoration; gene correction; gene function; gene therapy; graft vs host disease; humanized mouse; improved; in vivo; insertion/deletion mutation; interest; member; mortality; mutation correction; pediatric patients; promoter; repaired; stem cells; success; transgene expression; tumor

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.

项目概要 范可尼贫血 (FA) 是一种与进行性骨髓衰竭相关的破坏性遗传病 （BFM）、先天性异常和癌症易感性。 FA 患者体内任一部位均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 互补组 C (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，这是早期疾病的主要原因。 儿科患者的死亡率和继发性恶性肿瘤。同种异体干细胞移植是首选 治疗具有匹配供体的患者的 BMF。然而，移植患者的风险增加 移植物抗宿主病（GVHD）和继发性癌症。克服局限性的替代方法 同种异体干细胞移植涉及基因治疗以纠正患者干细胞的突变，然后 将校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。科学家利用这项技术精确纠正血液中的突变 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，该途径 仅在分裂细胞中活跃。然而，由于以下原因，来自 FA 患者的血液干细胞在细胞生长方面存在缺陷： 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，我们提出了一种称为同源独立靶向整合的替代方法 (HITI) 将编码功能性 FANCC 基因的完整 DNA 序列引入内源性 FANCC 启动子（控制 FANCC 基因表达的调节 DNA 序列）。该系统可以应用 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 称为非同源末端连接（NHEJ）的途径。与 HDR 不同，NHEJ 在所有细胞中都高度活跃，包括 缓慢/不分裂的细胞。因此，我们预计 HITI 介导的基因校正将在 FA 患者衍生的疾病中有效。 干细胞。我们已经开发了所有必要的系统来验证基因编辑效率和功能 在体内和体外编辑细胞。我们还通过敲除 FANCC 生成了 FA 的替代模型 人类 CD34+ 细胞。这些细胞显示出 FA-HSPC 的典型表型，从而为 优化我们的基因编辑系统。值得注意的是，尽管效率较低，但 HDR 校正小鼠 HSPC 的功能 在体外部分获救。因此，使用HITI的高编辑效率将充分挽救校正词干的功能 细胞。我们的建议将提供一种改进的方法来精确纠正具有高风险的患者 FANCC 突变 功效。我们的长期目标是开发一个全面的FA C组基因治疗临床前模型。 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 疾病并了解其发病机制。