Nongenotoxic conditioning for gene therapy and allogeneic transplantation in Fanconi anemia

摘要 范可尼贫血 (FA) 是一种遗传性骨髓 (BM) 衰竭性疾病，由 DNA 内在缺陷引起 修复导致急性髓细胞白血病和鳞状细胞癌等癌症的风险增加。 全球每年大约有 130,000 名出生的儿童受到 FA 的影响。目前，治疗该病的唯一方法是 FA 的血液学并发症是来自合适患者的同种异体 BM 或造血干细胞移植 (HSCT) HLA 捐献者。一个关键部分是使用某种形式的调节来准备接受者 BM，以消除 病变细胞并促进供体产品的植入。目前使用的所有 FA 预处理方案 依赖于使用烷基化化疗药物和/或放射治疗，这两者都与 发生继发性恶性肿瘤的风险增加，尤其是 FA 等 DNA 修复疾病。作为一个 另一种策略是，靶向造血干细胞 (HSC) 的抗体药物偶联物 (ADC) 是一种有前景的方法 促进基因修饰的自体或同种异体移植物植入的非基因毒性方法。最近的研究 已证明 ADC 与与免疫毒素偶联的 CD45 或 CD117 (c-Kit) 抗体的有效使用 皂素（SAP）。由于普遍的共识是在 FA 和其他治疗中应避免基因毒性调节 具有 DNA 修复缺陷的疾病，我们建议开发新方法来克服这些关键限制 适用于当前的基因治疗和 HSCT 方案。因此，在目标 1 中，我们将开发非基因毒性调理 使用 FANCA 敲除小鼠模型进行 FA 治疗方案，以最佳地消耗残留 HSC 并促进 基因修饰或同种异体细胞的植入。尽管尽可能安全地消除了宿主 HSC， 存在宿主HSC残留的风险，这可能导致术后残留的与疾病相关的造血功能 基因修饰细胞移植以及非清髓性、T 细胞耗尽的同种异体移植 造血干细胞移植。目标 2 将寻求一种新方法来消除基因治疗或同种异体 HSCT 后残留的 FA 细胞。 虽然目标 1 旨在避免异基因造血干细胞移植并发症，但并非所有 FA 患者都是基因治疗的良好候选者。 因此，在目标 3 中，我们将确定我们的新型非基因毒性调理方法是否可以耗尽宿主 HSC 并防止宿主免疫介导的 BM 移植排斥，从而允许同种异体 HSC 植入 Fanca-/- 老鼠。拟议的研究将开发一种全新的自体非基因毒性调理方法 HSC 基因治疗并作为同种异体 HSC 移植新方案的关键组成部分。此外， 我们描述了一种适用于基因治疗和同种异体移植的创新策略，以消除 残留的和未经校正的 FA 造血细胞可能在移植后发展成白血病细胞。

ABSTRACT Fanconi anemia (FA) is an inherited bone marrow (BM) failure disorder resulting from an intrinsic defect in DNA repair leading to an increased risk of cancers such as acute myeloid leukemia and squamous cell carcinoma. Approximately 130,000 children born worldwide each year are affected by FA. Currently, the only cure for the hematologic complications of FA is an allogeneic BM or hematopoietic stem cell transplant (HSCT) from a suitable HLA donor. A key component is preparing the recipient BM using some form of conditioning to both eliminate diseased cells and promote engraftment of donor product. All of the currently used conditioning regimens for FA rely on the use of alkylating chemotherapy drugs and/or irradiation, both of which are associated with an increased risk of developing secondary malignancies, especially in DNA repair disorders like FA. As an alternative strategy, antibody drug conjugates (ADCs) targeting hematopoietic stem cells (HSCs) are a promising nongenotoxic method of facilitating engraftment of gene-modified autologous or allogeneic grafts. Recent studies have shown the effective use of ADCs with either CD45 or CD117 (c-Kit) antibodies conjugated to the immunotoxin saporin (SAP). Since the general consensus is that genotoxic conditioning should be avoided in FA and other diseases with DNA repair defects, we propose to develop novel approaches to overcome these critical limitations for current gene therapy and HSCT protocols. Thus, in Aim 1, we will develop nongenotoxic conditioning regimens for FA using a FANCA knockout mouse model to optimally deplete residual HSCs and facilitate engraftment of gene-modified or allogeneic cells. Despite eliminating as many host HSCs as safely possible, there will be a risk of remaining host HSCs, which can result in residual disease-related hematopoiesis after transplantation of gene-modified cells and also in the setting of nonmyeloablative, T-cell depleted allogeneic HSCT. Aim 2 will pursue a novel approach to eliminate residual FA cells after gene therapy or allogeneic HSCT. While Aim 1 seeks to avoid allo-HSCT complications, not all FA patients will be good candidates for gene therapy. Thus, in Aim 3, we will determine whether our novel nongenotoxic conditioning approach can deplete host HSCs and prevent host immune-mediated BM graft rejection and thus permit allogeneic HSC engraftment in Fanca-/- mice. The proposed studies will develop an entirely novel approach of nongenotoxic conditioning for autologous HSC gene therapy and as a key component of a novel regimen for allogeneic HSC transplantation. In addition, we describe an innovative strategy, applicable to both gene therapy and allogeneic transplantation, to eliminate residual and uncorrected FA hematopoietic cells that may develop into leukemic cells post-transplant.