Nongenotoxic conditioning for gene therapy and allogeneic transplantation in Fanconi anemia

摘要 Fanconi贫血(FA)是一种遗传性骨髓(BM)衰竭疾病，由DNA固有缺陷引起 修复会增加患癌症的风险，如急性髓系白血病和鳞癌。 全世界每年约有130,000名出生的儿童受到FA的影响。目前，治疗这种疾病的唯一方法 血液学并发症的FA是由异基因骨髓或造血干细胞移植(HSCT) 人类白细胞抗原捐献者。一个关键的组成部分是使用某种形式的条件来准备接受者BM，以消除 并促进供体产品的植入。目前使用的所有FA的调理方案 依赖烷化化疗药物的使用和/或放射治疗，两者都与 发生继发性恶性肿瘤的风险增加，特别是在像FA这样的DNA修复障碍中。作为一种 另一种策略是，针对造血干细胞的抗体药物结合物(Adc)是一种很有前途的策略。 促进基因修饰的自体或同种异体移植物植入的非遗传毒性方法。最新研究 已经证明了结合免疫毒素的CD45或CD117(c-Kit)抗体可以有效地使用ADCs 皂苷(SAP)。由于普遍的共识是在FA和其他疾病中应该避免遗传毒性条件反射 对于存在DNA修复缺陷的疾病，我们建议开发新的方法来克服这些关键限制 用于目前的基因治疗和造血干细胞移植方案。因此，在目标1中，我们将开发非遗传毒性的条件反射 使用FANCA基因敲除小鼠模型最佳耗尽残留HSCs和促进FA的方案 基因修饰细胞或同种异体细胞的植入。尽管尽可能安全地消除了尽可能多的主机HSC， 存在残留宿主HSCs的风险，这可能导致在以下情况下残留与疾病相关的造血 基因修饰细胞的移植以及非清髓、T细胞耗尽的同种异体移植 HSCT。目的2将寻求一种新的方法来消除基因治疗或异基因造血干细胞移植后残留的FA细胞。 虽然Aim 1试图避免allo-HSCT并发症，但并不是所有的FA患者都适合进行基因治疗。 因此，在目标3中，我们将确定我们的新的非遗传毒性条件作用方法是否可以耗尽宿主HSCs。 防止宿主免疫介导的骨髓移植排斥反应，从而允许异基因HSC移植到Fanca-/- 老鼠。拟议的研究将开发一种全新的非遗传毒性自体调节方法。 HSC基因治疗和作为异基因HSC移植新方案的关键组成部分。此外, 我们描述了一种创新的策略，既适用于基因治疗，也适用于异基因移植，以消除 移植后残留的和未矫正的FA造血细胞可能发展为白血病细胞。

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.