Novel therapies for Fanconi anemia

描述（由申请人提供）：范可尼贫血（FA）是一种罕见的隐性综合征，其特征是骨髓衰竭、先天性异常和恶性肿瘤倾向。FA细胞在DNA修复方面存在缺陷，导致自发性染色体断裂增加。这一特征增加了FA细胞对DNA双功能交联剂（如MMC和DEB）的敏感性。与该应用直接相关的是，越来越多的证据（包括来自该提案的主要研究者（PI）的证据）表明FA细胞在非同源末端连接（NHEJ）和同源重组（HR）中存在特定缺陷。在体外遗传缺陷细胞中转基因表达FA基因可纠正FA细胞的表型异常。骨髓移植已经治愈了一些骨髓衰竭或恶性血液病患者。然而，与没有这种疾病的患者相比，由于预处理方案，毒性增加。已报告了两项针对FA患者的临床基因转移试验，其中一项由本提案的PI进行。在这项研究中，我们证明了基因转移本身不再是这种方法有效性的限制，但即使在疾病过程的早期，可以收集并用作基因转移靶点的造血干细胞（HSC）的数量和/或功能也存在显着缺陷。因此，需要新的治疗方法来治疗这种疾病。可以解决这种缺陷的一种方法是从诱导多能干细胞（iPS）中衍生HSC，该诱导多能干细胞（iPS）是从其他体细胞组织重编程的。用于治疗的iPS细胞的重要优点是此类细胞的无限增殖能力（允许产生大量HSC）和克隆此类细胞的能力，这允许在治疗性使用之前精确地单细胞校正基因突变和对操纵的细胞进行前瞻性分子表征。一组疾病特异性人iPS细胞的重编程已经通过该提议的PI完成，但是体细胞范可尼贫血细胞的重编程已经被证明是困难的。这可能与FA NHEJ或HR中的缺陷以及最近报道的p53 DNA损伤反应途径参与重编程有关。事实上，我们最近发现FA中DNA损伤和胞质分裂失败之间的联系，可能在骨髓衰竭表型中起关键作用。因此，总的来说，我们认为，理解FA DNA损伤反应，这是密切参与FA细胞表型，似乎也参与体细胞重编程，是至关重要的开发新的细胞，遗传和小分子为基础的治疗FA。在这个多研究者提案中的三个项目通过一个共同的目标联系在一起，即获得对FA细胞中涉及的重编程技术和DNA修复途径的更多理解，并应用这一新知识，利用最先进的基础技术快速开发FA再生障碍性贫血的新治疗方法。 公共卫生相关性：这项研究将开发新的方法来纠正遗传疾病。我们的目标是获得对范可尼贫血生物学的新见解，范可尼贫血是最常见的遗传性骨髓衰竭综合征。我们的研究将探索一种非常有前途的新型细胞类型的潜力，称为诱导多能干细胞（iPS），为范可尼贫血生物学提供独特的见解，并为这种严重疾病开发新的治疗方式。

DESCRIPTION (provided by applicant): Fanconi anemia (FA) is a rare recessive syndrome characterized by bone marrow failure, congenital anomalies and a predisposition to malignancy. FA cells have a defect in DNA repair that leads to increased spontaneous chromosomal breakage. This feature increases the sensitivity of FA cells to DNA bifunctional cross-linking agents such as MMC and DEB. Relating directly to this application, increasing evidence including from the Principal Investigators (PI) of this proposal suggests FA cells have specific defects in non-homologous end joining (NHEJ) and homologous recombination (HR). Transgenic expression of FA genes in genetically deficient cells in vitro corrects the phenotypic abnormalities of FA cells. Bone marrow transplantation has cured some patients of their bone marrow failure or hematologic malignancies. However, there is increased toxicity due to the conditioning regimens compared to patients without the disorder. Two clinical gene transfer trials for patients with FA have been reported, including one by a PI of this proposal. In this study, we demonstrated that gene transfer per se is no longer a limitation to the effectiveness of this approach, but that even early in the disease process there are significant deficiencies in the number and/or function of hematopoietic stem cells (HSCs) that can be collected and utilized as targets in gene transfer. Thus, new therapeutic approaches are needed to treat this disease. One approach that could address this deficiency is the derivation of HSC from induced pluripotent stem cells (iPS) reprogrammed from other somatic tissues. Important advantages of iPS cells for therapy are the unlimited proliferative capacity of such cells (allowing production of large numbers of HSC) and the ability to clone such cells, which allows precise single cell correction of genetic mutations and prospective molecular characterization of manipulated cells prior to therapeutic use. The reprogramming of a panel of disease specific human iPS cells has been accomplished by a PI of this proposal, but the reprogramming of somatic Fanconi anemia cells has proven difficult. This is likely related to the defects in FA NHEJ or HR and to recent reports of involvement of the p53 DNA damage response pathway in reprogramming. Indeed, we have recently found a link between DNA damage and cytokinesis failure in FA that may play a critical role in the bone marrow failure phenotype. Thus, overall, we believe that understanding FA DNA damage response, which is intimately involved in the FA cellular phenotype and appears to be involved also in somatic cell reprogramming, is critical to development of novel cell, genetic and small molecule-based therapies in FA. The three projects in this multi-investigator proposal are linked via a common goal of acquiring increased understanding of reprogramming technology and DNA repair pathways as involved in FA cells and application of this new knowledge for rapid development of new therapeutic approaches to the aplastic anemia of FA utilizing state-of-the-art basic technology. PUBLIC HEALTH RELEVANCE: This study will develop new methodologies to correct genetic disease. We aim to gain new insights into the biology of Fanconi anemia, the most common inherited bone marrow failure syndrome. Our study will explore the potential of a very promising novel cell type, called induced pluripotent stem cells (iPS), to provide unique insights into Fanconi anemia biology and to enable the development of novel treatment modalities for this severe disorder.