Novel Molecular and Cellular Therapies in Fanconi Anemia

描述（由申请人提供）：范可尼贫血（FA）是一种隐性疾病，其特征是几乎普遍的进行性骨髓衰竭和一系列严重的身体异常。儿童期死亡率很高，最常见的是在诊断后平均5年内死于发育不全并发症。然而，支持性治疗，如血小板和红细胞输注，使用雄激素刺激体内造血，以及使用hla相同的同胞移植（少数患者可用），延长了FA患者的预期寿命。然而，考虑到随后的兄弟姐妹中FA疾病的发生率，潜在的hla相同的兄弟姐妹供体的可得性小于30%。匹配的非亲属供体移植在这种疾病中仍然是高度实验性的，非亲属供体移植后的存活率低至30%。确定供体、移植物衰竭和移植物抗宿主病仍然是重大障碍。FA细胞在DMA修复中有缺陷，导致自发性染色体断裂增加。这一特性增加了FA细胞对DNA双功能交联剂（如丝裂霉素C （MMC）和二氧丁烷（DEB））的敏感性。FA的诊断现在依赖于检测体外暴露于DEB后染色体断裂的增加。同样，从FA患者身上培养的细胞对丝裂霉素c的细胞毒性表现出更高的敏感性。最近，来自FA患者的细胞被证明表现出G2期延长、对氧的敏感性增加、p53诱导缺陷和细胞凋亡增加。通过体细胞杂交，FA可分为至少11个互补基团。互补是基于对杂交细胞中交联剂的染色体敏感性的校正。在这11个互补群（A-C、D1、D2、E-G和L）中克隆了9个独立的基因并进行了鉴定。这些基因在各自的FA细胞中的转基因表达在体外纠正了DEB增加的染色体断裂和对MMC的敏感性增加。此外，在体外实验中，这些基因在FA患者骨髓祖细胞中的表达增加了细胞存活率。最近的研究表明，该疾病的临床进展可能受到基因间和基因内变异的影响，这表明补体分配和突变鉴定对未来的临床管理将越来越重要。在这个拟议的项目中，我们寻求开发一种全面的方法来收集干细胞，补体分配/突变分析，以及FA患者的基因修饰（通过基因治疗），以有效治疗补体组a， C和G的患者，这些患者占北美患者的-90%。这些研究利用了我们在逆转录病毒介导的基因转移方面的长期专业知识，辛辛那提儿童医院医疗中心（CCHMC）范可尼贫血综合护理诊所（FACCC）在严重发育不全发病前观察到的大量FA患者，以及利用互补组确定作为指导高通量突变分析方法的筛选方法的潜力。干细胞收集和基因治疗试验已被CCHMC的机构审查委员会批准，NIH重组DNA咨询委员会（RAC）和研究新药申请已被食品和药物管理局批准。这项拨款申请寻求资金来进行这些临床试验，并在CAP/CLIA环境中进一步发展逆转录病毒介导的互补分析，用于FA患者的临床管理。

DESCRIPTION (provided by applicant): Fanconi anemia (FA) is a recessive disease characterized by nearly universal progressive bone marrow failure and a constellation of serious physical anomalies. There is a high rate of fatality in childhood, most frequently from complications of aplasia within an average of 5 years from diagnosis. Supportive care, in terms of platelet and erythrocyte transfusions, the use of androgens to stimulate hematopoiesis in vivo, and the use of HLA-identical sibling transplants (available to a minority of patients) has extended the life- expectancy of FA patients, however. However, taking into account the incidence of FA disease in subsequent siblings, the availability of potential HLA-identical sibling donors is <30%. Matched unrelated donor transplants remains highly experimental in this disease with a survival after an unrelated donor transplant as low as 30%. Identification of donors, graft failure and graft versus host disease remain significant obstacles. FA cells have a defect in DMA repair that leads to increased spontaneous chromosomal breakage. This feature increases the sensitivity of FA cells to DNA bifunctional cross-linking agents such as mitomycin C (MMC) and diepoxybutane (DEB). The diagnosis of FA now relies upon detecting increased chromosomal breakage after exposure in vitro to DEB. Similarly, cells cultured from patients with FA display increased susceptibility to the cytotoxicity of mitomycin C. More recently, cells from patients with FA have been demonstrated to display G2 phase prolongation arrest, increased sensitivity to oxygen, defective p53 induction and increased apoptosis. FA can be classified into at least eleven complementation groups by somatic cell hybrids. The complementation is based upon correction of the chromosomal sensitivity to cross-linking agents in hybrid cells. Nine independent genes have been cloned and characterized within these 11 complementation groups (A-C, D1, D2 and E-G, and L). Transgenic expression of these genes in the respective FA cells in vitro corrects the increased chromosomal breakage from DEB and the increased sensitivity to MMC. In addition, expression of these genes in bone marrow progenitors from patients with FA increases cell survival in in vitro assays. Recent studies have demonstrated that clinical progression of the disease may be influenced by inter- and intra-genic variations, suggesting that complementation assignment and mutation identification will be increasingly important for clinical management in the future. In this proposed project, we seek to develop a comprehensive approach to stem cell collection, complementation assignment/mutation analysis, and genetic modification of FA patients (by gene therapy) in order to effectively treat patients of complementation groups A, C, and G, which make up -90% of North American patients. These studies take advantage of our long-standing expertise in retrovirus-mediated gene transfer, an emerging large group of FA patients that are being seen in the Cincinnati Children's Hospital Medical Center (CCHMC) Fanconi Anemia Comprehensive Care Clinic (FACCC) well before the onset of severe aplasia and the potential for using complementation group ascertainment as a screening method to direct high-throughput methods for mutation analysis. A stem cell collection and a gene therapy trial have been developed, approved by the Institutional Review Board of CCHMC, the NIH Recombinant DNA Advisory Committee (RAC) and Investigational New Drug applications have been approved for these trials by the Food and Drug Administration. This grant proposal seeks funding to conduct these clinical trials and further develop retrovirus-mediated complementation analysis in a CAP/CLIA environment for use in clinical management of FA patients.