Novel Molecular and Cellular Therapies in Fanconi Anemia

描述(申请人提供)：Fanconi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。儿童时期的死亡率很高，最常见的是在确诊后平均5年内死于再生障碍性贫血的并发症。然而，支持性治疗，在血小板和红细胞输注方面，使用雄激素刺激体内造血，以及使用人类白细胞抗原相合的同胞移植(仅对少数患者适用)延长了FA患者的预期寿命。然而，考虑到后续兄弟姐妹中FA病的发病率，潜在的HLA相合兄弟姐妹捐赠者的可获得性为30%。匹配的无血缘关系供者移植在这种疾病中仍然是高度实验性的，无血缘关系供者移植后的存活率低至30%。捐献者的识别、移植物失败和移植物抗宿主病仍然是重大障碍。FA细胞在DNA修复中存在缺陷，导致自发染色体断裂增加。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)和二环氧丁烷(DEB)的敏感性。现在FA的诊断依赖于在体外暴露于DEB后检测到染色体断裂增加。同样，FA患者培养的细胞对丝裂霉素C的细胞毒性敏感性增加。最近，FA患者的细胞被证明表现出G2期延长停滞，对氧气的敏感性增加，P53诱导缺陷和细胞凋亡增加。根据体细胞杂交的结果，FA至少可分为11个互补群。这种互补是基于纠正杂交细胞中染色体对交联剂的敏感性。在这11个互补基团(A-C、D_1、D_2、E-G和L)中，已克隆到9个独立基因。在体外，这些基因在各自的FA细胞中的转基因表达纠正了DEB增加的染色体断裂和对MMC增加的敏感性。此外，在体外实验中，这些基因在FA患者的骨髓祖细胞中的表达增加了细胞的存活率。最近的研究表明，疾病的临床进展可能受到基因间和基因内变异的影响，这表明互补分配和突变鉴定将在未来的临床治疗中发挥越来越重要的作用。在这个拟议的项目中，我们寻求开发一种综合的方法来收集干细胞、互补分配/突变分析和(通过基因治疗)FA患者的基因修改，以便有效地治疗A、C和G组互补患者，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，即辛辛那提儿童医院医疗中心(CCHMC)范科尼贫血综合护理诊所(FACCC)早在严重再生障碍性贫血发病之前就已发现的一大批FA患者，以及将互补组确定作为筛查方法指导高通量突变分析的可能性。CCHMC的机构审查委员会批准了干细胞采集和基因治疗试验，NIH重组DNA咨询委员会(RAC)批准了这些试验，食品和药物管理局(FDA)批准了研究新药申请。这项赠款提案寻求资金，以进行这些临床试验，并在CAP/CLIA环境中进一步开发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.