Gene therapy for Fanconi anemia

项目描述（申请人提供）：本项目的主要目的是开发用于范可尼贫血（FA）患者的造血干细胞（HSC）基因治疗。FA是一种常染色体隐性遗传综合征，其特征是先天性异常、恶性肿瘤易感性和骨髓衰竭，后者是发病和死亡的主要原因。来自未受影响的供体的异基因HSC移植是唯一被证实的治疗患有骨髓衰竭的患者的治疗方法。然而，无关供体的患者的移植与显著的毒性相关，并且这些患者的结果不如HLA匹配的同胞供体的患者成功。此外，最近的数据表明，移植物抗宿主病（GVHD）增加了头颈癌的发病率，并导致FA患者死亡率增加。使用自体造血干细胞的基因替代疗法是一种潜在的替代治疗方式，特别是因为基因校正的FA细胞具有存活优势。此外，FA细胞对低剂量的环磷酰胺高度敏感，这可用于增加遗传修饰细胞的比例，并消除未修饰的细胞。然而，迄今为止，FA的基因治疗受到基因转移效率低的限制，导致仅瞬时检测到转基因细胞，最终没有临床益处。这个问题的一部分是γ逆转录病毒载体的局限性，其需要细胞分裂和延长的细胞培养期以进行有效的转导。这对于FA是一个特别的问题，因为FA细胞具有增加的凋亡率，因此，它们在培养物中分裂和生长的能力显著降低。与γ逆转录病毒载体相反，慢病毒载体不需要细胞分裂进行转导，并且即使用非常短的转导方案也可以使干细胞增殖。使用小鼠和大型动物模型，我们最近已经证明了有效的慢病毒HSC基因转移与过夜转导协议。因此，我们建议：1）完成FANCA和FANCC慢病毒载体在人FA细胞和FANCA-/-和FABCC-/-小鼠中的临床前研究，并优化临床转导程序，2）确定输注基因校正细胞的安全性和可行性，3）确定基因校正细胞的持久性和体内生长优势，4）评估环磷酰胺是否可以增强基因校正的FA细胞的存活优势，和5）分析慢病毒整合位点并监测输注基因修饰的细胞后患者中优势克隆的发展。我们的最终目标是通过慢病毒介导的HSC基因转移来改善或治愈FA患者的造血症状，并开发适用于其他遗传性疾病和血液病的方法。

DESCRIPTION (provided by applicant): The major objective of this project is to develop hematopoietic stem cell (HSC) gene therapy for patients with Fanconi anemia (FA). FA is an autosomal recessive syndrome characterized by congenital abnormalities, predisposition to malignancy, and bone marrow failure, the latter being the major cause of morbidity and mortality. Allogeneic HSC transplantation from unaffected donors is the only proven curative treatment for patients suffering from marrow failure. However, transplantation for patients with unrelated donors has been associated with significant toxicity, and outcome for these patients has been less successful than for patients with an HLA-matched sibling donor. In addition, recent data suggest that graft- versus-host disease (GVHD) increases the incidence of head and neck cancers, and leads to increased mortality in patients with FA. Gene replacement therapy using autologous hematopoietic stem cells is a potential alternative treatment modality, particularly since gene-corrected FA cells have a survival advantage. In addition, FA cells are highly sensitive to low doses of cyclophosphamide, which could be used to increase the proportion of genetically modified cells and also eliminate unmodified cells. Gene therapy for FA, however, has to date been limited by low gene transfer efficiency resulting in only transient detection of genetically modified cells and, ultimately, no clinical benefit. Part of this problem has been the limitation of gammaretroviral vectors, which require cell division and extended cell culture periods for efficient transduction. This is a particular problem for FA, since FA cells have an increased rate of apoptosis, and thus, their ability to divide and grow in culture is significantly reduced. In contrast to gammaretroviral vector, lentiviral vectors do not require cell division for transduction and can transduce stem cells even with very short transduction protocols. Using mouse and large animal models, we have recently demonstrated efficient lentiviral HSC gene transfer with an overnight transduction protocol. Thus, we propose to 1) complete preclinical studies of FANCA and FANCC lentiviral vectors in human FA cells and in FANCA-/- and FABCC-/- mice and optimize procedures for clinical transduction, 2) determine safety and feasibility of infusing gene-corrected cells, 3) determine persistence and in vivo growth advantage of gene-corrected cells, 4) evaluate whether cyclophosphamide can enhance the survival advantage of gene- corrected FA cells, and 5) analyze lentiviral integration sites and monitor for the development of dominant clones in patients after infusion of gene-modified cells. Our ultimate goal is to improve or cure the hematopoietic symptoms in FA patients by lentivirus-mediated HSC gene transfer, and develop methodologies that would be applicable to other genetic disorders and hematological diseases.