Preclinical Gene Therapy of Fanconi Anemia with Transposon-Based Approaches

睡美人（SB）转座子构成了一种新型的、整合的、非病毒载体系统，其可能极大地革新基因治疗领域，主要原因有三：1）与整合病毒载体相比，其特征性的和更安全的整合模式，2）体内转染细胞的可能性，如已经在实验模型中所示，和3）在GMP条件下载体生产的简单性和因此的经济性。值得注意的是，SB系统刚刚进入美国癌症免疫治疗的临床试验。具有改进的基因转移特性的超活性SB转座酶/转座子系统的开发为用非病毒载体治疗罕见疾病开辟了新的前景。在这项提案中，我们的目标是开发这个平台，用于范可尼贫血（FA）的基因治疗，这是一种遗传不稳定综合征，由迄今为止在FA途径中描述的18个基因中的任何一个突变引起，这些基因在DNA损伤的修复中起作用。该疾病与由于造血祖细胞和干细胞（HPSC）的进行性耗竭导致的严重骨髓衰竭相关，对炎性细胞因子和内源性醛类的敏感性增加。因此，在FA HPSC的遗传校正后，这些细胞与体内未校正的细胞相比将发展出显著的增殖优势，这可以将患者从骨髓衰竭中拯救出来。在这个项目中，我们将开发SB转座子载体用于纠正Fanconi贫血-A患者（FA中最普遍的亚型），验证载体在FA成纤维细胞和从FA小鼠和FA患者分离的HPSC中的离体性能，并彻底表征这些基因修饰的细胞在移植后受体小鼠中的植入和造血重建能力。将进行全面的载体整合位点分析，以评估基因治疗方法的相对安全性。该项目将建立，评估和完善转座子介导的Fanconi贫血基因治疗的临床适用方案，与目前基于使用慢病毒载体的方法进行比较。

The Sleeping Beauty (SB) transposon constitutes a novel, integrative, non-viral vector system that may greatly innovate the field of gene therapy for three main reasons: 1) its characteristic and safer pattern of integration, as compared to integrating viral vectors, 2) the possibility of transfecting cells in vivo, as already shown in experimental models and 3) the simplicity and therefore the economy of vector production under GMP conditions. Significantly, the SB system has just entered clinical trials in the USA for the immunotherapy of cancer. The development of a hyperactive SB transposase/transposon system, with improved gene transfer properties has opened new perspectives for the treatment of rare diseases with non-viral vectors. In this proposal, we aim to develop this platform for gene therapy of Fanconi Anemia (FA), a genetic instability syndrome caused by mutations in any of the 18 genes described to date in the FA pathway that cooperate in the repair of DNA lesions. The disorder is associated with profound bone marrow failure due to the progressive depletion of hematopoietic progenitor and stem cells (HPSCs), with increased sensitivity to inflammatory cytokines and endogenous aldehydes. Therefore, upon the genetic correction of FA HPSCs, these cells will develop a marked proliferation advantage in comparison to uncorrected cells in vivo, which may rescue the patients from the bone marrow failure. In this project we will develop SB transposon vectors for the correction of Fanconi Anemia-A patients (the most prevalent subtype in FA), validate the performance of the vectors ex vivo in FA fibroblasts and HPSCs isolated from FA mice and FA patients, and thoroughly characterize the abilities of these gene-modified cells with respect to engraftment and hematopoietic reconstitution in recipient mice following transplantation. A comprehensive vector integration site analysis will be conducted to assess relative safety of the gene therapy approach. The project will establish, evaluate and refine clinically applicable protocols for transposon-mediated gene therapy of Fanconi Anemia in comparison to current approaches based on the use of lentiviral vectors.