Therapeutic gene targeting in human CD34+ cells.

治疗遗传性疾病的最终目标是永久纠正导致疾病的突变。该项目旨在建立一种新的治疗严重联合免疫缺陷（SCID）小鼠模型中遗传性血液病的疗法。该疗法基于同源重组（HR），并直接纠正染色体中的潜在突变。由于HR的低频率，这种方法只有在人工核酸酶可用的情况下才变得可行。通过这种核酸酶在DNA中产生位点特异性切割，通过激活细胞DNA修复途径显著刺激HR。为了纠正SCID的潜在点突变，我们将产生作为基于HR的DNA修复的模板的修复载体以及在突变位点切割DNA以刺激HR的定制核酸酶。造血干细胞HSC中的离体基因修复将通过用整合酶缺陷型慢病毒载体或基于不同腺病毒载体转移基因修复组分来实现。相关病毒血清型。将在剂量递增研究中确定基因修复的程度以及遗传毒性副作用的频率，以评价该系统用于假定临床应用的风险/受益比。

The ultimate goal in the treatment of inherited disorders is the permanent correction of the mutation that causes the disease. This project aims at establishing a novel therapy for the treatment of inherited hematological disorders in a mouse model for severe combined immunodeficiency (SCID). The therapy is based on homologous recombination (HR) and corrects the underlying mutation directly in the chromosome. Because of the low frequency of HR, such an approach has only become feasible with the availability of artificial nucleases. The creation of site-specific cuts in the DNA by such nuclease stimulates HR significantly by activating the cellular DNA repair pathways. In order to correct the underlying point mutation for SCID, we will generate repair vectors that serve as a template for HR-based DNA repair as well as custom nucleases that cleave the DNA at the site of the mutation to stimulate HR. Ex vivo gene repair in hematopoietic stem cells HSCs will be achieved by transferring the gene repair components with either integrase-deficient lentiviral vectors or vectors based on different adeno-associated virus serotypes. The extent of gene repair as well as the frequency of genotoxic side effects will be determined in dose escalation studies to evaluate the risk/benefit ratio of this system for putative clinical applications.