单基因遗传性疾病严重危害人类健康，目前还没有很好的治疗手段，基因治疗的出现为该类疾病的治疗提供了新的途径。基因治疗的关键因素是载体，而病毒载体是最常用的基因治疗载体之一。病毒载体通过将具有正常功能的基因导入有基因缺陷的细胞中来达到治疗疾病的目的，但很难实现对有缺陷的基因进行原位修复。此外，病毒载体所导入的外源基因在表达调控、表达持续时间以及在基因组中的定点整合等方面仍存在一些缺陷。近年来靶向基因组编辑技术的发展为单基因缺陷的原位修复提供了可能。针对目前病毒载体所存在的缺陷，本课题将结合靶向基因组编辑技术构建一种具有毒性低、容量大、对靶细胞具有高感染效率、在一个病毒载体中同时携带对基因组具有靶向切割作用的核酸酶及用于同源重组的供体DNA片段等特点的新型载体。最后通过单基因缺陷的动物模型评估新型病毒载体在疾病动物模型体内的实验治疗效果，为最终实现单基因遗传性疾病的基因原位修复奠定基础。

Monogenic diseases are serious threats on human health, which have no possible cure until the emergence of gene therapy. One of the key issues of gene therapy is vector selection and development. Unquestionably the most effective vector systems for gene delivery in use today are modified viruses. Most of the current gene therapy approaches on monogenic diseases use viral vector to deliver genes to appropriate target cells with the aim of producing a product that the patient lacks. However, the effects of this treatment are not permanent. In addition, serious adverse effects, e.g., immune response to in vivo administered viral vectors, and insertional activation of proto-oncogenes, can occur. An alternative to the above gene addition approach is gene correction, i.e., correcting the mutation instead of complementing the mutation, through homologous recombination (HR). This approach can minimize the oncogenic risk of gene therapy due to its targeted gene modification, and holds promise of long-term effects with one-shot treatment. In this project, we will construct a novel adenoviral vector with high capacity to carry simultaneously donor fragment and targeted nuclease (e.g., TALENs) to obtain efficient HR-mediated gene repair in situ based on targeted genome editing technique. In addition, the adenoviral vector will be modified to have lower toxicity, higher infection efficiency for target cells to further improve the safety and efficiency of the treatment. Lastly, the novel system will be evaluated in vivo using a mouse model with a point mutation in beta-glucuronidase gene. This study will provide proof-of-concept for TALENs-mediated gene repair treatment for monogenetic diseases.