Establishment of the Risk of AAV Vector Integration

描述(申请人提供)：重组腺相关病毒(RAAV)载体在许多靶组织中有效地运送治疗性基因。它们已经变得非常流行，并被广泛用于许多基因转移实验，包括代谢性疾病和遗传性疾病的基因治疗。由于rAAV载体是基于非致病病毒产生的，多年来一直被认为是最安全的载体之一。然而，最近关于小鼠和人类发生不可预测的逆转录病毒插入突变的报道，以及我们最近在对有限数量的rAAV整合事件的分析中发现，rAAV载体优先整合到活性基因中，这些都引起了人们对rAAV介导的插入突变的可能性的担忧。因此，重新定义rAAV介导的基因治疗的风险是非常重要的。我们在这方面的长期目标是确定rAAV载体整合的风险，并开发最安全的rAAV载体系统。为了实现这些目标，我们提出了三项探索性研究。首先，我们将开发一种新的高通量分离载体转导组织中rAAV载体插入位点的方法，并进行大规模分析，以深入了解rAAV载体生物学和rAAV载体整合的后果。其次，考虑到rAAV优先整合到活性基因中，我们假设肝细胞损伤后持续再生的细胞可能存在细胞周期相关基因的表达异常，这可能是rAAV载体整合的易感因素，导致肝细胞加速恶性转化。因此，我们将使用易患肝癌的病毒性肝炎的转基因小鼠模型来评估rAAV载体的协同致癌潜力。第三，我们将探索使用突变的Cre重组酶(保留与IoxP结合的能力，但没有催化活性)来开发非整合的rAAV载体系统。我们假设，突变的Cre的表达和在每个载体末端带有IoxP位点的rAAV载体的使用，将促进分子内的自循环，并最终抑制rAAV载体的基因组整合。我们将通过一系列的体外和体内实验来证明这一方法的原理。这些研究的结果将有助于深入理解rAAV载体整合的机制和插入突变的风险，并开发避免有害载体基因组整合的新技术。

DESCRIPTION (provided by applicant): Recombinant adeno-associated virus (rAAV) vectors efficiently deliver therapeutic genes in many target tissues. They have become very popular and are widely used for many gene transfer experiments including gene therapy of metabolic diseases and genetic disorders. Since rAAV vectors are generated based on a non-pathogenic virus, they have been considered as one of the safest vectors for years. However, recent reports of unpredictable retroviral insertional mutagenesis in mice and human subjects, and our recent findings in an analysis of a limited number of rAAV integration events showing that rAAV vectors preferentially integrate into active genes, have raised concerns about the potential for rAAV-mediated insertional mutagenesis. Therefore, it is very important to re-define the risk of rAAV-mediated gene therapy. Our long-term objectives in this regard are to establish the risk of rAAV vector integration and to develop the safest rAAV vector systems. To achieve these goals, we propose three exploratory studies. First, we will develop a novel high-throughput method for isolation of rAAV vector insertion sites in vector-transduced tissues and carry out a large-scale analysis for thorough understanding of rAAV vector biology and consequences of rAAV vector integration. Second, considering that rAAV preferentially integrate into active genes, we assume that continuously regenerating cells due to hepatocellular injury may have dysregulated expression of cell cycle-related genes, which may be susceptible to rAAV vector integration, leading to accelerated malignant transformation of hepatocytes. Therefore, we will assess synergistic procarcinogenic potential of rAAV vectors using transgenic mouse models for viral hepatitis predisposed to liver cancer. Third, we will explore the use of mutant Cre recombinases (retaining the ability of IoxP site binding but catalytically inactive) to develop a non-integrating rAAV vector system. We hypothesize that expression of a mutant Cre and the use of rAAV vectors with a IoxP site at each vector end, will facilitate intramolecular selfcircularization and ultimately inhibit rAAV vector genome integration. We will establish proof of principle of this approach with a series of in vitro and in vivo experiments. The results from these proposed studies will lead to profound understanding of the mechanisms of rAAV vector integration and the risk of insertional mutagenesis, and to development of new technologies to avoid unwanted vector genome integration.