AAV Vector Integration Frequency and Associated Genome Alterations

描述（由申请人提供）：由于过去10年的科学进步，治疗性基因递送的目标显着接近。基因治疗最显著的成功之一是治疗了几名患有X连锁严重联合免疫缺陷综合征（X-SCID）的儿童。然而，该疗法需要离体逆转录病毒转导和转导细胞的体内选择，使得该方法仅限于治疗少数疾病。用于基因递送的腺相关病毒（AAV）载体的开发是另一个显著的进步，并且已经导致基因递送效率的显著增加，使得体内基因治疗成为多种遗传疾病的可能性。由于AAV载体在治疗性基因传递方面具有如此大的前景，我们必须开始为可能影响载体设计和组织靶点选择的安全性研究奠定基础。由于整合频率和整合位点分布是一些X-SCID基因治疗患者中白血病发展的重要方面，因此这些是评估载体安全性的明显重点领域。AAV载体与逆转录病毒不同，因为它们完全依赖于宿主细胞蛋白进行载体基因组加工、转录和整合。这种依赖性提出了AAV载体在转导过程中是否影响正常细胞功能的问题。AAV载体清楚地影响细胞中的DSB修复途径，如通过它们在修复位点处并入的事实所证明的。一个重要的遗留问题是，AAV载体是否改变细胞修复途径，即使它们没有被纳入反应。了解哪些修复途径受到影响，以及AAV载体影响DNA修复的程度对于安全性研究的适当设计和分析至关重要。本文描述的实验首先集中在不同小鼠组织中的整合频率上，其次集中在AAV载体对细胞中DNA修复途径的影响上。最后，我们建议通过在良性基因组位置产生修复底物来改变整合位点的位置，所述修复底物比DNA DSB的致突变性小。<Relevance to public health>这项研究的重点是与用于治疗性基因递送的病毒载体管理相关的安全性问题，并将有助于推进遗传疾病的治疗。

DESCRIPTION (provided by applicant): The goal of therapeutic gene delivery is significantly closer due to scientific progress made in the last 10 years. One of the most notable successes of the gene therapy effort is the treatment of several children with X-linked severe combined immunodeficiency syndrome (X-SCID). However, the therapy required ex-vivo retroviral transduction, and in vivo selection of transduced cells making the approach limited to the treatment of a small number of diseases. The development of Adeno-Associated Virus (AAV) vectors for gene delivery is another significant advance and has resulted in substantial increases in the efficiency of gene delivery, making in vivo gene therapy a possibility for a variety of genetic diseases. Because AAV vectors hold such promise for therapeutic gene delivery, we must begin to lay the groundwork for safety studies that might influence vector design and the selection of tissue targets. Since both integration frequency and integration- site distribution were important aspects of the development of leukemia in some X-SCID gene therapy patients, these are obvious areas of focus for assessing vector safety. AAV vectors are different than retroviruses because they depend entirely on host cell proteins for vector genome-processing, transcription and integration. This dependence raises the question of whether AAV vectors influence normal cell functions during transduction. AAV vectors clearly influence DSB repair pathways in the cell as evidenced by the fact that they are incorporated at repair sites. An important remaining question is whether AAV vectors alter cellular repair pathways even when they are not incorporated in the reaction. Understanding which repair pathways are affected, and the extent that AAV vectors influence DNA repair is essential for the appropriate design and analysis of safety studies. Experiments described here focus first on integration frequencies in different mouse tissues, and second on the affect of AAV vectors on DNA repair pathways in the cell. Finally, we propose to alter integration site location by generating repair substrates at benign genomic locations that are less mutagenic than DNA DSBs. <Relevance to public health> This research focuses on safety issues related to the administration of viral vectors for therapeutic gene delivery and will help advance the treatment of genetic disease.