Vector Integration and Tracking

在接受干细胞基因治疗的19例SCID-XL患者中，有5例观察到白血病的发展 心理治疗。在所有5名患者中，白血病的发展是由于插入突变，即逆转录病毒- 介导附近原癌基因的激活。因此，仔细研究整合站点和 个体克隆对再繁殖的贡献将对所有基因治疗研究至关重要，并且 FDA要求对所有临床基因治疗中的逆转录病毒整合部位进行仔细监测。 学习。向量集成和跟踪核心D将为所有项目提供一个集中设施，以 高效识别泡沫病毒载体在复杂生物DNA样品中的整合位点 活体研究。我们将使用新开发的改良型非限制性LAM-PCR。核心将能够 为了处理来自小鼠、狗或人类的各种样本以产生用于穿梭载体或聚合酶链式反应的DNA- 基于方法。对于nrLAM-pcr，只需很少的dna就可以进行fv载体的整合定位。 扩增和延伸处理，为测序做准备。经D芯处理的样品将 可与用于试点实验的基于Sanger的测序和新测序相媲美 用于深度测序的方法学，例如焦磷酸测序，以识别所有可扩增的FV载体 给定样本中的集成站点。CORE D还将协助所有项目设计入门读物 特定于FV载体整合位点，以允许基于DNA的实时(RT)-PCR跟踪来评估 对被认为对进一步投资具有重要意义的单个克隆的贡献。此外，核心D将 通过通用网关接口(CGI)-Perl Web提供集中工具来分析集成站点 服务器。将支持人类(Hg19)、狗(CanFam2)和小鼠(MM9)基因组的当前版本。 核心还将通过Perl程序向调查人员提供支持，以关联FV载体整合 网站的数据来自已发表的数据库，包括原癌基因TS、微阵列数据和超- 代表了基因类别。生物信息学组件还将为这三个组件生成随机数据集 人类、小鼠和狗的基因组，以评估媒介前病毒附近代表过多的基因类别。 相关性： PPG的重点是测试最合适的逆转录病毒载体系统FV的有效性和安全性 治疗SCID-XL的载体。基于逆转录病毒的安全分析的一个不可或缺的方面是 整合位点分析和周围基因组元件的特征。的具体目标 核心D将提供一个仔细的标准化方法，定义当前和改进后的FV的安全性 在项目1-3中开发的载体。

The development of leukemia has been observed in 5 out of 19 SCID-Xl pafients treated by stem cell gene therapy. In all 5 pafients the development of leukemia was due to insertional mutagenesis, i.e. the retrovirus- mediated activafion of nearby proto-oncogenes. Thus, the careful study of integration sites and the contribufion of individual clones to repopulafion will be of crucial importance for all gene therapy studies, and the FDA has mandated the careful monitoring of retroviral integration sites in all clinical gene therapy studies. The Vector Integration and Tracking Core D will provide all projects a centralized facility to efficiently identify foamy virus (FV) vector integrafion sites in complex biological DNA samples from in vitro or in vivo studies. We will use recenfiy developed improved non-restricfion (nr)LAM-PCR. The core will be able to process a variety of samples from murine, dog, or human to generate DNA for shuttle vector or PCR- based methods. For nrLAM-PCR, as little as lOng of DNA can be used to carry out FV vector integrafion site amplificafion and extended processing in preparation for sequencing. The samples processed by Core D will be compafible with both Sanger-based sequencing for pilot experiments and new sequencing methodologies, such as pyrosequencing, for deep sequencing to identify all of the amplifiable FV vector integration sites in a given sample. Core D will also provide assistance to all projects to design primers specific to FV vector integrafion sites to allow for DNA-based real time (RT)-PCR tracking to assess the contribufion for individual clones that are deemed important for further invesfigation. In addifion, Core D will provide a centralized facility to analyze integrafion sites via a common gateway interface (CGI)-PERL web server. Current versions of the human (hg19), dog (canFam2) and mouse (mm9) genomes will be supported. The Core will also provide support to investigators through PERL programs to correlate FV vector integration sites with data from published databases including proto-oncogene TSS, microarray data and over- represented gene classes. The bioinformatics component will also generate random datasets for all three genomes, human, mouse and dog, to evaluate over-represented gene classes near vector proviruses. RELEVANCE: The focus of this PPG is to test the efficacy and safety of the most appropriate retrovirus vector system, FV vectors for the treatment of SCID-Xl. An indispensible aspect of retrovirus-based safety analysis is integration site profiling and characterization of the surrounding genomic elements. The specific aims of Core D will provide a careful standardized approach defining the safety of the current and improved FV vectors developed in Projects 1-3.