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。5例白血病均为插入突变所致，即逆转录病毒介导的邻近原癌基因激活。因此，对整合位点的仔细研究以及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，FDA已授权在所有临床基因治疗研究中仔细监测逆转录病毒整合位点。载体整合和跟踪核心D将为所有项目提供一个集中的设施，以有效地识别来自体外或体内研究的复杂生物DNA样本中的泡沫病毒(FV)载体整合位点。我们将使用最近开发的改进的非限制性(Nr)LAM-PCR。该核心将能够处理来自小鼠、狗或人类的各种样本，以产生用于穿梭载体或基于PCR的方法的DNA。对于nrLAM-PCR，只需很少的DNA就可以进行FV载体整合位点的扩增和延伸处理，为测序做准备。经Core D处理的样本将与用于中试实验的基于Sanger的测序和用于深度测序的新测序方法(例如焦磷酸测序)兼容，以识别给定样本中所有可扩增的FV载体整合位点。CORE D还将协助所有项目设计针对FV载体整合位点的特定引物，以实现基于DNA的实时(RT)-PCR跟踪，以评估被认为对进一步研究重要的单个克隆的贡献。此外，Core D将通过通用网关接口(CGI)-Perl Web服务器提供一个集中工具来分析集成站点。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程序向研究人员提供支持，将FV载体整合站点与已公布的数据库中的数据关联起来，包括原癌基因TS、微阵列数据和过度代表的基因类别。生物信息学部分还将为所有三个基因组(人、小鼠和狗)生成随机数据集，以评估载体前病毒附近过度代表的基因类别。

The development of leukemia has been observed in 5 out of 19 SCID-Xl patients treated by stem cell gene therapy. In all 5 patients the development of leukemia was due to insertional mutagenesis, i.e. the retrovirus- mediated activation of nearby proto-oncogenes. Thus, the careful study of integration sites and the contribution of individual clones to repopulation 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 integration sites in complex biological DNA samples from in vitro or in vivo studies. We will use recently developed improved non-restriction (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 integration site amplification and extended processing in preparation for sequencing. The samples processed by Core D will be compatible 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 integration sites to allow for DNA-based real time (RT)-PCR tracking to assess the contribution for individual clones that are deemed important for further investigation. In addition, Core D will provide a centralized facility to analyze integration 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.