Massively Parallel Analysis of Integration in Therapeutic Gene Transfer

描述（由申请人提供）：为了通过治疗性基因转移治疗遗传性疾病，通常需要将治疗性基因整合到宿主细胞的染色体中。然而，在接受SCID-X1基因治疗的患者中，这导致了临床不良事件，其中逆转录病毒载体的整合激活了细胞原癌基因，导致基因校正细胞的转化。因此，基因治疗界已经强烈关注基因转移载体在人类基因组中的整合问题。美国食品和药物管理局甚至要求对整合点进行分析，作为监测可能出现的不良事件的一个步骤。Bushman实验室已经建立了一个独特的技术集合，用于分析基于DNA条形码、焦磷酸测序和定制生物信息学工具的整合位点群体。使用这些高通量方法，可以在一天内生成多达105个碱基的整合位点序列。在一项已发表的研究中，我们生成并分析了人类基因组中40,000个独特的HIV DNA整合位点的位置。在这里，我们建议将这些方法应用于法国SCID-X1试验患者的系统分析。我们已经开始对一些法国SCID-X1患者的纵向DNA样本进行大规模平行测序研究，目的是了解转导细胞的进化、生态和最终命运。到目前为止，我们已经从80个患者样本中产生了约128,000个整合位点序列，总计约33,000,000个碱基的DNA序列。这是第一次，我们可以开始估计对基因校正细胞池有贡献的转导细胞克隆的数量。我们可以问基因校正细胞的种群是如何随时间变化的。在初步研究中，我们发现，在我们研究得最好的两位患者中，克隆多样性出现了令人不安的下降，这表明“克隆耗尽”。我们可以问，即使在没有临床不良事件的情况下，参与生长控制的基因的插入激活是否会导致克隆的生长。对于那些遭受不良事件并随后接受化疗的患者，我们将询问化疗治疗如何影响基因校正细胞池的大小、多样性和动态。我们还将分析动物模型中的不良事件，以及在新的临床试验中产生的长期整合位点。我们建议与法国SCID-X1小组密切合作，完成以下具体目标：确定SCID-X1患者样本中存在的整合位点总数。目标2。确定SCID-X1患者中整合位点的数量和分布如何随时间变化。目标3。在临床前模型和新的临床试验中确定整合位点的位置及其与遗传毒性的关系。

DESCRIPTION (provided by applicant): To treat genetic diseases by therapeutic gene transfer, it is usually necessary to integrated the therapeutic gene into a chromosome of the host cell. However, this has led to clinical adverse events in patients receiving gene therapy for SCID-X1, in which integration of retroviral vectors activated cellular proto-oncogenes, leading to transformation of gene-corrected cells. Thus the gene therapy community has become intensely focused on the question of where gene transfer vectors integrate in the human genome. The FDA has even mandated that integration sites be analyzed as a step in monitoring for possible adverse events. The Bushman laboratory has established a unique collection of technologies for analyzing integration site populations based on DNA bar coding, pyrosequencing, and custom bioinformatic tools. Using these high throughput methods, populations of integration site sequences can be generated of up to 105 bases of sequence in a single one day run. In one published study, we generated and analyzed the placement of 40,000 unique sites of HIV DNA integration in the human genome. Here we propose to apply these methods to systematic analysis of patients from the French SCID-X1 trial. We have initiated massively parallel sequencing studies of longitudinal DNA samples from some of the French SCID-X1 patients, with the goal of understanding the evolution, ecology, and ultimate fate of transduced cells. So far, we have generated ~128,000 integration site sequence reads from 80 patient samples for a total of ~33,000,000 bases of DNA sequence. For the first time, we can begin to estimate the numbers of transduced cell clones contributing to the gene-corrected cell pool. We can ask how populations of gene corrected cells change over time. In preliminary studies, we find a troubling decline in clone diversity in our two best-studied patients, suggesting "clone burn out". We can ask whether insertional activation of genes involved in growth control leads to outgrowth of clones even in the absence of clinical adverse events. For those patients who suffered adverse events and were subsequently treated by chemotherapy, we will ask how the chemotherapeutic treatment affected the size, diversity and dynamics of the gene-corrected cell pools. We will also analyze adverse events in animal models and, longer term, integration sites generated in new clinical trials. We propose to work in close collaboration with the French SCID-X1 team to complete the following Specific Aims: Aim 1. Determine the total numbers of integration sites present in samples from SCID-X1 patients. Aim 2. Determine how the number and distribution of integration sites changes over time in SCID-X1 patients. Aim 3. Determine integration site locations and relationship to genotoxicity in preclinical models and new clinical trials. PUBLIC HEALTH RELEVANCE: Success has been achieved with human gene therapy for SCID-X1, but adverse events due to insertional activation of proto-oncogenes and leukemia have caused severe setbacks. Here we propose to apply massively parallel pyrosequencing to analyzing vector integration in samples from the historic SCID-X1 trial.