Massively Parallel Analysis of Integration in Therapeutic Gene Transfer

描述（由申请人提供）：使用治疗性基因转移到造血干细胞中已成功治疗了遗传性疾病，如SCID-X1、WAS、ADA、ALD和β地中海贫血。 然而，并非所有患者都同样受益。 已经有几个不良事件，其中基因治疗载体在细胞原癌基因附近的整合与转录上调和白血病相关，并且在一些受试者中，基因治疗没有有效地改善基础病症。 我们已经进行了广泛的研究细胞群体在人类基因校正使用载体整合位点分布数据来推断祖细胞的行为。 为此，我们开发了新的深度测序和生物信息学方法，产生了大量关于成功和不成功结果的数据。 使用这些数据和持续的长期监测，我们可以解决一系列关于转导细胞的群体生物学的新问题，既可以更全面地了解人类基因校正的最佳策略，也可以了解人类造血系统本身的发育。 我们建议在长期患者监测期间采集和分析关于整合部位分布的数据。 为了扩大我们对血细胞发育的了解，我们还将获取和分析T细胞中VDJ重组产物的数据。 在我们的第一个目标中，我们将解决以下假设：1）具有完整LTR的载体（包括转录增强子）在驱动细胞克隆扩增中比没有它们的载体更有活性，但所有载体的某些类型的事件与插入激活和载体驱动一致。2）长期祖细胞功能根据疾病状态，载体类型，3）大多数转导的祖细胞事实上不仅定向于淋巴或骨髓谱系，而且甚至更具体地在每一种谱系内。 4)祖细胞间歇性地向外周提供细胞，并且这通过抗原应答细胞的增殖而进一步放大，以及5）干细胞活性随时间减弱，但仅非常缓慢。 在我们的第二个目标中，增加了TCR数据，我们将研究以下进一步的假设：1）SCID-X1或WAS基因校正后的TCR-β库大小接近在健康成人中观察到的那些。2）CD 4+和CD 8 + T细胞库主要含有不同的TCR-β重组产物，表明大多数情况下是独立的重组和定型~ 3）细胞分裂的数量可以从这些将祖细胞和成熟T细胞联系起来的数据中估计~ 4）应答抗原的增殖仅涉及特定的祖细胞亚群和TCR ~ 5）TCR测序可以是白血病中克隆扩增和通过化疗成功根除白血病克隆的敏感标记。 因此，拟议的研究将提供在优化基因治疗方法和理解人类造血方面有用的独特数据，并提供一种机制，支持FDA授权的基因校正受试者的长期随访。

DESCRIPTION (provided by applicant): Inherited diseases such as SCID-X1, WAS, ADA, ALD and beta-thalassemia have been treated successfully using therapeutic gene transfer into hematopoietic stem cells. However, not all patients have benefited equally. There have been several adverse events in which integration of gene therapy vectors near cellular proto-oncogenes was associated with upregulation of transcription and leukemia, and in some subjects the gene therapy did not effectively ameliorate the underlying condition. We have carried out extensive studies of cell populations during human gene correction using vector integration site distribution data to infer the behavior of progenitor cells. We developed novel deep sequencing and bioinformatic methods for this purpose, yielding a wealth of data on successful and unsuccessful outcomes. Using these data and ongoing long term monitoring, we can address a new set of questions on the population biology of transduced cells, both to understand more fully the optimal strategies for human gene correction, and to understand development of the human hematopoietic system itself. We propose to acquire and analyze data on integration site distributions during long term patient monitoring. To expand our picture of blood cell development, we will also acquire and analyze data on VDJ recombination products in T-cells. In our first Aim, we will address the following hypotheses: 1) vectors with intact LTRs (including transcriptional enhancers) are much more active in driving expansion of cell clones than vectors without them, but some types of events with all vectors are consistent with insertional activation and vector driving~ 2) long term progenitor function differs depending on disease state, vector type, conditioning regimen and clinical condition of the subject~ 3) most transduced progenitor cells are in fact committed not just to the lymphoid or myeloid lineages, but even more specifically within each~ 4) progenitors are contributing cells to the periphery episodically, and this is further magnified by proliferation of antigen responsive cells and 5) stem cell activity wanes over time, but only very slowly. In our second Aim, which adds in TCR data, we will investigate the following further hypotheses: 1) TCR-beta repertoire sizes after SCID-X1 or WAS gene correction approach those observed in healthy adults~ 2) CD4+ and CD8+ T-cell repertoires contain mostly different TCR-beta recombination products, indicating mostly independent recombination and commitment~ 3) the number of cell divisions can be estimated from these data that link progenitors and mature T-cells~ 4) proliferation in response to antigen involves only a specific subset of progenitors and TCRs~ 5) TCR sequencing can be a sensitive marker for clonal expansion in leukemia and successful eradication of the leukemic clone by chemotherapy. Thus the proposed study will provide unique data useful both in optimizing gene therapy methods and understanding human hematopoiesis, and also provide a mechanism for supporting FDA-mandated long-term follow up of gene-corrected subjects.