Massively Parallel Analysis of Integration in Therapeutic Gene Transfer

描述(申请人提供)：通过将治疗性基因转移到造血干细胞，成功地治疗了遗传性疾病，如SCID-X1、Was、ADA、ALD和β-地中海贫血。然而，并不是所有的患者都同样受益。有几个不良事件表明，整合接近细胞原癌基因的基因治疗载体与转录上调和白血病有关，在一些受试者中，基因治疗并不能有效改善潜在的疾病。我们使用载体整合位点分布数据对人类基因校正过程中的细胞群体进行了广泛的研究，以推断祖细胞的行为。为此，我们开发了新的深度测序和生物信息学方法，产生了关于成功和不成功结果的丰富数据。利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群体生物学的新问题，既可以更全面地了解人类基因纠正的最佳策略，也可以了解人类造血系统本身的发展。我们建议在长期患者监测期间获取和分析整合部位分布的数据。为了扩展我们的血细胞发展图景，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)具有完整的LTRS的载体(包括转录增强子)在驱动细胞克隆的扩张方面比没有它们的载体更加活跃，但具有所有载体的某些类型的事件与插入激活和载体驱动是一致的2)长期的祖细胞功能因受试者的疾病状态、载体类型、调节方案和临床情况而不同~3)大多数转导的祖细胞实际上不仅致力于淋巴系或髓系，而且更具体地说，在每个~4)祖细胞间歇性地向外周贡献细胞，而抗原反应细胞的增殖进一步放大了这一点，干细胞的活性随着时间的推移而减弱，但速度非常缓慢。在我们的第二个目标中，加入了TCR数据，我们将调查下列进一步的假设：1)SCID-X1或基因校正后的TCR-β谱系大小接近健康成年人观察到的大小~2)CD4+和CD8+T细胞谱系中大多包含不同的TCR-β重组产物，表明大部分是独立的重组和承诺~3)根据这些数据可以估计出连接祖细胞和成熟T细胞的细胞分裂数~4)抗原反应中的增殖只涉及一个特定的祖细胞亚集和TCRs~5)TCR测序可作为白血病克隆扩增和化疗成功根除白血病克隆的敏感标志。因此，这项拟议的研究将为优化基因治疗方法和了解人类造血提供独特的数据，并为支持FDA授权的基因校正受试者的长期随访提供一种机制。

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.