Massively Parallel Analysis of Integration in Therapeutic Gene Transfer

治疗性基因转移整合的大规模并行分析

• 批准号: 8755805
• 负责人: Frederic D Bushman
• 金额: $ 41.31万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-15 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8755805
• 关键词: Address; Adult; Adverse event; Antigen Receptors; Antigens; Automobile Driving; Behavior; Bioinformatics; Blood; Blood Cells; CD8B1 gene; Cell Fractionation; Cell division; Cell physiology; Cells; Cellular biology; Chromosomes; Clinical; Clonal Expansion; Cloning Vectors; Commit; DNA; Data; Data Analyses; Development; Disease; Enhancers; Event; Funding; Gene Transduction Agent; Gene Transfer; Gene-Modified; Genes; Genetic Recombination; Genetic Transcription; Growth; Hematopoiesis; Hematopoietic System; Hematopoietic stem cells; Human; Human Development; Human Microbiome; Immunologic Deficiency Syndromes; Individual; Inherited; Insertional Activations; Investigation; Label; Link; Longitudinal Studies; Lymphoid; Mature T-Lymphocyte; Measures; Methods; Monitor; Myelogenous; Outcome; Patient Monitoring; Patients; Population; Population Biology; Population Sizes; Proto-Oncogenes; Recovery; Regimen; Severe Adverse Event; Site; Staging; Statistical Methods; Stem cells; System; T-Lymphocyte; Time; Tracer; Up-Regulation; V(D)J Recombination; base; beta Thalassemia; cell population study; cell type; cellular transduction; chemotherapy; conditioning; congenital immunodeficiency; data acquisition; daughter cell; deep sequencing; follow-up; gene correction; gene therapy; insight; leukemia; mathematical model; novel; progenitor; public health relevance; response; success; therapeutic gene; tool; vector

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.

描述（由申请人提供）：通过将治疗性基因转移到造血干细胞中，已成功治疗 SCID-X1、WAS、ADA、ALD 和 β-地中海贫血等遗传性疾病。 然而，并非所有患者都平等受益。 已经发生了一些不良事件，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有关，并且在一些受试者中，基因治疗并没有有效改善潜在的病症。 我们利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，我们开发了新颖的深度测序和生物信息学方法，产生了有关成功和不成功结果的大量数据。 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获取和分析整合位点分布的数据。 为了扩展我们对血细胞发育的了解，我们还将获取并分析 T 细胞中 VDJ 重组产物的数据。 在我们的第一个目标中，我们将提出以下假设：1）具有完整 LTR（包括转录增强子）的载体在驱动细胞克隆扩增方面比没有它们的载体更加活跃，但所有载体的某些类型的事件与插入一致激活和载体驱动~ 2) 长期祖细胞功能根据疾病状态、载体类型、预处理方案和受试者的临床状况而有所不同~ 3) 大多数转导的祖细胞实际上不仅定向于淋巴或骨髓谱系，但更具体地说，在每个谱系中~ 4) 祖细胞偶尔向外周贡献细胞，并且抗原反应细胞的增殖进一步放大了这种情况，5) 干细胞活性随着时间的推移而减弱，但只是非常缓慢。 在我们的第二个目标中，添加了 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结果 ~ 2) CD4+ 和 CD8+ T 细胞库包含大部分不同的TCR-β重组产物，表明大部分是独立的重组和定型〜3）可以从这些连接祖细胞和成熟细胞的数据估计细胞分裂的次数T 细胞~ 4) 响应抗原的增殖仅涉及祖细胞和 TCR 的特定子集~ 5) TCR 测序可以成为白血病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，拟议的研究将提供独特的数据，可用于优化基因治疗方法和了解人类造血作用，并提供支持 FDA 规定的基因校正受试者长期随访的机制。