Vector Insertion and Mutagenesis in Human Hematopoiesis

人类造血中的载体插入和诱变

• 批准号: 7216799
• 负责人: CHRISTOF VON KALLE
• 金额: $ 35.32万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7216799
• 关键词: Acute T Cell Leukemia; Adverse effects; Applications Grants; Behavior; Biological Assay; Biological Models; Blood; Blood Cells; Bone Marrow; Bone Marrow Cells; Cell Count; Cell model; Cells; Child; Clinical; Clone Cells; Complementary DNA; Data; Databases; Disease; Engraftment; Equipment and supply inventories; Evolution; Frequencies; Future; Gene Expression; Gene Structure; Gene Transfer; Genes; Genetic; Genetic Transcription; Genomics; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Human; Human Genome; In Vitro; Incidence; Infusion procedures; Insertional Activations; Interleukin 2 Receptor; LMO2 gene; Maps; Marrow; Measures; Mediating; Methods; Modeling; Modification; Molecular; Molecular Profiling; Mus; Mutagenesis; Mutation; Nature; Oncogenes; Participant; Patients; Phenotype; Polymerase Chain Reaction; Population; Premalignant; Prevention strategy; Process; Protein Overexpression; Research; Retroviral Vector; Retroviridae; Risk; Role; Secondary to; Severe Combined Immunodeficiency; Site; Somatic Gene Therapy; Stem cells; Syndrome; System; T-Lymphocyte; Technology; Testing; Transplantation; Umbilical Cord Blood; Untranslated Regions; X-Linked Severe Combined Immunodeficiency; base; cellular transduction; cytokine; dosage; embryonic stem cell; gene correction; gene therapy; immune function; in vivo; insight; leukemia; pre-clinical; promoter; reconstitution; research study; size; vector

DESCRIPTION (provided by applicant): Because of their capacity to provide long-term in vivo reconstitution of the blood system, hematopoietic stem cells are an attractive target for somatic gene therapy. Retroviral gene transfer into cord blood or marrow cells has been used to correct genetic defects most successfully in patients with potentially fatal human severe combined immunodeficiency disease (SCID). In a total of 13 children with X-linked SCID worldwide, T cell and immune function could be restored to normal by transplantation of genetically corrected bone marrow cells expressing the human IL-2-receptor gamma chain (IL-2Rgamma) cDNA. Retroviral gene therapy of another form of SCID (ADA-SCID) has now also obtained significant levels of T cell correction. The emergence of two cases of a T-cell Acute Lymphocytic Leukemia like disease (T-ALL) that is most likely secondary to the genetic modification in the two youngest patients of the X-SCID gene therapy trial conducted by Fischer and colleagues has prompted new critical questions into the basis and generalizability of this phenomena in genetically modified hematopoietic cells. We have developed a new linear amplification mediated (LAM) PCR method that has allowed us to identify single retroviral insertion sites from freshly transduced cells in vitro, and in vivo, the proliferative expansion of single stem cell clones. This approach allowed us to discover retroviral insertion-activation of the LMO2 gene, a T-ALL associated oncogene, in the two patients suffering from the leukemic syndrome in the Fischer trial. Preliminary data obtained by the analysis of additional insertion sites from these and other patients suggests that a broad assessment of retroviral insertions before and after in vivo clone selection can be accomplished and that this will allow us to gain substantial additional insight into the identity, significance, and impact of retroviral insertions in gene corrected progenitor cells. Using material from all patients in these two clinical gene transfer trials, and insertion within the LMO2 locus as a model system, it is the overall aim of this proposal to determine the frequency of retrovirus insertion into the vicinity of the LMO2 locus in human repopulating cell clones and to track their fate in vivo (Aim 1), to understand the frequency of insertions into other critical cellular genes and gene structures (Aim 2), and to map the clonal composition of the inventory of genetically corrected hematopoietic cells in typical patients of the X-SCID gene therapy trials (Aim 3) in order to develop safe and efficient dosage and side effect prevention strategies for future gene therapy trials. Results from these studies of insertion sites in the human genome, and the role of LMO2 overexpression on the evolution of a lymphoproliferative phenotype will have great significance for the field.

描述（由申请人提供）：由于他们提供长期体内重建血液系统的能力，造血干细胞是体细胞基因疗法的有吸引力的靶标。逆转录病毒基因转移到脐带血或骨髓细胞中已被用来纠正潜在致命的人类严重严重合并免疫缺陷疾病（SCID）的患者中最成功的遗传缺陷。在全球范围内总共有13名患有X连锁SCID的儿童中，T细胞和免疫功能可以通过移植表达人IL-2受体伽马链（IL-2RGAMMA）cDNA的遗传校正的骨髓细胞来恢复正常。现在，SCID（ADA-SCID）的逆转录病毒基因治疗现在也获得了T细胞校正水平的显着水平。在Fischer和同事进行的两名最年轻的X-SCID基因治疗试验中，这两种最有可能是遗传修饰的T细胞急性淋巴细胞性白血病（T-ALL）的出现，这些病例很可能是遗传修饰的继发性。我们已经开发了一种新的线性扩增介导的（LAM）PCR方法，该方法使我们能够在体外和体内鉴定出来自新鲜转导的细胞的单个逆转录病毒插入位点，在体内，单个干细胞克隆的增殖膨胀。这种方法使我们能够在Fischer试验中发现了两名患有白血病综合征的患者的LMO2基因（一种T-ALL相关癌基因）的逆转录病毒插入激活。通过分析这些患者和其他患者的其他插入位点获得的初步数据表明，可以完成对体内克隆选择前后倒流病毒插入的广泛评估，这将使我们能够获得对基因矫正术中的递归插入的身份，意义和影响的实质性深入了解。 Using material from all patients in these two clinical gene transfer trials, and insertion within the LMO2 locus as a model system, it is the overall aim of this proposal to determine the frequency of retrovirus insertion into the vicinity of the LMO2 locus in human repopulating cell clones and to track their fate in vivo (Aim 1), to understand the frequency of insertions into other critical cellular genes and gene structures (Aim 2), and to绘制X-SCID基因治疗试验典型患者的遗传校正造血细胞库存的克隆组成（AIM 3），以制定安全有效的剂量和副作用预防策略，以预防未来基因治疗试验。这些对人基因组插入位点的研究的结果，以及LMO2过表达在淋巴增生表型进化中的作用将对该领域具有重要意义。