GENE THERAPY FOR THE TREATMENT OF CAMT

治疗 CAMT 的基因疗法

• 批准号: 7922605
• 负责人: Neil Josephson
• 金额: $ 42.21万
• 依托单位: BLOODWORKS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7922605
• 关键词: Affect; Allogenic; Autologous; Biological Assay; Blood Cell Count; Blood Cells; Breeding; CD34 gene; Cell Count; Cell Survival; Cells; Clinical Treatment; Clinical Trials; Code; Commit; Defect; Development; Disease; Doctor of Medicine; Ensure; Evaluation; Flow Cytometry; Gene Expression; Gene Transfer; Generations; Genes; Germ Cells; Germ Lines; Grant; Green Fluorescent Proteins; Growth; Harvest; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Human; Immunologics; Individual; Inherited; Intercistronic Region; Lymphoid; MPL gene; Marrow; Medicine; Megakaryocytes; Molecular; Mus; Myelogenous; Pancytopenia; Patients; Pattern; Phylogenetic Analysis; Protocols documentation; Reporter Genes; Research; Research Personnel; Retroviridae; Safety; Specificity; Spumavirus; Stem cell transplant; Stem cells; System; Techniques; Testing; Thrombocytopenia; Thrombopoietin; Tissue-Specific Gene Expression; Tissues; Transfusion; Transgenes; Transgenic Mice; Transgenic Organisms; Transplant Recipients; Transplantation; Work; base; cellular transduction; expression vector; gene correction; gene therapy; human MPL protein; improved; loss of function mutation; mouse model; peripheral blood; pre-clinical; progenitor; programs; promoter; prototype; restoration; retroviral-mediated; transduction efficiency; transgene expression; vector

Congenital Amegakaryocytic Thrombocytopenia (CAMT) is an inherited stem cell disorder caused by loss of function mutations in MPL,the receptor for thrombopoietin (TPO). Affected individuals are born thrombocytopenic and over months to years develop lethal pancytopenia unless they are successfully treated by allogeneic stem cell transplantation. Unfortunately, a significant number of CAMT patients do not have a suitable HLA matched donor. Hematopoietic stem cell (HSC) gene therapy offers the promise of treating all patients with CAMT by genetically modifying autologous stem cells through retroviral mediated transfer of a normal copy of the MPL gene. In this proposal we outline pre-clinical work for developing Prototype Foamy Virus (PFV) vectors and HSC gene transfer protocols to treat CAMT. Vector expression of MPL in CAMT patients needs to produce normal expression patterns to ensure stem cell survival and megakaryocyte development, without inducing dyshematopoiesis. Aim 1 proposes the development of vectors with tissue specific promoters. We will generate and test vectors with promoters that limit gene expression to primitive hematopoietic cells and those committed to the megakaryocyte lineage. In Aim 2 we will evaluate if Mpl gene addition can reverse the hematopoietic defects in a mouse model of CAMT. Using standard transgenic techniques an Mpl transgene under direction of the Mpl gene promoter has been added to the germ line cells of CAMT mice and we now have a stable breeding colony of gene corrected mice. In addition we will use PFV vectors, with the tissue specific promoters developed in Aim I, to transfer the Mpl gene into the HSCs of CAMT mice. Gene corrected mice will be evaluated for peripheral blood counts, megakaryocyte lineage development, and the restoration of the HSC compartment. We will check for vector safety by looking for morphological evidence of dyshematopoiesis and molecular evidence of clonal hematopoiesis in PFV vector corrected mice kept alive for at least 1 year. Aim 3 of the grant proposes using PFV vectors with the human MPL transgene and the promoters evaluated in Aims 1 and 2 to transduce human CD34+ cells from patients with CAMT. Transduced cells will be evaluated for both megakaryocyte development and clonogenic progenitor growth. We project that, as a result of the studies completed in this research plan, a clinical trial for the treatment of CAMT with PFV vectors will be proposed at the completion of this grant.

先天性无巨核细胞性血小板减少症（CAMT）是一种遗传性干细胞丢失引起的疾病， 血小板生成素（TPO）受体MPL功能突变的研究。受影响的人出生 血细胞减少，数月至数年发展为致命的全血细胞减少症，除非他们成功地 通过异基因干细胞移植治疗。不幸的是，大量的CAMT患者 没有合适的HLA配型供体。造血干细胞（HSC）基因治疗提供了希望 通过逆转录病毒基因修饰自体干细胞治疗所有CAMT患者 介导的MPL基因的正常拷贝的转移。在本提案中，我们概述了临床前工作， 开发原型泡沫病毒（PFV）载体和HSC基因转移方案以治疗CAMT。 MPL在CAMT患者中的载体表达需要产生正常的表达模式，以确保干细胞表达。 细胞存活和巨核细胞发育，而不诱导造血异常。目标1提出， 开发具有组织特异性启动子的载体。我们将产生并测试带有启动子的载体 这限制了原始造血细胞和巨核细胞的基因表达 脉在目标2中，我们将评估Mpl基因添加是否可以逆转小鼠中的造血缺陷。 CAMT模型使用标准的转基因技术，在Mpl基因的指导下， 启动子已经被添加到CAMT小鼠的生殖系细胞中，我们现在有一个稳定的繁殖 基因校正小鼠的群体。此外，我们将使用PFV载体，与组织特异性启动子 目的I中开发的，将Mpl基因转移到CAMT小鼠的HSC中。基因校正的小鼠将 评估外周血细胞计数、巨核细胞谱系发育和 HSC隔室。我们将通过寻找病原体的形态学证据来检查病媒安全性。 PFV载体校正小鼠造血功能障碍及克隆性造血的分子证据 至少活了一年。该授权的目的3提出使用具有人MPL转基因的PFV载体 以及在目的1和2中评估的启动子，以从CAMT患者中扩增人CD 34+细胞。 将对转导细胞的巨核细胞发育和克隆祖细胞进行评价 增长我们计划，作为本研究计划中完成的研究的结果， 在完成该补助金后，将提出用PFV载体治疗CAMT的建议。