VECTORS DIRECTING PERSISTENT MYELOID GENE EXPRESSION

指导持续性骨髓基因表达的载体

• 批准号: 6208597
• 负责人: John Peter O'Rourke
• 金额: $ 3.24万
• 依托单位: RESEARCH INST NATIONWIDE CHILDREN'S HOSP
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6208597
• 关键词: CD34 molecule; Retroviridae; SCID mouse; binding sites; cell differentiation; cell type; chromatin; embryo /fetus cell /tissue; flow cytometry; gene expression; gene induction /repression; gene therapy; genetic promoter element; green fluorescent proteins; hematopoietic stem cells; myeloid stem cell; northern blottings; nucleic acid repetitive sequence; polymerase chain reaction; technology /technique development; tissue /cell culture; transcription factor; transfection /expression vector; western blottings

Improvements have been made in retroviral mediated transduction of fetal tissues. However, in utero gene therapy, like other gene therapy applications, has been hampered by severely limited levels of transgene expression and a lack of persistent expression in differentiated cells due to promoter silencing mechanisms. Although some genetic therapy applications for genetic diseases of the hematopoietic system may not require cell specific transgene expression, for others targeted expression may be an essential prerequisite for treatment of these disorders. The long-term goal of the proposed research is the development novel retroyiral vectors which direct high level, persistent, and cell specific transgene expression in differentiated graniiloc and monocytes for use in our ongoing in utero gene therapy studies. We propose to investigate promoter regions from myeloid specific genes C/EBPepsilon and CD 11b to direct cell type specific transgene expression. To overcome the problem of low and transient transgene expression levels we propose to augment our cell specific expression vectors with tandem repeats of a myeloid enhancer region which may enhance promoter activity and/or chromatin insulators to eliminate chromatin silencin effects. These expression vectors will be investigated for their ability to direct high, protracted, myeloid specific transgene expression by transduction into human myeloid cell lines and primary human CD34+ cells followed by in vitro differentiation. Transplantation of transduced human CD 34+ cells into irradiated SCID mice to recapitulate human hematopoiesis and subsequent analysis of gene expression in differentiated hematopoietic cells will be performed. Finally, the vector will analyzed for their ability to direct high, protracted, myeloid specific transgene expression by injection of retroviral supernatant into fetal mice. The results of these studies will provide critical information regarding the feasibility of persistent, cell specific gene expression for use in the in utero treatment of inherited hematopoietic diseases.

在逆转录病毒介导的胎儿组织转导方面已经取得了进展。然而，与其他基因治疗应用一样，子宫内基因治疗一直受到转基因表达水平严重受限以及由于启动子沉默机制而在分化细胞中缺乏持续表达的阻碍。尽管一些造血系统遗传病的基因治疗应用可能不需要细胞特异性的转基因表达，但对另一些疾病来说，靶向表达可能是治疗这些疾病的必要前提。这项研究的长期目标是开发新的逆转录病毒载体，以指导在分化的颗粒和单核细胞中高水平、持续的和细胞特异性的转基因表达，用于我们正在进行的宫内基因治疗研究。我们建议研究髓系特异性基因C/EBPepsilon和CD11b的启动子区域，以指导细胞类型特异性转基因表达。为了克服转基因表达水平低和短暂的问题，我们建议用髓系增强子区的串联重复序列来增强我们的细胞特异性表达载体，这可能会增强启动子活性和/或染色质绝缘体，以消除染色质沉默效应。这些表达载体将通过将其导入人髓系细胞系和原代人CD34+细胞并进行体外分化来研究它们定向高、持久的髓系特异性转基因表达的能力。将转导的人CD34+细胞移植到受照射的SCID小鼠中，以重现人类的造血功能，并随后分析分化的造血细胞中的基因表达。最后，该载体将通过将逆转录病毒上清液注射到胎鼠体内来分析它们引导高水平、持久的髓系特异性转基因表达的能力。这些研究的结果将为持续的、细胞特异性的基因表达用于遗传性造血疾病的宫内治疗的可行性提供关键信息。