VECTORS DIRECTING PERSISTENT MYELOID GENE EXPRESSION

指导持续性骨髓基因表达的载体

• 批准号: 6526625
• 负责人: John Peter O'Rourke
• 金额: $ 4.62万
• 依托单位: UNIVERSITY OF NEW MEXICO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6526625
• 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.

逆转录病毒介导的胎儿组织转导已取得进展。然而，与其他基因治疗应用一样，子宫内基因治疗受到转基因表达水平严重有限以及由于启动子沉默机制导致分化细胞中缺乏持续表达的阻碍。尽管一些针对造血系统遗传疾病的基因治疗应用可能不需要细胞特异性转基因表达，但对于其他疾病，靶向表达可能是治疗这些疾病的必要先决条件。拟议研究的长期目标是开发新型逆转录载体，其指导分化的花核细胞和单核细胞中高水平、持久和细胞特异性的转基因表达，用于我们正在进行的子宫内基因治疗研究。 我们建议研究骨髓特异性基因 C/EBPepsilon 和 CD 11b 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且短暂的问题，我们建议用串联重复的骨髓增强子区域来增强我们的细胞特异性表达载体，这可以增强启动子活性和/或染色质绝缘体以消除染色质沉默效应。将研究这些表达载体通过转导到人骨髓细胞系和原代人CD34+细胞中，然后进行体外分化来指导高、持久、骨髓特异性转基因表达的能力。将转导的人类 CD 34+ 细胞移植到受辐射的 SCID 小鼠中以重现人类造血作用，并随后对分化的造血细胞中的基因表达进行分析。 最后，通过将逆转录病毒上清液注射到胎儿小鼠体内，分析载体引导高、持久、骨髓特异性转基因表达的能力。这些研究的结果将提供有关持久性细胞特异性基因表达用于遗传性造血疾病子宫内治疗的可行性的关键信息。