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.

在逆转录病毒介导的胎儿组织转导中已经改善。然而，在子宫基因疗法中，与其他基因治疗的应用一样，由于启动子沉默机制而导致的分化细胞中的转基因表达严重有限，并且缺乏持续的表达。尽管某些基因治疗应用于造血系统的遗传疾病可能不需要细胞特异性转基因表达，但对于其他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的缺失媒介载体，该载体在分化的Graniiloc和单核细胞中直接高水平，持久性和细胞特异性转基因表达，以用于我们正在进行的子宫基因治疗研究中。 我们建议研究来自髓样特异性基因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。为了克服低和短暂的转基因表达水平的问题，我们建议用髓样增强子区域的串联重复序列增强细胞特异性表达载体，从而增强启动子活性和/或染色质绝缘剂，以消除染色质沉默。这些表达矢量将通过转导向人髓样细胞系和原代人CD34+细胞，然后进行体外分化，从而研究其直接，持久的髓样特异性转基因表达的能力。将转导的人Cd 34+细胞移植到受辐照的SCID小鼠中，以概括人造血，随后对分化造血细胞中基因表达进行分析。 最后，通过将逆转录病毒上清液注射到胎儿小鼠中，将分析载体的直接高，长期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续的细胞特异性基因表达可行性的关键信息，用于在子宫内治疗遗传性造血疾病中。