VECTORS DIRECTING PERSISTENT MYELOID GENE EXPRESSION

在逆转录病毒介导的胎儿组织转导方面取得了进展。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限制和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。虽然一些用于造血系统遗传性疾病的基因治疗应用可能不需要细胞特异性转基因表达，但对于其他靶向表达可能是治疗这些疾病的必要先决条件。这项研究的长期目标是开发新的逆转录病毒载体，在分化的粒细胞和单核细胞中指导高水平、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基因治疗研究。我们建议研究骨髓特异性基因C/EBPepsilon和cd11b的启动子区域，以指导细胞类型特异性的转基因表达。为了克服低和瞬时转基因表达水平的问题，我们建议用骨髓增强子区域的串联重复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/或染色质绝缘子，以消除染色质沉默蛋白效应。这些表达载体将通过转导到人骨髓细胞系和原代人CD34+细胞并进行体外分化来指导高水平、长时间、髓系特异性转基因表达的能力。将转导的人cd34 +细胞移植到辐照的SCID小鼠中以重现人造血，并随后分析分化的造血细胞中的基因表达。最后，通过将逆转录病毒上清液注射到胎鼠体内，分析该载体对高、持久、髓系特异性转基因表达的指导能力。这些研究的结果将为持续的、细胞特异性的基因表达在子宫内治疗遗传性造血疾病的可行性提供关键信息。

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.