Du Z, Ricordi C, Inverardi L, Podack E, Pastori R L
Diabetes Research Institute, University of Miami School of Medicine, FL 33136, USA.
Hum Gene Ther. 1998 Jul 20;9(11):1551-60. doi: 10.1089/hum.1998.9.11-1551.
Graft-versus-host disease (GVHD) is a feared complication of allogeneic bone marrow transplantation. Research in rodent models has linked perforin and Fas ligand (FasL), two components of independent lytic pathways, with the induction of GVHD. In this study we characterized two hammerhead ribozymes that cleave their target perforin and Fas ligand RNAs with high efficiency in CTLL-2 cells. The perforin and Fas ligand ribozymes were expressed from a tRNA-directed RNA polymerase III promoter that was inserted in an episomal multicopy plasmid derived from papilloma virus. Chimeric anti-perforin and anti-FasL tRNA-ribozymes had sequences engineered in order to have specific secondary structure effects. These sequence modifications allow the formation of a 5' --> 3' stem structure and also place the ribozyme in a flexible bulge region that keeps the ribozyme separated from the tRNA domain. Northern and RT in situ PCR analyses showed high levels of transcription and efficient transportation to the cytoplasm. The expression of perforin and FasL in CTLL-2 cells was significantly reduced as assessed by RNA and protein analyses.
移植物抗宿主病（GVHD）是同种异体骨髓移植令人担忧的并发症。对啮齿动物模型的研究已将穿孔素和Fas配体（FasL）这两种独立裂解途径的成分与GVHD的诱导联系起来。在本研究中，我们鉴定了两种锤头状核酶，它们在CTLL-2细胞中能高效切割其靶标穿孔素和Fas配体RNA。穿孔素和Fas配体核酶由插入源自乳头瘤病毒的游离多拷贝质粒中的tRNA指导的RNA聚合酶III启动子表达。嵌合抗穿孔素和抗FasL tRNA-核酶经过序列工程设计，以产生特定的二级结构效应。这些序列修饰允许形成5'→3'茎结构，并且还将核酶置于一个灵活的凸起区域，使核酶与tRNA结构域分离。Northern印迹和RT原位PCR分析显示转录水平高且能有效转运至细胞质。通过RNA和蛋白质分析评估，CTLL-2细胞中穿孔素和FasL的表达显著降低。
