Adenoviral-mediated Delivery of anti RET Hammerhead Ribozymes Inhibits Thyroid Medullary Carcinoma Cell Proliferation.

RET原癌基因的激活突变导致多发性内分泌瘤（MEN） 2A、MEN2B、家族性甲状腺髓样癌（MTC）。下调RET表达可能是癌症治疗的重要策略。事实上，我们之前报道过靶向突变RET的核酶抑制了转染突变RET的NIH 3T3细胞的转化。然而，大多数突变序列不符合锤头核酶切割的要求。在这项研究中，我们首先设计了锤头核酶来切割正常的RET转录物。我们选择了两个目标位点，分别针对细胞内和细胞外区域的核酶切割。体外裂解实验表明，这些核酶能特异性、高效地裂解人工靶序列。为了实现高效的基因传递，我们开发了由巨细胞病毒启动子驱动编码这些核酶的重组腺病毒。用腺病毒感染人MTC细胞株TT。在表达活性核酶的腺病毒感染细胞中，观察到RET在RNA和蛋白水平上的表达降低。与此同时，表达活性核酶的腺病毒感染TT细胞的细胞增殖也受到抑制。为了证明核酶的特异性，对携带ret/PTC-1重排（ret的胞内结构域与无关蛋白融合）的TPC-1细胞的感染表明，只有靶向胞内结构域的核酶对细胞增殖有显著但不太显著的影响。最后，在表达活性核酶的腺病毒感染的TT细胞中，caspase-3活性升高，表明凋亡途径参与了核酶的作用。这些结果为通过选择性mRNA降解抑制RET及其潜在的治疗应用提供了新的途径。

Activating mutations of the RET proto-oncogene cause multiple endocrine neoplasia (MEN) 2A, MEN2B, familial medullary thyroid carcinoma (MTC). Down-regulation of RET expression may be an important strategy for cancer therapy. Indeed, we previously reported ribozyme targeting mutant RET suppress transformation of NIH 3T3 cells transfected with mutant RET. However, the most of mutant sequences are not compatible with requirement for cleavage by hammerhead ribozyme. In this study, we first designed hammerhead ribozymes to cleave the normal RET transcript. We select two target sites for cleavage of ribozymes targeting intracellular and extracellular domain, respectively. In vitro cleavage assay demonstrates that these ribozymes can cleave the artificial target sequence specifically and efficiently. In order to achieve efficient gene delivery, we developed recombinant adenoviruses encoding these ribozymes driven by the cytomegalovirus promoter. Human MTC cell line TT was infected with the adenoviruses. Reduction of RET expression in RNA and protein level was observed in active ribozyme-expressing adenovirus-infected cells. Concordantly, cell proliferation was also suppressed in the active ribozyme expressing adenovirus-infected TT cells. In order to demonstrate specificity of the ribozymes, infection into TPC-1 cells which harbor ret/PTC-1 rearrangement (where intracellular domain of RET is fused with irrelevant protein) exhibit that only ribozyme targeting intracellular domain has significant but much less prominent effect on the cell proliferation. Finally, caspase-3 activity was elevated in active ribozyme-expressing adenovirus infected TT cells, indicating that apoptotic pathways are involved in the effects of the ribozymes. These results provide a new avenue for inhibition of RET by selective mRNA degradation with its potential therapeutic application.