Gene therapy for hepatitis C with gene cutter

HCV基因切割器由核酶RNA及其载体（asialoorosomucoid-poly-L-lysin; ASOR-PLL）组成。当大鼠静脉注射生物素化的poly-C （RNA）和ASOR-PLL复合物时，仅在肝细胞中检测到生物素化的poly-C，这表明RNA通过载体特异性递送到肝细胞。采用原位反转录法（原位RT）在切片标本上检测培养的人肝细胞HCV RNA。此外，该核酶还能在肝细胞中特异性切割HCV RNA的核苷酸序列。我们将HCV感染的培养人肝细胞移植到大鼠脾脏，建立了HCV感染动物模型。利用该模型对基因切割的能力进行了测试。简单地说，将HCV基因切割器静脉注射到大鼠体内。注射后10 min取脾，石蜡包埋含移植肝细胞的脾。在切片标本上进行原位RT，检测核酶对HCV RNA的特异性裂解。在注射HCV基因切割器的大鼠中未观察到HCV RNA的RT。另一方面，在未注射基因切割器的对照大鼠中观察到HCV RNA的RT。这些结果表明，我们的HCV基因切割器可以在动物模型中特异性地切割HCV RNA。研究了一种聚合物载体，以建立一种有效的系统，将核酶引入HCV复制的肝细胞细胞质中。初步结果表明，寡聚dna可以通过聚合物载体转运到细胞质中。

HCV gene cutter consisted of ribozyme RNA and its carrier (asialoorosomucoid-poly-L-lysin ; ASOR-PLL). When a complex of biotinylated poly-C (RNA) and ASOR-PLL was injected intravenously to rat, biotinylated poly-C was detected only in hepatocyte, indicating specific delivery of RNA to hepatocyte by the carrier.HCV RNA in cultured human hepatocyte was detected on sliced specimen by in-situ reverse transcription (in-situ RT). Further, nucleotide sequence specific cleavage of HCV RNA in hepatocyte by the ribozyme could be confirmed with the in-situ RT.We made an animal model of HCV infection by transplanting HCV infected cultured human hepatocytes to the spleen of a rat. Ability of the gene cutter was tested using this model. Briefly, HCV gene cutter was infected intravenously to the rat. The spleen was removed 10 min. after the injection, and then the spleen which included transplanted hepatocytes were embedded in paraffin. In-situ RT was performed on a sliced specimen to detect the specific cleavage of HCV RNA by the ribozyme. RT of HCV RNA was not observed in the rat which was injected with the HCV gene cutter. On the other hand, RT of HCV RNA was observed in a control rat which was not injected with the gene cutter. These results indicate that our HCV gene cutter can cleave HCV RNA specifically in the animal model.A polymer vector was studied to develop an effective system to introduce ribozyme into cytoplasm of hepatocyte where HCV replicates. Preliminary results indicated that oligo-DNA can be transported into the cytoplasm with the polymer vector.