Macejak D G, Jensen K L, Jamison S F, Domenico K, Roberts E C, Chaudhary N, von Carlowitz I, Bellon L, Tong M J, Conrad A, Pavco P A, Blatt L M
Ribozyme Pharmaceuticals Incorporated, Boulder, CO 80301, USA.
Hepatology. 2000 Mar;31(3):769-76. doi: 10.1002/hep.510310331.
Ribozymes are catalytic RNA molecules that can be designed to cleave specific RNA sequences. To investigate the potential use of synthetic stabilized ribozymes for the treatment of chronic hepatitis C virus (HCV) infection, we designed and synthesized hammerhead ribozymes targeting 15 conserved sites in the 5' untranslated region (UTR) of HCV RNA. This region forms an internal ribosome entry site that allows for efficient translation of the HCV polyprotein. The 15 synthetic ribozymes contained modified nucleotides and linkages that stabilize the molecules against nuclease degradation. All 15 ribozymes were tested for their ability to reduce expression in an HCV 5' UTR/luciferase reporter system and for their ability to inhibit replication of an HCV-poliovirus (HCV-PV) chimera. Treatment with several ribozymes resulted in significant down-regulation of HCV 5' UTR/luciferase reporter expression (range 40% to 80% inhibition, P <.05). Moreover, several ribozymes showed significant inhibition (>90%, P <.001) of chimeric HCV-PV replication. We further show that the inhibitory activity of ribozymes targeting site 195 of HCV RNA exhibits a sequence-specific dose response, requires an active catalytic ribozyme core, and is dependent on the presence of the HCV 5' UTR. Treatment with synthetic stabilized anti-HCV ribozymes has the potential to aid patients who are infected with HCV by reducing the viral burden through specific targeting and cleavage of the viral genome.
核酶是一类催化性RNA分子，可设计用于切割特定的RNA序列。为了研究合成稳定化核酶在治疗慢性丙型肝炎病毒（HCV）感染中的潜在用途，我们设计并合成了针对HCV RNA 5'非翻译区（UTR）中15个保守位点的锤头状核酶。该区域形成一个内部核糖体进入位点，可实现HCV多聚蛋白的高效翻译。这15种合成核酶含有修饰的核苷酸和连接键，可使分子稳定，抵抗核酸酶降解。对所有15种核酶进行了测试，检测它们在HCV 5'UTR/荧光素酶报告系统中降低表达的能力以及抑制HCV-脊髓灰质炎病毒（HCV-PV）嵌合体复制的能力。用几种核酶处理导致HCV 5'UTR/荧光素酶报告基因表达显著下调（抑制范围为40%至80%，P<.05）。此外，几种核酶对嵌合型HCV-PV复制表现出显著抑制作用（>90%，P<.001）。我们进一步表明，靶向HCV RNA第195位点的核酶的抑制活性呈现序列特异性剂量反应，需要有活性的催化核酶核心，并且依赖于HCV 5'UTR的存在。用合成稳定化抗HCV核酶进行治疗有可能通过特异性靶向和切割病毒基因组来减轻病毒载量，从而帮助HCV感染患者。
