Anti-HPV RNA Interference Using Modified RNA's

描述(申请人提供)：人类乳头瘤病毒(HPV)是导致男性和女性肛门发育不良和癌症的主要原因。HPVE6和E7蛋白是乳头瘤病毒致病能力的基础。我们已经使用针对HPV-18E6和E7的短干扰双链RNA(RNA干扰)成功地导致了HeLa细胞的生长停滞和衰老。在SW756细胞(第二个HPV-18转化的细胞株)中也得到了类似的结果，但在C33A细胞(非HPV转化的宫颈癌细胞株)中却没有得到类似的结果。我们的结果表明，抗E6/E7 RNA干扰可以降低E6和E7的表达，这种降低E6和E7的表达既可以用于治疗HPV癌前感染，也可以用于治疗HPV相关的癌症。 RNA干扰治疗应用的一个障碍是双链磷酸二酯RNA进入细胞的低效。为了在不使用脂质体的情况下促进治疗性干扰RNA进入细胞，我们已经开始评估硼磷和硫代磷酸骨架RNA进入细胞和诱导RNA干扰的能力。我们的数据表明，在HeLa细胞中，硼磷酸盐和硫代磷酸骨架RNA都可以用来诱导对绿色荧光蛋白表达的RNA干扰。硼磷酸盐修饰的单链siRNA也是活性的。我们预测，硼磷酸盐和硫代磷酸核糖核酸将有效进入细胞，并将诱导RNA干扰E6和E7的表达，导致HeLa细胞在培养过程中生长停滞和衰老。 RNA干扰对许多疾病的治疗具有重要的早期前景，包括HPV感染和HPV相关恶性肿瘤。这项工作是评估基于硼磷酸盐和硫代磷酸盐的RNA干扰作为一种治疗方式的第一步。

DESCRIPTION (provided by applicant): Human papillomaviruses (HPVs) are the major cause of anogenital dysplasias and carcinomas in both men and women. The HPV E6 and E7 proteins underlie the ability of papillomaviruses to cause disease. We have used short interfering double strand RNAs directed against HPV-18 E6 and E7 (RNA interference) to successfully cause growth arrest and senescence in HeLa cells. Similar results have been obtained in SW756 cells (a second HPV-18-transformed cell line), but not in C33A cells (a non-HPV-transformed cervical carcinoma cell line). Our data indicate that anti-E6/E7 RNA interference can reduce E6 and E7 expression, and that the reduction of E6 and E7 expression could be used to treat both pre-malignant HPV infections and HPV-related cancers. A barrier to the therapeutic use of RNA interference is the inefficiency with which double strand phosphodiester RNAs enter cells. To promote the entry of therapeutic interfering RNAs into cells without the use of lipofection reagents, we have begun to evaluate boranophosphate- and phosphorothioate-backbone RNAs for their abilities to enter cells and to induce RNA interference. Our data indicate that both boranophosphate- and phosphorothioate-backbone RNAs can be used to induce RNA interference of green fluorescent protein expression in HeLa cells. Boranophosphate-modified single-stranded siRNAs are also active. We predict that boranophosphate and phosphorothioate RNAs will enter cells efficiently, and will induce RNA interference with E6 and E7 expression, resulting in growth arrest and senescence of HeLa cells in culture. RNA interference holds significant early promise for the treatment of many disorders, including HPV infections and HPV-related malignancies. This work is an initial step in the evaluation of boranophosphateand phosphorothioate-based RNA interference as a therapeutic modality.