RESUMEN
Small interfering RNA (siRNA) shows great promise in cancer therapy, but its effectiveness in vivo still remains a crucial issue for its transition into the clinics. Although the successful use of polyethylene glycol (PEG)ylated lipidic delivery systems have already been reported, most of the formulation procedures used are labour intensive and also result in unstable end products. We have previously developed a simple yet efficient hydration-of-freeze-dried-matrix (HFDM) method to entrap siRNA within lipid particles, in which the products exhibited superior stability. Here, we show that these HFDM-formulated particles are stable in the presence of serum and can deliver siRNA efficiently to tumours after intravenous administration. Using these particles, around 50% knockdown of the target gene expression was observed in tumours. With the use of siRNA targeting the E6/7 oncogenes expressed in cervical cancer, we showed a 50% reduction in tumour size. This level of tumour growth suppression was comparable to that achieved from cisplatin at the clinically used dose. Overall, our results demonstrate the feasibility of using HFDM-formulated particles to systematically administer E6/7-targeted siRNA for cervical cancer treatment. The simplicity of preparation procedure along with superior product stability obtained from our method offers an innovative approach for the in vivo delivery of siRNA.
Asunto(s)
Antineoplásicos/administración & dosificación , Cisplatino/administración & dosificación , Lípidos/química , Proteínas Oncogénicas Virales/antagonistas & inhibidores , Proteínas E7 de Papillomavirus/antagonistas & inhibidores , ARN Interferente Pequeño/administración & dosificación , Proteínas Represoras/antagonistas & inhibidores , Neoplasias del Cuello Uterino/terapia , Animales , Antineoplásicos/uso terapéutico , Cuello del Útero/efectos de los fármacos , Cuello del Útero/metabolismo , Cuello del Útero/patología , Cisplatino/uso terapéutico , Terapia Combinada , Femenino , Silenciador del Gen , Ratones , Ratones Endogámicos C57BL , Neoplasias , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus/genética , ARN Interferente Pequeño/metabolismo , Proteínas Represoras/genética , Transfección , Neoplasias del Cuello Uterino/tratamiento farmacológicoRESUMEN
Cutaneous human papillomavirus (HPV) types are commonly found in normal skin, and some of them have been suspected to play a role in the development of non-melanoma skin cancer. This present study is divided into three sections, the aims of this study were to examine if certain HPV-types persist over time and if HPV-types are shared within families. From the first part of the study, swab samples from foreheads were collected for three longitudinal studies from one family with a newborn baby. Five specific HPV-types were isolated from the family with a newborn, with HPV-5 and FA67 being found at various time points and prevalence rates in all four members of the family. Part 2 consisted of a followed up study from two families with a 6 years interval. Six of the family members were found to have at least one of the HPV-types identified in the family 6 years earlier. Many of the HPV-types identified were shared within the families studied. Part 3 of this study involved weekly samples from four healthy females for 4 months. Among the four healthy individuals, 11%, 65%, and 56% of the weekly samples were HPV-DNA positive with one individual HPV-negative. All specimens were tested for HPV-DNA by PCR using the broad range HPV-type primer pair FAP59/64. The positive samples were HPV-type determined by cloning and sequencing. Specific cutaneous HPV-types persist over long periods of time in healthy skin in most individuals investigated and certain HPVs are shared between family members.
Asunto(s)
Papillomaviridae/clasificación , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/virología , Enfermedades Cutáneas Virales/virología , Piel/virología , Adulto , Niño , Preescolar , Salud de la Familia , Femenino , Experimentación Humana , Humanos , Recién Nacido , Masculino , Datos de Secuencia Molecular , Papillomaviridae/genética , Adulto JovenRESUMEN
In this study, we investigated the suppressive effect of a short hairpin RNA delivered by a lentiviral vector (LV-shRNA) against human papillomavirus (HPV) type 18 E6 on the expression of the oncogenes E6 and E7 in cervical cancer HeLa cells both in vitro and in vivo. The LV-shRNA effectively delivered the shRNA to HeLa cells and lead to a dose-dependent reduction of E7 protein and the stabilization of E6 target proteins, p53 and p21. Low-dose infection of HeLa cells with LV-shRNA caused reduced cell growth and the induction of senescence, whereas a high-dose infection resulted in specific cell death via apoptosis. Transplant of HeLa cells infected with a low dose of LV-shRNA into Rag-/- mice significantly reduced the tumor weight, whereas transplant of cells infected with a high dose resulted in a complete loss of tumor growth. Systemic delivery of LV-shRNA into mice with established HeLa cell lung metastases led to a significant reduction in the number of tumor nodules. Our data collectively suggest that lentiviral delivery is an effective way to achieve stable suppression of E6/E7 oncogene expression and induce inhibition of tumor growth both in vitro and in vivo. These results encourage further investigation of this form of RNA interference as a promising treatment for cervical cancer.
Asunto(s)
Proteínas Oncogénicas Virales/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas Represoras/genética , Neoplasias del Cuello Uterino/terapia , Animales , Apoptosis/genética , Northern Blotting , Western Blotting , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Supervivencia Celular/genética , Femenino , Expresión Génica/genética , Vectores Genéticos/genética , Células HeLa , Humanos , Lentivirus/genética , Ratones , Proteínas Oncogénicas Virales/metabolismo , Proteínas E7 de Papillomavirus , Reacción en Cadena de la Polimerasa/métodos , Proteínas Represoras/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
RNA interference (RNAi)-based gene silencing is widely used in laboratories for gene function studies and also holds a great promise for developing treatments for diseases. However, in vivo delivery of RNAi therapy remains a key issue. Lentiviral vectors have been employed for stable gene transfer and gene therapy and therefore are expected to deliver a stable and durable RNAi therapy. But this does not seem to be true in some disease models. Here, we showed that lentivirus delivered short-hairpin RNA (shRNA) against human papillomavirus (HPV) E6/E7 oncogenes were effective for only 2 weeks in a cervical cancer model. However, using this vector to carry two copies of the same shRNA or two shRNAs targeting at two different but closely related genes (HPV E6 and vascular endothelial growth factor) was more effective at silencing the gene targets and inhibiting cell or even tumor growth than their single shRNA counterparts. The cancer cells treated with dual shRNA were also more sensitive to chemotherapeutic drugs than single shRNA-treated cells. These results suggest that a multi-shRNA strategy may be a more attractive approach for developing an RNAi therapy for this cancer.