Lisa Putral
Research interests
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InterestsRNAi, Liposomes, Antigen Presentation, microRNA
Publications
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5.21Impact points
Sensitive quantification of somatic mutations using molecular inversion probes.
Analytical chemistry. 09/2011; 83(21):8215-21.
Somatic mutations in DNA can serve as cancer specific biomarkers and are increasingly being used to direct treatment. However, they can be difficult to detect in tissue biopsies because there is often only a minimal amount of sample and the mutations are often masked by the presence of wild type all... [more] Somatic mutations in DNA can serve as cancer specific biomarkers and are increasingly being used to direct treatment. However, they can be difficult to detect in tissue biopsies because there is often only a minimal amount of sample and the mutations are often masked by the presence of wild type alleles from nontumor material in the sample. To facilitate the sensitive and specific analysis of DNA mutations in tissues, a multiplex assay capable of detecting nucleotide changes in less than 150 cells was developed. The assay extends the application of molecular inversion probes to enable sensitive discrimination and quantification of nucleotide mutations that are present in less than 0.1% of a cell population. The assay was characterized by detecting selected mutations in the KRAS gene, which has been implicated in up to 25% of all cancers. These mutations were detected in a single multiplex assay by incorporating the rapid flow cytometric readout of multiplexable DNA biosensors.
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4.75Impact points
Systemic delivery of E6/7 siRNA using novel lipidic particles and its application with cisplatin in cervical cancer mouse models.
Gene therapy. 01/2011; 18(1):14-22.
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 formul... [more] 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.
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3.28Impact points
Development of a Novel Method for Formulating Stable siRNA-Loaded Lipid Particles for In vivo Use.
Pharmaceutical research. 12/2008;
PURPOSE: A simple yet novel method was developed to prepare stable PEGylated siRNA-loaded lipid particles which are suitable for in vivo use. METHODS: PEGylated siRNA-loaded lipid particles were formulated by hydration of a freeze-dried matrix. The effect of various formulation parameters on the siz... [more] PURPOSE: A simple yet novel method was developed to prepare stable PEGylated siRNA-loaded lipid particles which are suitable for in vivo use. METHODS: PEGylated siRNA-loaded lipid particles were formulated by hydration of a freeze-dried matrix. The effect of various formulation parameters on the size and homogeneity of resulting particles was studied. Particles prepared using this method were compared to those prepared using an established post-insertion procedure for the entrapment efficiency, stability, in vitro biological activity as well as in vivo biodistribution. RESULTS: Using this hydration method, a particle size of less than 200 nm can be obtained with high siRNA entrapment efficiency (>90%) and high gene-silencing efficiency. Following intravenous administration into mice, these particles achieved a similar degree of accumulation in subcutaneous tumours but displayed less liver uptake compared to the post-insertion formulations. Importantly, in contrast to post-insertion preparations, particles made by hydration method retained 100% of their gene-silencing efficiency after storage at room temperature for 1 month. CONCLUSIONS: This paper describes a simple method of formulating PEGylated siRNA-loaded lipid particles. Given the ease of preparation, long term stability and favourable characteristics for in vivo delivery, our work represents an advance in lipid formulation of siRNA for in vivo use.
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siRNA and shRNA as anticancer agents in a cervical cancer model.
Methods in molecular biology (Clifton, N.J.). 02/2008; 442:159-72.
We describe the protocols of using siRNAs, or shRNAs delivered by a lentiviral vector, as a means to silence cancer-causing genes. We use cervical cancer as a model to demonstrate the inhibition of the human papillomavirus (HPV) oncogenes E6 and E7 in cervical cancer cells by RNAi and inhibition of ... [more] We describe the protocols of using siRNAs, or shRNAs delivered by a lentiviral vector, as a means to silence cancer-causing genes. We use cervical cancer as a model to demonstrate the inhibition of the human papillomavirus (HPV) oncogenes E6 and E7 in cervical cancer cells by RNAi and inhibition of the cell growth in vitro and tumor growth in mouse models. The protocols include methods on siRNA and shRNA design, production of lentiviral-vectored shRNA, transfection or transduction of cervical cancer cells with siRNA or shRNA, and detection of the inhibitory effects of siRNA or shRNA both in vitro and in vitro.
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3.45Impact points
The development and future of oligonucleotide-based therapies for cervical cancer.
Current opinion in molecular therapeutics. 05/2007; 9(2):126-31.
Cervical cancer is an attractive model in which to test gene-specific therapies, because elimination of the HPV oncogenes E6 and E7 may result in cancer cell senescence. Oligonucleotide-based therapies tested over the years include antisense oligonucleotides, ribozymes and, more recently, small inte... [more] Cervical cancer is an attractive model in which to test gene-specific therapies, because elimination of the HPV oncogenes E6 and E7 may result in cancer cell senescence. Oligonucleotide-based therapies tested over the years include antisense oligonucleotides, ribozymes and, more recently, small interfering RNA (siRNA)-based treatments. The development and use of these technologies are reviewed. siRNA-based therapies have been touted as potential treatments for cancers, genetic disorders and viral infections and have a number of advantages over antisense and ribozyme technologies. As with the older technologies, in vitro testing of siRNAs against cervical cancer has shown promising results, however, the issues that held up the clinical development of ribozymes and antisense are currently also challenging the siRNA field; these are target selection, specificity and delivery. If these issues can be overcome, a range of new and potent therapies for cervical cancer could become available.
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3.13Impact points
Inhibition of cervical cancer cell growth in vitro and in vivo with lentiviral-vector delivered short hairpin RNA targeting human papillomavirus E6 and E7 oncogenes.
Cancer gene therapy. 12/2006; 13(11):1023-32.
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 delive... [more] 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.
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4.53Impact points
RNA interference against human papillomavirus oncogenes in cervical cancer cells results in increased sensitivity to cisplatin.
Molecular pharmacology. 12/2005; 68(5):1311-9.
Targeted inhibition of oncogenes in tumor cells is a rational approach toward the development of cancer therapies based on RNA interference (RNAi). Tumors caused by human papillomavirus (HPV) infection are an ideal model system for RNAi-based cancer therapies because the oncogenes that cause cervica... [more] Targeted inhibition of oncogenes in tumor cells is a rational approach toward the development of cancer therapies based on RNA interference (RNAi). Tumors caused by human papillomavirus (HPV) infection are an ideal model system for RNAi-based cancer therapies because the oncogenes that cause cervical cancer, E6 and E7, are expressed only in cancerous cells. We investigated whether targeting HPV E6 and E7 oncogenes yields cancer cells more sensitive to chemotherapy by cisplatin, the chemotherapeutic agent currently used for the treatment of advanced cervical cancer. We have designed siRNAs directed against the HPV E6 oncogene that simultaneously targets both E6 and E7, which results in an 80% reduction in E7 protein and reactivation of the p53 pathway. The loss of E6 and E7 resulted in a reduction in cellular viability concurrent with the induction of cellular senescence. Interference was specific in that no effect on HPV-negative cells was observed. We demonstrate that RNAi against E6 and E7 oncogenes enhances the chemotherapeutic effect of cisplatin in HeLa cells. The IC50 for HeLa cells treated with cisplatin was 9.4 microM, but after the addition of a lentivirus-delivered shRNA against E6, the IC50 was reduced almost 4-fold to 2.4 microM. We also observed a decrease in E7 expression with a concurrent increase in p53 protein levels upon cotreatment with shRNA and cisplatin over that seen with individual treatment alone. Our results provide strong evidence that loss of E6 and E7 results in increased sensitivity to cisplatin, probably because of increased p53 levels.
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2.10Impact points
RNA interference for the treatment of cancer.
Drug news & perspectives. 19(6):317-24.
RNA interference (RNAi) is the latest new technology in the field of genetic medicine in which specific genes can be turned off, or silenced, so as to affect a therapeutic outcome. It can be highly specific, works in the nanomolar range and is far more effective than the antisense approaches popular... [more] RNA interference (RNAi) is the latest new technology in the field of genetic medicine in which specific genes can be turned off, or silenced, so as to affect a therapeutic outcome. It can be highly specific, works in the nanomolar range and is far more effective than the antisense approaches popular 10-15 years ago. Here we review the field and explore the potential role of RNAi in cancer therapy, highlighting recent progress and examining the hurdles that must be overcome before this promising technology is ready for clinical use.
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RNA interference against human papillomavirus oncogenes in cervical cancer cells results in increased sensitivity to cisplatin
Targeted inhibition of oncogenes in tumor cells is a rational approach toward the development of cancer therapies based on RNA interference (RNAi). Tumors caused by human papillomavirus (HPV) infection are an ideal model system for RNAi-based cancer therapies because the oncogenes that cause cervica... [more] Targeted inhibition of oncogenes in tumor cells is a rational approach toward the development of cancer therapies based on RNA interference (RNAi). Tumors caused by human papillomavirus (HPV) infection are an ideal model system for RNAi-based cancer therapies because the oncogenes that cause cervical cancer, E6 and E7, are expressed only in cancerous cells. We investigated whether targeting HPV E6 and E7 oncogenes yields cancer cells more sensitive to chemotherapy by cisplatin, the chemotherapeutic agent currently used for the treatment of advanced cervical cancer. We have designed siRNAs directed against the HPV E6 oncogene that simultaneously targets both E6 and E7, which results in an 80% reduction in E7 protein and reactivation of the p53 pathway. The loss of E6 and E7 resulted in a reduction in cellular viability concurrent with the induction of cellular senescence. Interference was specific in that no effect on HPV-negative cells was observed. We demonstrate that RNAi against E6 and E7 oncogenes enhances the chemotherapeutic effect of cisplatin in HeLa cells. The IC50 for HeLa cells treated with cisplatin was 9.4 mu M, but after the addition of a lentivirus-delivered shRNA against E6, the IC50 was reduced almost 4-fold to 2.4 mu M. We also observed a decrease in E7 expression with a concurrent increase in p53 protein levels upon cotreatment with shRNA and cisplatin over that seen with individual treatment alone. Our results provide strong evidence that loss of E6 and E7 results in increased sensitivity to cisplatin, probably because of increased p53 levels.
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The development and future of oligonucleotide-based therapies for cervical cancer
Cervical cancer is an attractive model in which to test gene-specific therapies, because elimination of the HPV oncogenes E6 and E7 may result in cancer cell senescence. Oligonucleotide-based therapies tested over the years include antisense oligonucleotides, ribozymes and, more recently, small inte... [more] Cervical cancer is an attractive model in which to test gene-specific therapies, because elimination of the HPV oncogenes E6 and E7 may result in cancer cell senescence. Oligonucleotide-based therapies tested over the years include antisense oligonucleotides, ribozymes and, more recently, small interfering RNA ( siRNA)based treatments. The development and use of these technologies are reviewed. siRNA-based therapies have been touted as potential treatments for cancers, genetic disorders and viral infections and have a number of advantages over antisense and ribozyme technologies. As with the older technologies, in vitro testing of siRNAs against cervical cancer has shown promising results, however, the issues that held up the clinical development of ribozymes and antisense are currently also challenging the siRNA field; these are target selection, specificity and delivery. If these issues can be overcome, a range of new and potent therapies for cervical cancer could become available.
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TREATMENT OF CERVICAL CANCER USING RNA-INTERFERENCE AGAINST HUMAN PAPILLOMAVIRUS ONCOGENES
Human papillomavirus (HPV) is the infectious agent responsible for the development of cervical cancer, with type 16 responsible for 50% of all cases. Cervical cancer remains the number one cancer killer of women under 50 worldwide, resulting in more than 270,000 deaths each year. Tumours caused by h... [more] Human papillomavirus (HPV) is the infectious agent responsible for the development of cervical cancer, with type 16 responsible for 50% of all cases. Cervical cancer remains the number one cancer killer of women under 50 worldwide, resulting in more than 270,000 deaths each year. Tumours caused by human HPV infection are an ideal model system for RNA interference-based cancer therapies as the oncogenes which cause cervical cancer, E6 and E7, are only expressed in cervical cancer cells. Furthermore, E6 and E7 are absolutely required for the ongoing proliferation of cervical cancer cells, as it has been demonstrated that upon their suppression, cells undergo apoptosis or senescence. In this study, siRNAs were designed that were directed against the HPV E6 and E7 oncogenes that simultaneously targeted both E6 and E7 with a single siRNA. These siRNAs were shown to reduce E7 protein levels by 80%. Following siRNA treatment, it was observed that there was reactivation of the p53 tumour suppressor pathway. The loss of E6 and E7 in cervical cancer cell lines resulted in a reduction in cellular viability concurrent with the induction of cellular senescence. RNA interference was specific as no effect on HPV-negative cells was observed. It was also shown in this study that RNAi against E6 and E7 oncogenes enhances the chemotherapeutic effect of cisplatin in HeLa cells following a combined treatment, resulting in a four-fold increase in cytotoxicity for the combined treatment as compared to cisplatin alone. Upon co-treatment with cisplatin and shRNA expressed from a plasmid, a decrease in E7 expression was observed with a concurrent increase in p53 protein levels over that seen with either treatment alone. These results provide strong evidence that loss of E6 and E7 results in increased sensitivity to cisplatin, and that this effect may occur as a result of increased p53 protein levels. This research also investigated the potential for a liposome-based system to facilitate the delivery of siRNA to tumours in vivo. It was demonstrated that liposomes produced by a lyophilisation method entrap nucleic acids with high efficiency (greater than 60%), can fully protect nucleic acids from digestion with nucleases, and are taken up by more than 90% of cells in culture. It was also demonstrated that these liposomes are able to accumulate at tumour sites in vivo in immune-competent mice. Despite excellent cellular uptake, siRNAs entrapped within liposomes were not transfection-competent, suggesting an inability of siRNAs to dissociate from liposomes, localize in the cytoplasm and become available to the silencing machinery. This research lays the groundwork for further development of effective liposomes for the in vivo delivery of siRNA nucleotides.
Following (2)
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Aaron Irving
Monash University -
Lyana Khairuddin
Diamantina Institute, UQ
Topics (3)
