Gene silencing in primary and metastatic tumors by small interfering RNA delivery in mice: Quantitative analysis using melanoma cells expressing firefly and sea pansy luciferases

Abstract

Silencing of oncogenes or other genes contributing to tumor malignancy or progression by RNA interference (RNAi) offers a promising approach to treating tumor patients. To achieve RNAi-based tumor therapy, a small interfering RNA (siRNA) or siRNA-expressing vector needs to be delivered to tumor cells, but little information about its in vivo delivery has been reported. In this study, we examined whether the expression of the target gene in tumor cells can be suppressed by the delivery of RNAi effectors to primary and metastatic tumor cells. To quantitatively evaluate the RNAi effects in tumor cells, mouse melanoma B16-BL6 cells were stably transfected with both firefly (a model target gene) and sea pansy (an internal standard gene) luciferase genes to obtain B16-BL6/dual Luc cells. The target gene expression in subcutaneous primary tumors of B16-BL6/dual Luc cells was significantly suppressed by direct injection of the RNAi effectors followed by electroporation. The expression in metastatic hepatic tumors was also significantly reduced by an intravenous injection of either RNAi effector by the hydrodynamics-based procedure. These results indicate that the both RNAi effectors have a potential to silence target gene in tumor cells in vivo when successfully delivered to tumor cells.

Introduction

RNA interference (RNAi) is a post-transcriptional gene silencing event in which short double-stranded RNA (siRNA) degrades target mRNA in a sequence-specific manner [1], [2], [3]. After the discovery that the use of short double-stranded RNA can induce RNAi in mammalian cells without a sequence-nonspecific response [4], [5], RNAi has been widely used as an experimental tool to suppress specific gene expression for research involving gene function. This is because RNAi is attractive for its speed, usefulness, and lower cost, compared with conventional strategies to suppress gene function, such as gene knock-out by homologous recombination, and is generally more powerful than antisense strategies [6], [7]. Moreover, RNAi is expected to be used as a therapeutic tool in the treatment of various diseases, such as cancer, viral infections, and neurodegenerative disorders [8], [9].

Tumor cells have at least two major abnormalities: dysregulation of the cell cycle leading to uncontrolled growth, and resistance to death resulting from abnormalities in one or more genes that mediate apoptosis. Therefore, silencing these genes by RNAi offers a therapeutic treatment for cancer patients. In the last 5 years, there have been a number of reports in the literature describing how induction of RNAi suppresses these genes and decreases the malignancy of tumor cells in vitro. However, a few reports have described the successful treatment of tumors by the induction of RNAi in vivo [10], [11], [12], [13]. Poor delivery of RNAi effectors may be the major reason for the limited success in tumor therapy by RNAi in vivo in contrast to the many successes in vitro. One reason for this is that RNAi effectors are water soluble macromolecules and so they have difficulty in crossing cellular membranes and their gene silencing effect is limited in the cells that have received RNAi effectors [14], [15]. Regarding the delivery of RNAi effectors, the authors reporting successful in vivo tumor therapy administrated naked siRNA, siRNA or siRNA-expressing vector complexed with a cationic carrier. Duxbury et al. [13] and Verma et al. [10] did not evaluate the delivery of siRNA to tumor cells in detail but they examined the therapeutic effects produced by silencing the target genes. To inhibit tumor growth by siRNA targeting to vascular endothelial growth factor, Filleur et al. [11] investigated the effect of the routes of administration of siRNA on the gene silencing efficiency in subcutaneous tumors. Zhang et al. [12] administered siRNA-expressing plasmid DNA (pDNA) encapsulated in liposomes coated with antibody targeting the brain tumor and succeeded in suppressing gene expression in the tumor. However, the delivery of RNAi effectors to tumor cells and intratumoral gene silencing in vivo has received little attention in any of these previous studies.

RNAi can be induced by the delivery of siRNA or siRNA-expressing vector, which works as a platform to produce siRNA within the target cell for a relatively long period. Some groups have developed vector-based siRNA expression systems [16], [17], [18], [19], which showed effective RNAi induction. The molecular weight of siRNA-expressing vector is several hundred-fold greater than that of siRNA and must be delivered to the nucleus of target cells, unlike siRNA which can work if it is present in the cytoplasm [14]. Therefore, siRNA-expressing vector is more problematic than siRNA as far as delivery to the target cells is concerned. However, siRNA-expressing vector has the advantages of a sustained effect and ease in regulating its function compared with siRNA. Moreover, siRNA-expressing vectors that have an on-off switch for siRNA have also been reported [20]. Both RNAi effectors have advantages and disadvantages, and so should be used as the situation demands.

In either case of siRNA or siRNA-expressing vectors, the delivery of RNAi effectors to the tumor cells is the key factor for treating tumor patients with RNAi, because the gene silencing effect is limited in the cells that receive RNAi effectors. To improve RNAi-based tumor therapy, efficient RNAi effector delivery systems and delivery methods need to be developed. We considered that a model system in which the gene silencing effect can be sensitively evaluated may be a powerful tool for achieving successful in vivo RNAi-based tumor therapy. As used in previous studies involving the simultaneous administration of target gene and RNAi effectors [21], we think that clones of tumor cells stably expressing reporter genes, such as luciferase and green fluorescent protein, will be useful for examining whether siRNA or siRNA-expressing vector is effective in reducing target gene expression in tumor cells in vivo. Some research groups have introduced marker genes to detect tumor cells in vivo [22], [23]. We selected firefly (model target gene of RNAi) and sea pansy luciferases (indicator of tumor cell number) for detecting in vivo RNAi, because (i) these luciferases can be detected with a high degree of sensitivity and (ii) they can be simultaneously and quantitatively measured using a simple luminometric assay. Therefore, mouse melanoma B16-BL6 cells were stably transfected with firefly and sea pansy luciferases to obtain B16-BL6/dual Luc cells. This cell clone has advantages in evaluating in vivo intratumoral gene silencing effect in that we can investigate the effect easily, sensitively, and quantitatively. A subcutaneous primary tumor model and a hepatic metastasis model were selected as the targets of the delivery. There are no literature reports about gene silencing in metastatic tumors in the liver. As delivery methods, we used naked siRNA or pDNA without any viral or cationic carriers. We investigated whether the gene transfer methods reported to result in high transgene expression in the liver or muscle can deliver RNAi effectors into tumor cells inoculated in the liver or under the skin. Our data suggest one solution to the question of how the in vivo intratumoral delivery of siRNA can be achieved.