Since the initial discovery of a functional RNA interference (RNAi) system in mammals, significant effort has been devoted to the development of therapeutics that utilize this pathway (de Fougerolles et al., 2007
). While progress has been made towards the design and delivery of short interfering (si) and short hairpin (sh) RNAs for therapeutic gene silencing, accumulating evidence indicates that modulation of miRNA activity also represents an attractive strategy. miRNAs potently influence cellular behavior through the regulation of extensive gene expression networks (Baek et al., 2008
; Selbach et al., 2008
). Therapeutic modulation of a single miRNA may therefore affect many pathways simultaneously to achieve clinical benefit. Thus far, most translational in vivo
studies targeting miRNAs have aimed to inhibit miRNA function through the use of antisense reagents such as antagomirs, locked nucleic acid (LNA) oligomers, and other modified oligonucleotides (Elmen et al., 2008
; Esau et al., 2006
; Krutzfeldt et al., 2005
). While the in vivo
use of synthetic oligonucleotide inhibitors is promising and will no doubt remain a fruitful area of investigation, the therapeutic delivery of miRNAs has certain advantages, especially in cancer. It has been demonstrated that most tumors are characterized by globally diminished miRNA expression (Gaur et al., 2007
; Lu et al., 2005
) and that experimental impairment of miRNA processing enhances cellular transformation and tumorigenesis (Kumar et al., 2007
). Additionally, common oncogenic lesions can result in widespread miRNA repression (Chang et al., 2008
). Thus, miRNA delivery might allow the therapeutic restitution of physiological programs of regulation lost in cancer and other disease states.
Therapeutic miRNA delivery may have unique technical advantages as well. First, the risk of off-target gene silencing is likely to be lower than that associated with artificial RNAi triggers since physiologic gene expression networks have evolved to accommodate the regulatory effects of endogenous miRNAs. Second, as compared to siRNAs or shRNAs that target a single transcript, the regulation of hundreds of targets in multiple pathways by miRNAs may reduce the emergence of resistant clones in diseases such as cancer since many simultaneous mutations would be required to subvert the effects of miRNA expression. At the same time, however, miRNA-based therapies will require thorough pre-clinical validation as these broad effects may in some cases have toxic consequences. Finally, it has been previously shown that the miRNA biogenesis pathway can be competitively inhibited by the expression of certain shRNAs, resulting in toxic effects following delivery of these transcripts (Grimm et al., 2006
). This may be due to inefficient processing and/or nuclear-cytoplasmic transport of shRNA sequences which are not evolutionarily adapted for precise handling by this pathway. Supporting this notion, shRNA-associated toxicity can be mitigated by placing a shRNA into a miRNA-like context (McBride et al., 2008
). In this study we demonstrated high expression of an exogenously supplied natural miRNA without toxic effects on endogenous miRNA biogenesis.
The results described herein demonstrate for the first time that therapeutic delivery of a miRNA can result in tumor suppression even in a setting where the initiating oncogene is not targeted. This establishes the principle that miRNAs may be useful as anti-cancer agents through their ability to broadly regulate cancer cell proliferation and survival. Furthermore, this study design involved the treatment of existing tumors with a miRNA, a paradigm closely related to the clinical scenarios in which such therapies would be employed. Finally, we demonstrate highly specific effects of miRNA delivery on tumor cells without affecting surrounding normal tissue. Although the molecular basis of this specificity requires further investigation, it is likely that the high physiologic expression of miR-26a in normal hepatocytes confers tolerance to exogenous administration of this miRNA. In contrast, the specific reduction of miR-26a in neoplastic cells and their sensitivity to its restored expression underscores the contribution of loss-of-function of this miRNA to tumorigenesis in this setting. It is noteworthy that large scale cloning efforts have documented expression of miR-26a in most mouse and human tissues (Landgraf et al., 2007
), while in situ hybridization data from zebrafish has documented ubiquitous expression with especially high levels in the head, spinal cord, and gut (Wienholds et al., 2005
). The widespread expression of this miRNA is consistent with our observation that systemic AAV-mediated delivery of miR-26a is well-tolerated by many tissues. Overall, our demonstration that miR-26a delivery potently suppresses even a severe, multifocal model of carcinogenesis in the absence of measurable toxicity provides proof-of-principle that the systemic administration of miRNAs may be a clinically viable anti-cancer therapeutic strategy.
In this study, we elected to use an AAV-based vector system, an especially attractive platform for regulatory RNA delivery (Giering et al., 2008
; Grimm and Kay, 2007
; McCarty, 2008
). When delivered in viral vectors, miRNAs are continually transcribed, allowing sustained high level expression in target tissues. The use of tissue-specific promoters could restrict this expression to particular cell types of interest. Compared to retroviral delivery systems, DNA viruses such as AAV carry substantially diminished risk of insertional mutagenesis since viral genomes persist primarily as episomes (Schnepp et al., 2003
). Further, the availability of multiple AAV serotypes allows efficient targeting of many tissues of interest (Gao et al., 2002
; McCarty, 2008
). Finally, the general safety of AAV has been well documented, with clinical trials using this platform already underway (Carter, 2005
; Maguire et al., 2008
; Park et al., 2008
). Despite these advantages, prior studies have achieved mixed results when attempting to use AAV vectors to transduce HCC cells in vivo
(Peng et al., 2000
; Shen et al., 2008
). Our successful use of AAV to treat an animal model of HCC may indicate that tumor cells are highly sensitive to restored expression of miRNAs, resulting in strong tumor suppression even with a relatively low number of vector genomes introduced into each cell. The use of self-complementary vectors may have further enhanced tumor cell transduction and therapeutic miRNA expression in the present study. Finally, while our findings are most consistent with miR-26a influencing cancer cell proliferation and survival through a cell-autonomous mechanism, transduction of tumor-associated stromal cells may have also contributed to the observed therapeutic effects through a yet unknown mechanism.
We have previously documented that miR-26 family members suppress tumorigenesis in c-Myc-driven B lymphoma cells (Chang et al., 2008
) and now extend these findings to the tet-o-MYC
; LAP-tTA HCC model. Our demonstration that a genetically complex human liver cancer cell line is also susceptible to miR-26a-mediated anti-proliferative effects suggests that the tumor suppressive activities of this miRNA are not limited to Myc-initiated malignancies. Several additional lines of evidence further support this notion. Work from our group and others has revealed a role for miR-26a in the p53 tumor suppressor network as this miRNA is upregulated in a p53-dependent manner following DNA damage (Chang et al., 2007
; Xi et al., 2006
). Additionally, a profiling study of human anaplastic thyroid cancers (ATC) identified miR-26a as consistently downregulated and demonstrated that transient transfection of this miRNA significantly impairs proliferation of ATC cells in vitro
(Visone et al., 2007
). Moreover, the miR-26a-1-encoding locus at 3p21.3 is contained within a sub-megabase interval that is frequently deleted in small cell lung carcinomas, renal cell carcinomas, and breast carcinomas (Kashuba et al., 2004
). These observations suggest broad anti-tumorigenic properties of miR-26 family members in diverse settings. Nevertheless, if future work reveals that the effectiveness of miR-26 delivery is restricted to settings of Myc dysregulation, therapeutic delivery of this miRNA may still be beneficial for a large number of cancer subtypes since hyperactivity of Myc is one of the most common attributes of human cancer cells.
Although miR-26a delivery confers dramatic tumor protection, it is likely that many equally or more effective miRNAs with therapeutic potential remain to be functionally characterized. The approach employed in this study provides an experimental framework to identify additional favorable candidates. We suggest that the most promising miRNAs will, like miR-26a, be highly expressed in a wide variety of normal tissues, be underexpressed in the disease state being studied, and, when evaluated using in vitro or in vivo models, demonstrate specific phenotypic effects in disease cells while sparing normal cells. While there clearly remains significant work to be done both in identifying such miRNAs and optimizing their controlled delivery, our findings highlight the therapeutic promise of this approach.