MicroRNAs (miRNAs) are emerging as potential cancer therapeutics, but effective delivery mechanisms to tumor sites are a roadblock to utility. Here we show that systemically delivered, synthetic miRNA mimics in complex with a novel neutral lipid emulsion are preferentially targeted to lung tumors and show therapeutic benefit in mouse models of lung cancer. Therapeutic delivery was demonstrated using mimics of the tumor suppressors, microRNA-34a (miR-34a) and let-7, both of which are often down regulated or lost in lung cancer. Systemic treatment of a Kras-activated autochthonous mouse model of non-small cell lung cancer (NSCLC) led to a significant decrease in tumor burden. Specifically, mice treated with miR-34a displayed a 60% reduction in tumor area compared to mice treated with a miRNA control. Similar results were obtained with the let-7 mimic. These findings provide direct evidence that synthetic miRNA mimics can be systemically delivered to the mammalian lung and support the promise of miRNAs as a future targeted therapy for lung cancer.

MicroRNAs have been increasingly implicated in human cancer and interest has grown about the potential to use microRNAs to combat cancer. Lung cancer is the most prevalent form of cancer worldwide and lacks effective therapies. Here we have used both in vitro and in vivo approaches to show that the let-7 microRNA directly represses cancer growth in the lung. We find that let-7 inhibits the growth of multiple human lung cancer cell lines in culture, as well as the growth of lung cancer cell xenografts in immunodeficient mice. Using an established orthotopic mouse lung cancer model, we show that intranasal let-7 administration reduces tumor formation in vivo in the lungs of animals expressing a G12D activating mutation for the K-ras oncogene. These findings provide direct evidence that let-7 acts as a tumor suppressor gene in the lung and indicate that this miRNA may be useful as a novel therapeutic agent in lung cancer.

Tumor cells use preexisting prosurvival signaling pathways to evade the damaging and cytotoxic effects of anticancer agents. Radiation therapy is a primary form of cytotoxic anticancer treatment, but agents that successfully modify the radiation response in vivo are lacking. MicroRNAs (miRNA) are global gene regulators that play critical roles in oncogenesis and have been found to regulate prosurvival pathways. However, there is little understanding of how cellular miRNA expression affects the response of a cancer to cytotoxic therapy and ultimately outcome. The let-7 family of miRNAs regulates expression of oncogenes, such as RAS, and is specifically down-regulated in many cancer subtypes. In fact, low levels of let-7 predict a poor outcome in lung cancer. Here, we report that the let-7 family of miRNAs is overrepresented in a class of miRNAs exhibiting altered expression in response to radiation. More strikingly, we also can create a radiosensitive state when the select let-7 family of miRNAs is overexpressed in vitro in lung cancer cells and in vivo in a Caenorhabditis elegans model of radiation-induced cell death, whereas decreasing their levels causes radioresistance. In C. elegans, we show that this is partly through control of the protooncogene homologue let-60/RAS and genes in the DNA damage response pathway. These findings are the first direct evidence that miRNAs can suppress resistance to anticancer cytotoxic therapy, a common feature of cancer cells, and suggest that miRNAs may be a viable tool to augment current cancer therapies. [Cancer Res 2007;67(23):11111-6]

MicroRNAs (miRNAs) have recently emerged as an important new class of cellular regulators that control various cellular processes and are implicated in human diseases, including cancer. Here, we show that loss of let-7 function enhances lung tumor formation in vivo, strongly supporting the hypothesis that let-7 is a tumor suppressor. Moreover, we report that exogenous delivery of let-7 to established tumors in mouse models of non-small cell lung cancer (NSCLC) significantly reduces tumor burden. These results demonstrate the therapeutic potential of let-7 in NSCLC and point to miRNA replacement therapy as a promising approach in cancer treatment.

A sequence-specific ribozyme (M1GS RNA) derived from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the overlapping exon 3 region of the mRNAs encoding the major transcription regulatory proteins IE1 and IE2 of human cytomegalovirus. A reduction of more than 80% in the expression levels of IE1 and IE2 and a reduction of about 150-fold in viral growth were observed in human cells that stably expressed the ribozyme. In contrast, a reduction of less than 10% in the IE1͞IE2 expression and viral growth was observed in cells that either did not express the ribozyme or produced a ''disabled'' ribozyme that carried mutations that abolished its catalytic activity. Examination of the expression of several other viral early and late genes in the cells that expressed the M1GS ribozyme further revealed an overall reduction of at least 80% in their expression. These results are consistent with the notion that the antiviral effects in these cells are due to the fact that the ribozyme specifically inhibits the expression of IE1 and IE2 and, consequently, abolishes the expression of viral early and late genes as well as viral growth. Our study is the first, to our knowledge, to use M1GS ribozyme for inhibiting human cytomegalovirus replication and demonstrates the utility of this ribozyme for antiviral applications. Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that causes mild or subclinical diseases in immunocompetent adults but may lead to severe morbidity or mortality in neonates and immunocompromised individuals (1). Infection by this virus accounts for one of the most common opportunistic diseases in patients with AIDS, CMV retinitis. The emergence of drug-resistant strains of HCMV has posed a need for the development of new drugs and novel treatment strategies (2, 3).Antisense nucleic acid molecules, including conventional antisense oligonucleotides and antisense ribozymes, are promising gene-targeting agents for specific inhibition of gene expression (4-8). Antisense molecules have been used as anti-HCMV agents to inhibit the expression of HCMV-essential genes and abolish viral replication (4, 9-12). External guide sequences (EGSs; refs. 13 and 14) are antisense oligoribonucleotides that have been used in conjunction with either ribonuclease P (RNase P) or the catalytic RNA subunit of RNase P from Escherichia coli (M1 RNA; ref. 15) to diminish the expression of several genes both in E. coli (16,17) and in mammalian cells (14,(18)(19)(20)(21). The EGS-based technology takes advantage of RNase P or M1 RNA to cleave a targeted mRNA when the EGS hybridizes to the target RNA (refs. 13 and 14; Fig. 1A). The EGSs, when expressed separately from the enzyme, have been shown recently to be effective in inhibiting the gene expression of herpes simplex virus 1 (HSV-1) and influenza virus and, in addition, in abolishing the replication of influenza virus (20,21). To increase the targeting efficiency, the EGS can be covalently linked to M1 RNA (i.e., its 3Ј end) to generate a sequence-specific...

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An in vitro selection procedure was used to select RNase P ribozyme variants that efficiently cleaved the sequence of the mRNA encoding thymidine kinase of herpes simplex virus 1. Of the 45 selected variants sequenced, 25 ribozymes carried a common mutation at nucleotides 224 and 225 of RNase P catalytic RNA from Escherichia coli (G 224 G 225 3 AA). These selected ribozymes exhibited at least 10 times higher cleavage efficiency (k cat /K m ) than that derived from the wild type ribozyme. Our results suggest that the mutated A 224 A 225 are in close proximity to the substrate and enhance substrate binding of the ribozyme. When these ribozyme variants were expressed in herpes simplex virus 1-infected cells, the levels of thymidine kinase mRNA and protein were reduced by 95-99%. Our study provides the first direct evidence that RNase P ribozyme variants isolated by the selection procedure can be used for the construction of gene-targeting ribozymes that are highly effective in tissue culture. These results demonstrate the potential for using RNase P ribozymes as gene-targeting agents against any mRNA sequences, and using the selection procedure as a general approach for the engineering of RNase P ribozymes.RNA enzymes are being developed as promising gene-targeting reagents to specifically cleave RNA sequences of choice (1-3). For example, both hammerhead and hairpin ribozymes have been shown to cleave viral mRNA sequences and inhibit viral replication in cells infected with human viruses, while a ribozyme derived from a group I intron has been used to repair mutant mRNAs in cells (4 -9). Thus, ribozymes can be used as a tool in both basic and clinical research, such as in studies of tumorigenesis and antiviral gene therapy.RNase P is a ribonucleoprotein complex responsible for the 5Ј maturation of tRNAs (10, 11). It catalyzes a hydrolysis reaction to remove a 5Ј leader sequence from tRNA precursors (ptRNA) 1 and several other small RNAs . In Escherichia coli, RNase P consists of a catalytic RNA subunit (M1 RNA) and a protein subunit (C5 protein) (10, 11). In the presence of a high concentration of salt, such as 100 mM Mg 2ϩ , M1 RNA acts as a catalyst and cleaves ptRNAs in vitro in the absence of C5 protein (12). Extensive studies with both phylogenetic and biochemical analyses have established models for the secondary and threedimensional structures of RNase P catalytic RNAs (13-16). These models provide a framework to identify the putative active site and substrate binding site, and to study the mechanism of RNase P catalytic RNAs.Studies on substrate recognition by RNase P have revealed that a small model substrate can be cleaved efficiently by M1 ribozyme (Fig. 1A). This model substrate contains a structure equivalent to the acceptor stem, the T-stem, the 3Ј CCA sequence, and the 5Ј leader sequence of a ptRNA molecule. Accordingly, M1 catalytic RNA can cleave a mRNA sequence if the mRNA substrate forms a hybrid complex with its complementary sequence (external guide sequence) (Fig. 1A) (17). Moreover, a sequen...

SummaryMicroRNAs (miRNAs) are a large class of ~ 22-nucleotide non-coding RNAs that facilitate mRNA cleavage and translation repression through the RNA interference pathway. Until recently, miRNAs have been exclusively found in eukaryotic organisms. A nonimmunogenic molecule requiring minimal genomic investment, these RNAs may offer an efficient means for viruses to modulate both their own and the host's gene expression during a productive viral infection. In this study we report that human cytomegalovirus (HCMV) expresses miRNAs during its productive lytic infection of four clinically relevant human cell types: fibroblast, endothelial, epithelial and astrocyte cells. The sequences of the miRNAs, expressed from the UL23 and US24 loci of the viral genome, were conserved among all HCMV strains examined and in chimpanzee cytomegalovirus. Furthermore, their expression was detected from both a laboratoryadapted strain and a clinical isolate of HCMV. The conservation of these miRNAs and their expression in different cell types suggests that they represent an evolutionarily primitive feature in the viral genome, and that virus-encoded miRNAs may be more common than previously believed.