Cheng CJ, Bahal R, Babar IA, et al.
MicroRNA silencing for cancer therapy targeted tot he tumour microenvironment.

MicroRNAs (miRs) are short, noncoding RNA molecules that regulate the expression of target genes. OncomiRs are miRs that are overexpressed in certain cancers and play a role in the onset and development of these tumors, which display oncomiR addiction. Inhibition of oncomiRs by antimiRs is therefore an attractive and evolving therapeutic strategy. Various approaches to silence the aberrantly expressed miRs have been used, including peptide nucleic acids (PNAs), which are nucleic acid analogues consisting of nucleobases connected by intramolecular amide bonds. The PNA backbone stabilizes the antimiR by preventing nuclease degradation and increases the binding affinity for the complementary target sequence.

Notwithstanding recent improvements in RNA-based treatments, the effectiveness of current antimiR therapy is hindered by non-specific organ targeting, clearance in the reticuloendothelial system, and endolysosomal trafficking. Dr. Christopher Cheng and colleagues from Yale University have overcome these drawbacks by constructing a novel antimiR delivery system, which specifically targets tumors, facilitates entry into cancer cells via a nonendocytic pathway, and avoids clearance by the liver. The researchers exploited the acidic microenvironment of tumors and the unique properties of a peptide with a low pH-induced transmembrane structure (pHLIP), which forms an α-helix under acidic conditions and facilitates translocation of membrane-impermeable molecules, such as PNA-antimiRs, into cells.

To evaluate their new delivery platform, they used a mouse model of an inducible miR-155–addicted lymphoma whereby miR-155 expression is stimulated in hematologic tissues to promote tumorigenesis, which can be reversed by adding doxycycline. They first demonstrated that pHLIP, labeled with a fluorochrome, localized to inducible lymphoid tumors in nude mice, and also localized correctly in mice with disseminated lymphadenopathy. The liver was not affected, but some of the peptide accumulated in the kidneys where it was cleared by renal excretion.

These promising results prompted the researchers to create a pHLIP-antimiR-155 to target lymphoma. The construct consisted of the pHLIP peptide linked at the C-terminus to the PNA antimiR by a disulfide bond. The acidic pH (approximately 6) of the tumor microenvironment promoted insertion of the construct into the lipid bilayer of the plasma membrane and delivered the antimiR to the intracellular cytosol where the disulfide bond was cleaved to release the free antimiR, which could then silence the aberrant miR-155.

The in vivo therapeutic efficacy of the construct was assessed using their lymphoma mouse models. Intravenous administration of pHLIP-antimiR-155 resulted in a significant reduction in tumor growth and an increased survival advantage, which was similar to that of the positive control mice that had been treated with doxycycline or CHOP to suppress miR-155 expression. Additionally, metastasis of neoplastic lymphocytes to other organs, including the liver, spleen, and lymph nodes, was suppressed. The onset of splenomegaly and the development of lymphadenopathy were also significantly delayed. Importantly, the treated mice showed no clinical signs of distress, toxicity, or renal damage, and this was confirmed by studies on healthy mice using the highest dose of pHLIP-antimiR-155. These control mice exhibited no significant impairment of liver and kidney function and had normal white blood cell counts, as well as body and organ mass. PNA antimiRs are cleared by the reticuloendothelial system, which leads to accumulation in the liver, but mice treated with pHLIP-antimiR-155 showed levels approximately tenfold less in their livers compared with mice treated with antimiR-155 alone.

The mechanisms of tumor induction and subsequent addiction to miR-155 are not well characterized. Similarly, the pathways that facilitate tumor regression once miR-155 has been removed are still unknown. To investigate these aspects, the researchers performed RNA sequencing on miR-155–addicted lymphoid tumors and compared the data to regressing tumors. More than 2,000 genes were significantly up- or down-regulated when miR-155 was silenced, and the majority of these have been associated with cancer or cell adhesion and migration pathways. A notable example is the BACH1 gene, which codes for a transcription factor, and which was validated as a miR-155 target in clinical studies on mouse models.

In Brief

The novel pHLIP-antimiR delivery platform described in this study represents a significant advance in targeted RNA therapy. It has several advantages, including 1) specificity, since it targets the acidic microenvironment of the tumor and is ineffective in cells with a normal, neutral pH; 2) low systemic toxicity; 3) efficient delivery of the cargo antimiR, which facilitates the use of low doses of the therapeutic construct; and 4) versatility, since delivery is not dependent on the sequence of the antimiR and the system can thus be exploited to target multiple cancer types. Additionally, this approach has therapeutic potential for other pathological conditions in which a localized acidic environment is produced, such as ischemia, myocardial infarction, stroke, tissue trauma, and sites of inflammation and infection.

Competing Interests

Dr. Coetzer indicated no relevant conflicts of interest.