PHLIP® Targeting and Delivery of PNA to Silence MicroRNA in Tumor Cells§

Abstract

MicroRNAs (miRNAs) are short non-coding RNAs that, when expressed in different tissue and cell types, suppress the expression of complementary genes. Certain miRNAs, called oncomiRs, play a causal role in the onset and maintenance of cancer when overexpressed. Tumors that depend on these miRNAs are said to display oncomiR addiction. Inhibition of oncomiRs using antisense oligomers (i.e. antimiRs) is an evolving therapeutic strategy. The efficacy of current antimiR technologies in vivo is, however, hindered by barriers to targeting and delivery into tumor cells.pHLIPs are peptides that insert across cell membranes in acidic environments, delivering cargoes into the cytoplasm, and releasing them by disulfide cleavage. We have used a pHLIP as an antimiR delivery platform that targets the acidic tumor microenvironment, evades systemic clearance by the liver, and facilitates cell entry via a nonendocytic pathway. We found that the disulfide attachment of peptide nucleic acid (PNA) antimiRs to the inserting end of a pHLIP peptide produced a construct that could transport these antimiRs across plasma membranes under acidic conditions such as those found in solid tumors (pH ∼6). This conjugate effectively inhibited the miR-155 oncomiR in vitro and in vivo by targeting lymphoid tumors in mouse models. In a large B-cell lymphoma mouse model, silencing miR-155 reduced tumor burden, prevented lymphocyte metastases, and derepressed targets of miR-155 identified using RNA-seq analysis, with no apparent toxicity to the mice. This study introduces a new paradigm in the targeted delivery of polar molecules, such as antimiR PNAs, as anticancer drugs, which can have broad applicability in the field of targeted drug delivery.§Nature, 2014, accepted. Supported by NIH grants CA133890, CA131301, GM073857, ES005775, CA148996, HL007974, and the Yale Comprehensive Cancer Center.