Abstract
Peptide nucleic acids have emerged over the past two decades as a promising class of nucleic acid mimics because of their strong binding affinity and sequence selectivity toward DNA and RNA, and resistance to enzymatic degradation by proteases and nucleases. While they have been shown to be effective in regulation of gene expression in vitro, and to a small extent in vivo, their full potential for molecular therapy has not yet been fully realized due to poor cellular uptake. Herein, we report the development of cell-permeable, guanidine-based peptide nucleic acids targeting the epidermal growth factor receptor (EGFR) in preclinical models as therapeutic modality for head and neck squamous cell carcinoma (HNSCC) and nonsmall cell lung cancer (NSCLC). A GPNA oligomer, 16 nucleotides in length, designed to bind to EGFR gene transcript elicited potent antisense effects in HNSCC and NSCLC cells in preclinical models. When administered intraperitoneally in mice, EGFRAS-GPNA was taken-up by several tissues including the xenograft tumor. Systemic administration of EGFRAS-GPNA induced antitumor effects in HNSCC xenografts, with similar efficacies as the FDA-approved EGFR inhibitors: cetuximab and erlotinib. In addition to targeting wild-type EGFR, EGFRAS-GPNA is effective against the constitutively active EGFR vIII mutant implicated in cetuximab resistance. Our data reveals that GPNA is just as effective as a molecular platform for treating cetuximab resistant cells, demonstrating its utility in the treatment of cancer.
Highlights
Peptide nucleic acids have emerged over the past two decades as a promising class of nucleic acid mimics because of their strong binding affinity and sequence selectivity toward DNA and RNA, and resistance to enzymatic degradation by proteases and nucleases
To assess cellular uptake in live cells, an N-terminal FITC-tagged EGFRAS-guanidine-based peptide nucleic acids (GPNAs) oligomer was incubated with nonsmall cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC) cells in 10% serum-containing medium and imaged with confocal fluorescent microscopy without fixing (Figures 1C and S4, Supporting Information)
We carried out a line-scan analysis of Het 1A cells stained with the Endoplasmic reticulum (ER) marker and transfected with FITC-tagged EGFRAS-GPNA and found that the two fluorescent signals localized to the same regions within the cells (Supplementary Figure S6)
Summary
Peptide nucleic acids have emerged over the past two decades as a promising class of nucleic acid mimics because of their strong binding affinity and sequence selectivity toward DNA and RNA, and resistance to enzymatic degradation by proteases and nucleases. Attempts to improve enzymatic stability have led to the development of several classes of synthetic oligonucleotides;[5] one such promising class is peptide nucleic acids (PNAs).[6] PNAs are DNA and RNA analogues in which the sugar phosphodiester backbone is replaced by N-(2-aminoethyl) glycine units to which the nucleobases are attached through a flexible carboxymethylene linker.[7] The charge-neutral backbone enables PNAs to form highly stable duplexes with complementary DNA and RNA strands,[8] while the unnatural polyamide linkage renders them impervious to recognition and degradation by proteases and nucleases.[9] Together, these properties make PNAs attractive as antisense reagents for molecular therapy Though their ability to regulate gene expression has been demonstrated in cell culture,[10−15] and to a small extent in vivo,[16−19] their potential application as therapeutics have not yet been fully realized due to poor cellular uptake.[20] To overcome this limitation, we have prepared a chiral class of PNAs called guanidine-based peptide nucleic acids (GPNAs).[21] GPNA contains an arginine side-chain with an R-configuration at the αbackbone (Figure 1A). The results presented here have important implications for in vivo gene regulation and for the future treatment of head and neck cancer, as well as a number of other cancer types including lung and stomach, associated with overexpression of EGFR
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