Ku80-Targeted pH-Sensitive Peptide-PNA Conjugates Are Tumor Selective and Sensitize Cancer Cells to Ionizing Radiation.

The development of therapeutic agents that specifically target cancer cells while sparing healthy tissue could be used to enhance the efficacy of cancer therapy without increasing its toxicity. Specific targeting of cancer cells can be achieved through the use of pH-low insertion peptides (pHLIP), which take advantage of the acidity of the tumor microenvironment to deliver cargoes selectively to tumor cells. We developed a pHLIP-peptide nucleic acid (PNA) conjugate as an antisense reagent to reduce expression of the otherwise undruggable DNA double-strand break repair factor, KU80, and thereby radiosensitize tumor cells. Increased antisense activity of the pHLIP-PNA conjugate was achieved by partial mini-PEG sidechain substitution of the PNA at the gamma position, designated pHLIP-αKu80(γ). We evaluated selective effects of pHLIP-αKu80(γ) in cancer cells in acidic culture conditions as well as in two subcutaneous mouse tumor models. Fluorescently labeled pHLIP-αKu80(γ) delivers specifically to acidic cancer cells and accumulates preferentially in tumors when injected i.v. in mice. Furthermore, pHLIP-αKu80(γ) selectively reduced KU80 expression in cells under acidic conditions and in tumors in vivo. When pHLIP-αKu80(γ) was administered to mice prior to local tumor irradiation, tumor growth was substantially reduced compared with radiation treatment alone. Furthermore, there was no evidence of acute toxicity associated with pHLIP-αKu80(γ) administration to the mice. These results establish pHLIP-αKu80(γ) as a tumor-selective radiosensitizing agent. IMPLICATIONS: This study describes a novel agent, pHLIP-αKu80(γ), which combines PNA antisense and pHLIP technologies to selectively reduce the expression of the DNA repair factor KU80 in tumors and confer tumor-selective radiosensitization.

Poly(Lactic-co-Glycolic Acid) Nanoparticle Delivery of Peptide Nucleic Acids In Vivo.

Many important biological applications of peptide nucleic acids (PNAs) target nucleic acid binding in eukaryotic cells, which requires PNA translocation across at least one membrane barrier. The delivery challenge is further exacerbated for applications in whole organisms, where clearance mechanisms rapidly deplete and/or deactivate exogenous agents. We have demonstrated that nanoparticles (NPs) composed of biodegradable polymers can encapsulate and release PNAs (alone or with co-reagents) in amounts sufficient to mediate desired effects in vitro and in vivo without deleterious reactions in the recipient cell or organism. For example, poly(lactic-co-glycolic acid) (PLGA) NPs can encapsulate and deliver PNAs and accompanying reagents to mediate gene editing outcomes in cells and animals, or PNAs alone to target oncogenic drivers in cells and correct cancer phenotypes in animal models. In this chapter, we provide a primer on PNA-induced gene editing and microRNA targeting-the two PNA-based biotechnological applications where NPs have enhanced and/or enabled in vivo demonstrations-as well as an introduction to the PLGA material and detailed protocols for formulation and robust characterization of PNA/DNA-laden PLGA NPs.

Peptide Nucleic Acids and Gene Editing: Perspectives on Structure and Repair.

Unusual nucleic acid structures are salient triggers of endogenous repair and can occur in sequence-specific contexts. Peptide nucleic acids (PNAs) rely on these principles to achieve non-enzymatic gene editing. By forming high-affinity heterotriplex structures within the genome, PNAs have been used to correct multiple human disease-relevant mutations with low off-target effects. Advances in molecular design, chemical modification, and delivery have enabled systemic in vivo application of PNAs resulting in detectable editing in preclinical mouse models. In a model of ß-thalassemia, treated animals demonstrated clinically relevant protein restoration and disease phenotype amelioration, suggesting a potential for curative therapeutic application of PNAs to monogenic disorders. This review discusses the rationale and advances of PNA technologies and their application to gene editing with an emphasis on structural biochemistry and repair.

Suppressing miR-21 activity in tumor-associated macrophages promotes an antitumor immune response.

microRNA-21 (miR-21) is the most commonly upregulated miRNA in solid tumors. This cancer-associated microRNA (oncomiR) regulates various downstream effectors associated with tumor pathogenesis during all stages of carcinogenesis. In this study, we analyzed the function of miR-21 in noncancer cells of the tumor microenvironment to further evaluate its contribution to tumor progression. We report that the expression of miR-21 in cells of the tumor immune infiltrate, and in particular in macrophages, was responsible for promoting tumor growth. Absence of miR-21 expression in tumor- associated macrophages (TAMs), caused a global rewiring of their transcriptional regulatory network that was skewed toward a proinflammatory angiostatic phenotype. This promoted an antitumoral immune response characterized by a macrophage-mediated improvement of cytotoxic T-cell responses through the induction of cytokines and chemokines, including IL-12 and C-X-C motif chemokine 10. These effects translated to a reduction in tumor neovascularization and an induction of tumor cell death that led to decreased tumor growth. Additionally, using the carrier peptide pH (low) insertion peptide, we were able to target miR-21 in TAMs, which decreased tumor growth even under conditions where miR-21 expression was deficient in cancer cells. Consequently, miR-21 inhibition in TAMs induced an angiostatic and immunostimulatory activation with potential therapeutic implications.