A comparative analysis of peptide-delivered antisense antibiotics using diverse nucleotide mimics.

Antisense oligomer (ASO)-based antibiotics that target mRNAs of essential bacterial genes have great potential for counteracting antimicrobial resistance and for precision microbiome editing. To date, the development of such antisense antibiotics has primarily focused on using phosphorodiamidate morpholino (PMO) and peptide nucleic acid (PNA) backbones, largely ignoring the growing number of chemical modalities that have spurred the success of ASO-based human therapy. Here, we directly compare the activities of seven chemically distinct 10mer ASOs, all designed to target the essential gene acpP upon delivery with a KFF-peptide carrier into Salmonella. Our systematic analysis of PNA, PMO, phosphorothioate (PTO)-modified DNA, 2'-methylated RNA (RNA-OMe), 2'-methoxyethylated RNA (RNA-MOE), 2'-fluorinated RNA (RNA-F), and 2'-4'-locked RNA (LNA) is based on a variety of in vitro and in vivo methods to evaluate ASO uptake, target pairing and inhibition of bacterial growth. Our data show that only PNA and PMO are efficiently delivered by the KFF peptide into Salmonella to inhibit bacterial growth. Nevertheless, the strong target binding affinity and in vitro translational repression activity of LNA and RNA-MOE make them promising modalities for antisense antibiotics that will require the identification of an effective carrier.

Antitumor efficacy of a sequence-specific DNA-targeted γPNA-based c-Myc inhibitor.

Targeting oncogenes at the genomic DNA level can open new avenues for precision medicine. Significant efforts are ongoing to target oncogenes using RNA-targeted and protein-targeted platforms, but no progress has been made to target genomic DNA for cancer therapy. Here, we introduce a gamma peptide nucleic acid (γPNA)-based genomic DNA-targeted platform to silence oncogenes in vivo. γPNAs efficiently invade the mixed sequences of genomic DNA with high affinity and specificity. As a proof of concept, we establish that γPNA can inhibit c-Myc transcription in multiple cell lines. We evaluate the in vivo efficacy and safety of genomic DNA targeting in three pre-clinical models. We also establish that anti-transcription γPNA in combination with histone deacetylase inhibitors and chemotherapeutic drugs results in robust antitumor activity in cell-line- and patient-derived xenografts. Overall, this strategy offers a unique therapeutic platform to target genomic DNA to inhibit oncogenes for cancer therapy.

High-order framework nucleic acid for targeted-delivery of antisense peptide nucleic acids to overcome drug resistance.

A sophisticated high-order framework nucleic acid (FNA) was engineered for the targeted delivery and responsive release of environment tolerant antisense peptide nucleic acids (asPNAs). The dendritic FNA-asPNAs system was constructed via simple one-pot modular assembly and demonstrated a good synergistic effect with chemotherapy on drug resistant cancer cells.

Peptide Nucleic Acids: Recent Developments in the Synthesis and Backbone Modifications.

Nucleic acid represents the ideal drug candidate for protein targets that are hard to target or against which drug development is not easy. Peptide nucleic acids (PNAs) are synthesized by attaching modified peptide backbones generally derived from repetitive N-2-aminoethyl glycine units in place of the regular phosphodiester backbone and represent synthetic impersonator of nucleic acids that offers an exciting research field due to their fascinating spectrum of biotechnological, diagnostic and potential therapeutic applications. The semi-rigid peptide nucleic acid backbone serves as a nearly-perfect template for attaching complimentary base pairs on DNA or RNA in a sequence-dependent manner as described by Watson-Crick models. PNAs and their analogues are endowed with exceptionally high affinity and specificity for receptor sites, essentially due to their polyamide backbone's uncharged and flexible nature. The present review compiled various strategies to modify the polypeptide backbone for improving the target selectivity and stability of the PNAs in the body. The investigated biological activities carried out on PNAs have also been summarized in the present review.

Anti-seed PNAs targeting multiple oncomiRs for brain tumor therapy.

Glioblastoma (GBM) is one of the most lethal malignancies with poor survival and high recurrence rates. Here, we aimed to simultaneously target oncomiRs 10b and 21, reported to drive GBM progression and invasiveness. We designed short (8-mer) γ-modified peptide nucleic acids (sγPNAs), targeting the seed region of oncomiRs 10b and 21. We entrapped these anti-miR sγPNAs in nanoparticles (NPs) formed from a block copolymer of poly(lactic acid) and hyperbranched polyglycerol (PLA-HPG). The surface of the NPs was functionalized with aldehydes to produce bioadhesive NPs (BNPs) with superior transfection efficiency and tropism for tumor cells. When combined with temozolomide, sγPNA BNPs administered via convection-enhanced delivery (CED) markedly increased the survival (>120 days) of two orthotopic (intracranial) mouse models of GBM. Hence, we established that BNPs loaded with anti-seed sγPNAs targeting multiple oncomiRs are a promising approach to improve the treatment of GBM, with a potential to personalize treatment based on tumor-specific oncomiRs.

Towards a templated reaction that translates RNA in cells into a proaptotic peptide-PNA conjugate.

Nucleic acid-templated chemistry opens the intriguing prospect of triggering the synthesis of drugs only in diseased cells. Herein, we explore the feasibility of using RNA-templated chemical reactions for the activation of a known Smac peptidomimetic compound (SMC), which has proapoptotic activity. Two peptide nucleic acid (PNA) conjugates were used to enable conditional activation of a masked SMC by reduction of an azide either by Staudinger reduction or catalytic photoreduction using a ruthenium complex. The latter provided ~135 nM SMC-PNA on as little as 10 nM (0.01 eq.) template. For the evaluation of the templated azido-SMC reduction system in cellulo, a stable HEK 293 cell line was generated, which overexpressed a truncated, non-functional form of the XIAP mRNA target. We furthermore describe the development of electroporation protocols that enable a robust delivery of PNA conjugates into HEK 293 cells. The action of the reactive PNA conjugates was evaluated by viability and flow cytometric apoptosis assays. In addition, electroporated probes were re-isolated and analyzed by ultra-high performance liquid chromatography (UPLC). Unfortunately, the ruthenium-PNA conjugate proved phototoxic, and treatment of cells with PNA-linked reducing agent and the azido-masked SMC conjugate did not result in a greater viability loss than treatment with scrambled sequence controls. Intracellular product formation was not detectable. A control experiment in total cellular RNA isolate indicated that the templated reaction can in principle proceed in a complex system. The results of this first-of-its-kind study reveal the numerous hurdles that must be overcome if RNA molecules are to trigger the synthesis of pro-apoptotic drugs inside cells.

Simultaneous Targeting of Multiple oncomiRs with Phosphorothioate or PNA-Based Anti-miRs in Lymphoma Cell Lines.

PURPOSE: MicroRNAs (miRNAs) are short (~ 22 nts) RNAs that regulate gene expression via binding to mRNA. MiRNAs promoting cancer are known as oncomiRs. Targeting oncomiRs is an emerging area of cancer therapy. OncomiR-21 and oncomiR-155 are highly upregulated in lymphoma cells, which are dependent on these oncomiRs for survival. Targeting specific miRNAs and determining their effect on cancer cell progression and metastasis have been the focus of various studies. Inhibiting a single miRNA can have a limited effect, as there may be other overexpressed miRNAs present that may promote tumor proliferation. Herein, we target miR-21 and miR-155 simultaneously using nanoparticles delivered two different classes of antimiRs: phosphorothioates (PS) and peptide nucleic acids (PNAs) and compared their efficacy in lymphoma cell lines. METHODS: Poly-Lactic-co-Glycolic acid (PLGA) nanoparticles (NPs) containing PS and PNA-based antimiR-21 and -155 were formulated, and comprehensive NP characterizations: morphology (scanning electron microscopy), size (differential light scattering), and surface charge (zeta potential) were performed. Cellular uptake analysis was performed using a confocal microscope and flow cytometry analysis. The oncomiR knockdown and the effect on downstream targets were confirmed by gene expression (real time-polymerase chain reaction) assay. RESULTS: We demonstrated that simultaneous targeting with NP delivered PS and PNA-based antimiRs resulted in significant knockdown of miR-21 and miR-155, as well as their downstream target genes followed by reduced cell viability ex vivo. CONCLUSIONS: This project demonstrated that targeting miRNA-155 and miR-21 simultaneously using nanotechnology and a diverse class of antisense oligomers can be used as an effective approach for lymphoma therapy.

Combined Treatment of Bronchial Epithelial Calu-3 Cells with Peptide Nucleic Acids Targeting miR-145-5p and miR-101-3p: Synergistic Enhancement of the Expression of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene encodes for a chloride channel defective in Cystic Fibrosis (CF). Accordingly, upregulation of its expression might be relevant for the development of therapeutic protocols for CF. MicroRNAs are deeply involved in the CFTR regulation and their targeting with miRNA inhibitors (including those based on Peptide Nucleic Acids, PNAs)is associated with CFTR upregulation. Targeting of miR-145-5p, miR-101-3p, and miR-335-5p with antisense PNAs was found to be associated with CFTR upregulation. The main objective of this study was to verify whether combined treatments with the most active PNAs are associated with increased CFTR gene expression. The data obtained demonstrate that synergism of upregulation of CFTR production can be obtained by combined treatments of Calu-3 cells with antisense PNAs targeting CFTR-regulating microRNAs. In particular, highly effective combinations were found with PNAs targeting miR-145-5p and miR-101-3p. Content of mRNAs was analyzed by RT-qPCR, the CFTR production by Western blotting. Combined treatment with antagomiRNAs might lead to maximized upregulation of CFTR and should be considered in the development of protocols for CFTR activation in pathological conditions in which CFTR gene expression is lacking, such as Cystic Fibrosis.

MicroRNA21 inhibition affects porcine oocyte maturation and alters protein expression critical for metabolic pathway function.

MicroRNA21 (MIR21) abundance in porcine oocytes and cumulus cells increases during in vitro maturation. The mechanism by which MIR21 regulates oocyte maturation and the effect on the developmental competence of subsequent embryos remains unclear. The objective of this study was to assess the function of MIR21 during porcine oocyte maturation and its effect on embryonic development. Treatment with peptide nucleic acid MIR21 inhibitor (MIR21-PNA), designed to specifically bind to and prevent MIR21 activity during in vitro oocyte maturation, decreased cumulus cell expansion, and the oocyte ability to achieve metaphase II maturation stage when compared to control groups. Following parthenogenetic activation, the cleavage rate at 48 h in the MIR21-PNA group was decreased (p ≤ 0.03) relative to the control groups. Additionally, liquid chromatography-mass spectrometry (LC-MS/MS) of oocyte and cumulus cell total protein following MIR21-PNA treatment during in vitro maturation identified changes in signaling pathways with primary involvement of glucose metabolism (GM) pathways. Furthermore, there was no difference (p = 0.21) in oocyte maturation of control and MIR21-PNA treated oocytes when cultured in pyruvate lacking medium. Finally, MIR21-PNA treatment decreased (p = 0.04) glutathione and increased (p = 0.07) reactive oxygen species production in the oocyte. These data suggest that MIR21 influences porcine oocyte maturation by regulating GM pathways in the cumulus-oocyte complex.

Antisense Peptide Nucleic Acid-Diaminobutanoic Acid Dendron Conjugates with SbmA-Independent Antimicrobial Activity against Gram-Negative Bacteria.

Precision antisense antibacterial agents may be developed into novel antibiotics in the fight against multidrug-resistant Gram-negative bacteria. In this study, a series of diaminobutanoic acid (DAB) dendrons are presented as novel carriers for the delivery of antisense antibacterial peptide nucleic acids (PNAs). The dendron-PNA conjugates targeting the essential acpP gene exhibit specific antisense antimicrobial bactericidal activity against Escherichia coli and Klebsiella pneumoniae at one-digit micromolar concentrations, while showing low toxicity to human cells. One compound selected from a structure-activity relationship series showed high stability in mouse and human serum (t1/2 ≫ 24 h) as well as in vivo activity against a multidrug-resistant, extended spectrum beta-lactamase-producing E. coli in a murine peritonitis model. The compound was also well tolerated in mice upon i.v. administration up to a dose of 20 mg/kg, and in vivo fluorescence imaging indicated clearance via renal excretion with slight accumulation in the kidneys and liver. Thus, DAB-based dendrons constitute a promising new chemistry platform for development of effective delivery agents for antibacterial drugs with possible in vivo use.

Treatment of Human Glioblastoma U251 Cells with Sulforaphane and a Peptide Nucleic Acid (PNA) Targeting miR-15b-5p: Synergistic Effects on Induction of Apoptosis.

Glioblastoma multiforme (GBM) is a lethal malignant tumor accounting for 42% of the tumors of the central nervous system, the median survival being 15 months. At present, no curative treatment is available for GBM and new drugs and therapeutic protocols are urgently needed. In this context, combined therapy appears to be a very interesting approach. The isothiocyanate sulforaphane (SFN) has been previously shown to induce apoptosis and inhibit the growth and invasion of GBM cells. On the other hand, the microRNA miR-15b is involved in invasiveness and proliferation in GBM and its inhibition is associated with the induction of apoptosis. On the basis of these observations, the objective of the present study was to determine whether a combined treatment using SFN and a peptide nucleic acid interfering with miR-15b-5p (PNA-a15b) might be proposed for increasing the pro-apoptotic effects of the single agents. To verify this hypothesis, we have treated GMB U251 cells with SFN alone, PNA-a15b alone or their combination. The cell viability, apoptosis and combination index were, respectively, analyzed by calcein staining, annexin-V and caspase-3/7 assays, and RT-qPCR for genes involved in apoptosis. The efficacy of the PNA-a15b determined the miR-15b-5p content analyzed by RT-qPCR. The results obtained indicate that SFN and PNA-a15b synergistically act in inducing the apoptosis of U251 cells. Therefore, the PNA-a15b might be proposed in a "combo-therapy" associated with SFN. Overall, this study suggests the feasibility of using combined treatments based on PNAs targeting miRNA involved in GBM and nutraceuticals able to stimulate apoptosis.

Affibody-Mediated PNA-Based Pretargeted Cotreatment Improves Survival of Trastuzumab-Treated Mice Bearing HER2-Expressing Xenografts.

Treatment of patients with human epidermal growth factor receptor 2 (HER2)-expressing tumors using the monoclonal antibody trastuzumab increases survival. The Affibody-based peptide nucleic acid (PNA)-mediated pretargeted radionuclide therapy has demonstrated efficacy against HER2-expressing xenografts in mice. Structural studies suggest that Affibody molecules and trastuzumab bind to different epitopes on HER2. The aim of this study was to test the hypothesis that a combination of PNA-mediated pretargeted radionuclide therapy and trastuzumab treatment of HER2-expressing xenografts can extend survival compared with monotherapies. Methods: Mutual interference of the primary pretargeting probe ZHER2:342-SR-HP1 and trastuzumab in binding to HER2-expressing cell lines was investigated in vitro. Experimental therapy evaluated the survival of mice bearing HER2-expressing SKOV-3 xenografts after treatment with vehicle, trastuzumab only, pretargeting using Affibody-PNA chimera ZHER2:342-SR-HP1 and complementary probe 177Lu-HP2, and combination of trastuzumab and pretargeting. The ethical permit limited the study to 90 d. The animals' weights were monitored during the study. After study termination, samples of liver and kidneys were evaluated by a veterinary pathologist for toxicity signs. Results: The presence of a large molar excess of trastuzumab had no influence on the affinity of ZHER2:342-SR-HP1 binding to HER2-expressing cells in vitro. The affinity of trastuzumab was not affected by a large excess of ZHER2:342-SR-HP1 The median survival of mice treated with trastuzumab (75.5 d) was significantly longer than the survival of mice treated with a vehicle (59.5 d). Median survival of mice treated with pretargeting was not reached by day 90. Six mice of 10 in this group survived, and 2 had complete remission. All mice in the combination treatment group survived, and tumors in 7 mice had disappeared at study termination. There was no significant difference between animal weights in the different treatment groups. No significant pathologic alterations were detected in livers and kidneys of treated animals. Conclusion: Treatment of mice bearing HER2-expressing xenografts with the combination of trastuzumab and Affibody-mediated PNA-based radionuclide pretargeting significantly increased survival compared with monotherapies. Cotreatment was not toxic for normal tissues.

Dual-Modality Poly-l-histidine Nanoparticles to Deliver Peptide Nucleic Acids and Paclitaxel for In Vivo Cancer Therapy.

Cationic polymeric nanoformulations have been explored to increase the transfection efficiency of small molecules and nucleic acid-based drugs. However, an excessive positive charge density often leads to severe cell and tissue-based toxicity that restricts the clinical translation of cationic polymeric nanoformulations. Herein, we investigate a series of cationic poly(lactic-co-glycolic acid) (PLGA)-histidine-based nanoformulations for enhanced cytoplasmic delivery with minimal toxicity. PLGA/poly-l-histidine nanoparticles show promising physico-biochemical features and transfection efficiency in a series of in vitro and cell culture-based studies. Further, the use of acetone/dichloromethane as a solvent mixture during the formulation process significantly improves the morphology and size distribution of PLGA/poly-l-histidine nanoparticles. PLGA/poly-l-histidine nanoformulations undergo clathrin-mediated endocytosis. A contrast-matched small-angle neutron scattering experiment confirmed poly-l-histidine's distribution on the PLGA nanoformulations. PLGA/poly-l-histidine formulations containing paclitaxel as a small molecule-based drug and peptide nucleic acids targeting microRNA-155 as nucleic acid analog are efficacious in in vitro and in vivo studies. PLGA/poly-l-histidine NPs significantly decrease tumor growth in PNA-155 (∼6 fold) and paclitaxel (∼6.5 fold) treatment groups in a lymphoma cell line derived xenograft mice model without inducing any toxicity. Hence, PLGA/poly-l-histidine nanoformulations exhibit substantial transfection efficiency and are safe to deliver reagents ranging from small molecules to synthetic nucleic acid analogs and can serve as a novel platform for drug delivery.

Therapeutic Potential of Chemically Modified, Synthetic, Triplex Peptide Nucleic Acid-Based Oncomir Inhibitors for Cancer Therapy.

miRNA-155 (miR-155) is overexpressed in various types of lymphomas and leukemias, suggesting that targeting miR-155 could be a potential platform for the development of precision medicine. Here, we tested the anticancer activity of novel, chemically modified, triplex peptide nucleic acid (PNA)-based antimiRs compared with the current state-of-the-art conventional full-length antimiRs. Next-generation modified PNAs that bound miR-155 by Watson-Crick and Hoogsteen domains possessed superior therapeutic efficacy in vivo and ex vivo compared with conventional full-length anti-miR-155. The efficacy of anti-miR-155 targeting in multiple lymphoma cell lines was comprehensively corroborated by gene expression, Western blot analysis, and cell viability-based functional studies. Finally, preclinical testing in vivo in xenograft mouse models containing lymphoma cell lines demonstrated that treatment with the miR-155-targeting next-generation antimiR resulted in a significant decrease in miR-155 expression, followed by reduced tumor growth. These findings support the effective therapeutic application of chemically modified triplex PNAs to target miR-155 to treat lymphoma. Overall, the present proof-of-concept study further implicates the potential for next-generation triplex gamma PNAs to target other miRNAs for treating cancer. SIGNIFICANCE: This study demonstrates the utility of novel oncomiR inhibitors as cancer therapeutics, providing a new approach for targeting miRNAs and other noncoding RNAs.

Bifacial PNAs Destabilize MALAT1 by 3' A-Tail Displacement from the U-Rich Internal Loop.

We report herein a new class of synthetic reagents for targeting the element for nuclear expression (ENE) in MALAT1, a long noncoding RNA upregulated in many cancers. The cis-acting ENE contains a U-rich internal loop (URIL) that forms an 11 base UAU-rich triplex stem with the truncated 3' oligo-A tail of MALAT1, protecting the terminus from exonuclease digestion and greatly extending transcript lifetime. Bifacial peptide nucleic acids (bPNAs) similarly bind URILs via base triple formation between two uracil bases and a synthetic base, melamine. We synthesized a set of low molecular weight bPNAs composed of α-linked peptide, isodipeptide, and diketopiperazine backbones and evaluated their ENE binding efficacy in vitro via oligo-A strand displacement and consequent exonuclease sensitivity. Degradation was greatly enhanced by bPNA treatment in the presence of exonucleases, with ENE half-life plunging to 6 min from >24 h. RNA digestion kinetics could clearly distinguish between bPNAs with similar URIL affinities, highlighting the utility of functional assays for evaluating synthetic RNA binders. In vitro activity was mirrored by a 50% knockdown of MALAT1 expression in pancreatic cancer (PANC-1) cells upon treatment with bPNAs, consistent with intracellular digestion triggered by a similar ENE A-tail displacement mechanism. Pulldown from PANC-1 total RNA with biotinylated bPNA enriched MALAT1 > 4000× , supportive of bPNA-URIL selectivity. Together, these experiments establish the feasibility of native transcript targeting by bPNA in both in vitro and intracellular contexts. Reagents such as bPNAs may be useful tools for the investigation of transcripts stabilized by cis-acting poly(A) binding RNA elements.

Synergistic effects of the combined treatment of U251 and T98G glioma cells with an anti­tubulin tetrahydrothieno[2,3­c]pyridine derivative and a peptide nucleic acid targeting miR­221­3p.

In the development of novel and more effective anticancer approaches, combined treatments appear to be of great interest, based on the possibility of obtaining relevant biological or therapeutic effects using lower concentrations of single drugs. Combination therapy may prove to be of utmost significance in the management of glioblastoma (GBM), a lethal malignancy that accounts for 42% of cancer cases of the central nervous system, with a median survival rate of 15 months. As regards novel therapeutic approaches, the authors have recently demonstrated that peptide nucleic acids (PNAs) that target microRNA (miRNA/miR)­221 are very active in inducing the apoptosis of glioma cells. Furthermore, in a recent study, the authors described two novel series of tubulin polymerization inhibitors based on the 4,5,6,7­tetrahydrothieno[2,3­c]pyridine and 4,5,6,7­tetrahydrobenzo[b]thiophene scaffold, which exerted a potent anti­proliferative effect on a variety of tumor cell lines. The present study aimed to verify the activity on glioblastoma cancer cell lines of one of the most active compounds tested, corresponding to 2­(3', 4', 5'­trimethoxyanilino)­3­cyano/alkoxycarbonyl­6­substituted­4 5,6,7­tetrahydrothiene[2,3­c] pyridine (compound 3b), used in combination with an anti­miR­221­3p PNA, already demonstrated to be able to induce high levels of apoptosis. To the best of our knowledge, the results obtained herein demonstrate for the first time a 'combination therapy' performed by the combined use of a PNA targeting miR­221 and the tetrahydrothiene[2,3­c]pyridine derivative 3b, supporting the concept that the combined treatment of GBM cells with a PNA against a specific upregulated oncomiRNA (in the present study a PNA targeting miR­221­3p was used) and anti­tubulin agents (in the present study derivative 3b was used) is an encouraging strategy which may be used to enhance the efficacy of anticancer therapies and at the same time, to reduce side­effects.

Global RNA profiles show target selectivity and physiological effects of peptide-delivered antisense antibiotics.

Antisense peptide nucleic acids (PNAs) inhibiting mRNAs of essential genes provide a straight-forward way to repurpose our knowledge of bacterial regulatory RNAs for development of programmable species-specific antibiotics. While there is ample proof of PNA efficacy, their target selectivity and impact on bacterial physiology are poorly understood. Moreover, while antibacterial PNAs are typically designed to block mRNA translation, effects on target mRNA levels are not well-investigated. Here, we pioneer the use of global RNA-seq analysis to decipher PNA activity in a transcriptome-wide manner. We find that PNA-based antisense oligomer conjugates robustly decrease mRNA levels of the widely-used target gene, acpP, in Salmonella enterica, with limited off-target effects. Systematic analysis of several different PNA-carrier peptides attached not only shows different bactericidal efficiency, but also activation of stress pathways. In particular, KFF-, RXR- and Tat-PNA conjugates especially induce the PhoP/Q response, whereas the latter two additionally trigger several distinct pathways. We show that constitutive activation of the PhoP/Q response can lead to Tat-PNA resistance, illustrating the utility of RNA-seq for understanding PNA antibacterial activity. In sum, our study establishes an experimental framework for the design and assessment of PNA antimicrobials in the long-term quest to use these for precision editing of microbiota.

Assisting PNA transport through cystic fibrosis human airway epithelia with biodegradable hybrid lipid-polymer nanoparticles.

Cystic fibrosis (CF) is characterized by an airway obstruction caused by a thick mucus due to a malfunctioning Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein. The sticky mucus restricts drugs in reaching target cells limiting the efficiency of treatments. The development of new approaches to enhance drug delivery to the lungs represents CF treatment's main challenge. In this work, we report the production and characterization of hybrid core-shell nanoparticles (hNPs) comprising a PLGA core and a dipalmitoylphosphatidylcholine (DPPC) shell engineered for inhalation. We loaded hNPs with a 7-mer peptide nucleic acid (PNA) previously considered for its ability to modulate the post-transcriptional regulation of the CFTR gene. We also investigated the in vitro release kinetics of hNPs and their efficacy in PNA delivery across the human epithelial airway barrier using an ex vivo model based on human primary nasal epithelial cells (HNEC) from CF patients. Confocal analyses and hNPs transport assay demonstrated the ability of hNPs to overcome the mucus barrier and release their PNA cargo within the cytoplasm, where it can exert its biological function.

Targeting Ovarian Cancer Stem Cells by Dual Inhibition of HOTAIR and DNA Methylation.

Ovarian cancer is a chemoresponsive tumor with very high initial response rates to standard therapy consisting of platinum/paclitaxel. However, most women eventually develop recurrence, which rapidly evolves into chemoresistant disease. Persistence of ovarian cancer stem cells (OCSCs) at the end of therapy has been shown to contribute to resistant tumors. In this study, we demonstrate that the long noncoding RNA HOTAIR is overexpressed in HGSOC cell lines. Furthermore, HOTAIR expression was upregulated in OCSCs compared with non-CSC, ectopic overexpression of HOTAIR enriched the ALDH+ cell population and HOTAIR overexpression increased spheroid formation and colony-forming ability. Targeting HOTAIR using peptide nucleic acid-PNA3, which acts by disrupting the interaction between HOTAIR and EZH2, in combination with a DNMT inhibitor inhibited OCSC spheroid formation and decreased the percentage of ALDH+ cells. Disrupting HOTAIR-EZH2 with PNA3 in combination with the DNMTi on the ability of OCSCs to initiate tumors in vivo as xenografts was examined. HGSOC OVCAR3 cells were treated with PNA3 in vitro and then implanted in nude mice. Tumor growth, initiation, and stem cell frequency were inhibited. Collectively, these results demonstrate that blocking HOTAIR-EZH2 interaction combined with inhibiting DNA methylation is a potential approach to eradicate OCSCs and block disease recurrence.

Treatment of human airway epithelial Calu-3 cells with a peptide-nucleic acid (PNA) targeting the microRNA miR-101-3p is associated with increased expression of the cystic fibrosis Transmembrane Conductance Regulator () gene.

Since the identification of microRNAs (miRNAs) involved in the regulation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, miRNAs known to down-regulate the expression of the CFTR and associated proteins have been investigated as potential therapeutic targets. Here we show that miR-101-3p, targeting the 3'-UTR sequence of the CFTR mRNA, can be selectively inhibited by a peptide nucleic acid (PNA) carrying a full complementary sequence. With respect to clinical relevance of microRNA targeting, it is expected that reduction in concentration of miRNAs (the anti-miRNA approach) could be associated with increasing amounts of target mRNAs. Consistently to this hypothesis, we report that PNA-mediated inhibition of miR-101-3p was accompanied by CFTR up-regulation. Next Generation Sequencing (NGS) was performed in order to verify the effects of the anti-miR-101-3p PNA on the Calu-3 miRNome. Upon inhibition of miR-101-3p we observed a fold change (FC) expression <2 of the majority of miRNAs (403/479, 84.13%), whereas we identified a list of dysregulated miRNAs, suggesting that specific miRNA inhibition (in our case miR-101-3p) might be accompanied by alteration of expression of other miRNAs, some of them known to be involved in Cystic Fibrosis (CF), such as miR-155-5p and miR-125b-5p.