Therapeutic and diagnostic applications of antisense peptide nucleic acids.

Peptide nucleic acids (PNAs) are synthetic nucleic acid analogs with a neutral N-(2-aminoethyl) glycine backbone. PNAs possess unique physicochemical characteristics such as increased resistance to enzymatic degradation, ionic strength and stability over a wide range of temperatures and pH, and low intrinsic electrostatic repulsion against complementary target oligonucleotides. PNA has been widely used as an antisense oligonucleotide (ASO). Despite the favorable characteristics of PNA, in comparison with other ASO technologies, the use of antisense PNA for novel therapeutics has lagged. This review provides a brief overview of PNA, its antisense mechanisms of action, delivery strategies, and highlights successful applications of PNA, focusing on anti-pathogenic, anti-neurodegenerative disease, anti-cancer, and diagnostic agents. For each application, several studies are discussed focusing on the different target sites of the PNA, design of different PNAs and the therapeutic outcome in different cell lines and animal models. Thereafter, persisting limitations slowing the successful integration of antisense PNA therapeutics are discussed in order to highlight actionable next steps in the development and optimization of PNA as an ASO.

Why don't dental teams routinely discuss dentine hypersensitivity during consultations? A qualitative study informed by the Theoretical Domains Framework.

AIM: Although dentine hypersensitivity is widespread, can cause substantial pain and impact quality of life, it is not routinely discussed during dental consultations. This qualitative study aimed to develop an understanding of the barriers and facilitators to these discussions. MATERIALS AND METHODS: Using the Theoretical Domains Framework to shape the topic guide, N = 7 online focus groups were organized with a total N = 40 participants comprising experienced dentists, dental foundation trainees and dental care professionals. Inductive and deductive thematic analyses of the anonymized, transcribed focus group conversations were undertaken. RESULTS: An attitude-behaviour gap was observed in dental teams' accounts. Although they saw it as part of their professional role to routinely discuss sensitivity, and believed that such conversations were 'an easy win', in practice they experienced several behavioural barriers that hindered these conversations from taking place. These included competing priorities, a perceived lack of seriousness and assessment of dentine hypersensitivity and practical issues such as time. CONCLUSIONS: Systemic (e.g., lack of time and training, professional culture) and behavioural (e.g., dental teams' belief that conversations should take place only with patients likely to be adherent) barriers to dentine hypersensitivity conversations explain why these conversations do not routinely take place.

Multivalent Lactobionic Acid and N-Acetylgalactosamine-Conjugated Peptide Nucleic Acids for Efficient In Vivo Targeting of Hepatocytes.

Peptide nucleic acids (PNAs) are used/applied in various studies to target genomic DNA and RNA to modulate gene expression. Non-specific targeting and rapid elimination always remain a challenge for PNA-based applications. Here, the synthesis, characterization, in vitro and in vivo study of di lactobionic acid (diLBA) and tris N-acetyl galactosamine (tGalNAc) conjugated PNAs for liver-targeted delivery are reported. For proof of concept, diLBA, and tGalNAc conjugated PNAs (anti-miR-122 PNAs) were synthesized to target microRNA-122 (miR-122) which is over-expressed in the hepatic tissue. Different lengths of anti-miR-122 PNAs conjugated with diLBA and tGalNAc are tested. Cell culture and in vivo analyses to determine biodistribution, efficacy, and toxicity profile are performed. This work indicates that diLBA conjugates show significant retention in hepatocytes in addition to tGalNAc conjugates after in vivo delivery. Full-length PNA conjugates show significant downregulation of miR-122 levels and subsequent de-repression of its downstream targets with no evidence of toxicity. The results provide a robust framework for ligand-conjugated delivery systems for PNAs that can be explored for broader biomedical applications.

In vivo correction of cystic fibrosis mediated by PNA nanoparticles.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We sought to correct the multiple organ dysfunction of the F508del CF-causing mutation using systemic delivery of peptide nucleic acid gene editing technology mediated by biocompatible polymeric nanoparticles. We confirmed phenotypic and genotypic modification in vitro in primary nasal epithelial cells from F508del mice grown at air-liquid interface and in vivo in F508del mice following intravenous delivery. In vivo treatment resulted in a partial gain of CFTR function in epithelia as measured by in situ potential differences and Ussing chamber assays and correction of CFTR in both airway and GI tissues with no off-target effects above background. Our studies demonstrate that systemic gene editing is possible, and more specifically that intravenous delivery of PNA NPs designed to correct CF-causing mutations is a viable option to ameliorate CF in multiple affected organs.

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.

The Challenges and Opportunities in the Development of MicroRNA Therapeutics: A Multidisciplinary Viewpoint.

microRNAs (miRs) are emerging as attractive therapeutic targets because of their small size, specific targetability, and critical role in disease pathogenesis. However, <20 miR targeting molecules have entered clinical trials, and none progressed to phase III. The difficulties in miR target identification, the moderate efficacy of miR inhibitors, cell type-specific delivery, and adverse outcomes have impeded the development of miR therapeutics. These hurdles are rooted in the functional complexity of miR's role in disease and sequence complementarity-dependent/-independent effects in nontarget tissues. The advances in understanding miR's role in disease, the development of efficient miR inhibitors, and innovative delivery approaches have helped resolve some of these hurdles. In this review, we provide a multidisciplinary viewpoint on the challenges and opportunities in the development of miR therapeutics.

Formulation and fluoride content of dentifrices: a review of current patterns.

Introduction Consumer oral hygiene products play a key role in improving and maintaining population oral health. The oral personal care market is rapidly diversifying; a growing number of dentifrices marketed a 'natural' and fluoride-free are entering mainstream retailers, which may have implications for the oral health of the population 'with regards to caries risk.Aims To investigate the range of fluoride concentrations, flavour formulations and delivery mechanisms of dentifrices available on the UK market.Methods A cross-sectional survey was used to catalogue dentifrices sold in a range of supermarkets, high-street pharmacy and health chains, and specialist online retailers. In addition, a standard search engine was used to examine dentifrice brands being sold in the UK. The fluoride content was recorded as parts per million (ppm) and the product name data were analysed for key terms using Microsoft Excel. Excluded from the survey were mouthwashes, rinses and non-dentifrice whitening products.Results Five hundred different toothpaste, tooth powder and tablet products from 95 different brands were recorded. Sixty percent of these contained a fluoride concentration of 1,000 ppm or above. Forty-five percent of all products had the recommended adult concentration of at least 1,350 ppm. Almost one-third (31%) contained no fluoride and 4% of products did not specify the absence, presence or concentration of fluoride.Conclusions This study has quantified and confirmed the increasingly diverse range of dentifrices for sale in the UK. A large number of fluoride-free products exist within a growing 'natural' and 'organic market'. The study also gives oral health professionals an insight into the diverse types of products available to consumers in order to appropriately advise patients on caries prevention.

Green Synthesis of Nanomaterials.

Nanotechnology is considered one of the paramount forefronts in science over the last decade. Its versatile implementations and fast-growing demand have paved the way for innovative measures for the synthesis of higher quality nanomaterials. In the early stages, traditional synthesis methods were utilized, and they relied on both carcinogenic chemicals and high energy input for production of nano-sized material. The pollution produced as a result of traditional synthesis methods induces a need for environmentally safer synthesis methods. As the downfalls of climate change become more abundant, the scientific community is persistently seeking solutions to combat the devastation caused by toxic production methods. Green methods for nanomaterial synthesis apply natural biological systems to nanomaterial production. The present review highlights the history of nanoparticle synthesis, starting with traditional methods and progressing towards green methods. Green synthesis is a method just as effective, if not more so, than traditional synthesis; it provides a sustainable approach to nanomaterial manufacturing by using naturally sourced starting materials and relying on low energy processes. The recent use of active molecules in natural biological systems such as bacteria, yeast, algae and fungi report successful results in the synthesis of various nanoparticle systems. Thus, the integration of green synthesis in scientific research and mass production provides a potential solution to the limitations of traditional synthesis methods.

Emerging Therapeutic Modalities against COVID-19.

The novel SARS-CoV-2 virus has quickly spread worldwide, bringing the whole world as well as the economy to a standstill. As the world is struggling to minimize the transmission of this devastating disease, several strategies are being actively deployed to develop therapeutic interventions. Pharmaceutical companies and academic researchers are relentlessly working to investigate experimental, repurposed or FDA-approved drugs on a compassionate basis and novel biologics for SARS-CoV-2 prophylaxis and treatment. Presently, a tremendous surge of COVID-19 clinical trials are advancing through different stages. Among currently registered clinical efforts, ~86% are centered on testing small molecules or antibodies either alone or in combination with immunomodulators. The rest ~14% of clinical efforts are aimed at evaluating vaccines and convalescent plasma-based therapies to mitigate the disease's symptoms. This review provides a comprehensive overview of current therapeutic modalities being evaluated against SARS-CoV-2 virus in clinical trials.

Antisense Oligonucleotides: An Emerging Area in Drug Discovery and Development.

Antisense oligonucleotides (ASOs) bind sequence specifically to the target RNA and modulate protein expression through several different mechanisms. The ASO field is an emerging area of drug development that targets the disease source at the RNA level and offers a promising alternative to therapies targeting downstream processes. To translate ASO-based therapies into a clinical success, it is crucial to overcome the challenges associated with off-target side effects and insufficient biological activity. In this regard, several chemical modifications and diverse delivery strategies have been explored. In this review, we systematically discuss the chemical modifications, mechanism of action, and optimized delivery strategies of several different classes of ASOs. Further, we highlight the recent advances made in development of ASO-based drugs with a focus on drugs that are approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for clinical applications. We also discuss various promising ASO-based drug candidates in the clinical trials, and the outstanding opportunity of emerging microRNA as a viable therapeutic target for future ASO-based therapies.

Role of Lipid-Based and Polymer-Based Non-Viral Vectors in Nucleic Acid Delivery for Next-Generation Gene Therapy.

The field of gene therapy has experienced an insurgence of attention for its widespread ability to regulate gene expression by targeting genomic DNA, messenger RNA, microRNA, and short-interfering RNA for treating malignant and non-malignant disorders. Numerous nucleic acid analogs have been developed to target coding or non-coding sequences of the human genome for gene regulation. However, broader clinical applications of nucleic acid analogs have been limited due to their poor cell or organ-specific delivery. To resolve these issues, non-viral vectors based on nanoparticles, liposomes, and polyplexes have been developed to date. This review is centered on non-viral vectors mainly comprising of cationic lipids and polymers for nucleic acid-based delivery for numerous gene therapy-based applications.

Anti-tumor Activity of miniPEG-γ-Modified PNAs to Inhibit MicroRNA-210 for Cancer Therapy.

MicroRNAs (miRs) are frequently overexpressed in human cancers. In particular, miR-210 is induced in hypoxic cells and acts to orchestrate the adaptation of tumor cells to hypoxia. Silencing oncogenic miRs such as miR-210 may therefore offer a promising approach to anticancer therapy. We have developed a miR-210 inhibition strategy based on a new class of conformationally preorganized antisense γ peptide nucleic acids (γPNAs) that possess vastly superior RNA-binding affinity, improved solubility, and favorable biocompatibility. For cellular delivery, we encapsulated the γPNAs in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs). Our results show that γPNAs targeting miR-210 cause significant delay in growth of a human tumor xenograft in mice compared to conventional PNAs. Further, histopathological analyses show considerable necrosis, fibrosis, and reduced cell proliferation in γPNA-treated tumors compared to controls. Overall, our work provides a chemical framework for a novel anti-miR therapeutic approach using γPNAs that should facilitate rational design of agents to potently inhibit oncogenic microRNAs.

Nanotechnology for delivery of peptide nucleic acids (PNAs).

Over the past three decades, peptide nucleic acids have been employed in numerous chemical and biological applications. Peptide nucleic acids possess enormous potential because of their superior biophysical properties, compared to other oligonucleotide chemistries. However, for therapeutic applications, intracellular delivery of peptide nucleic acids remains a challenge. In this review, we summarize the progress that has been made in delivering peptide nucleic acids to intracellular targets. In addition, we emphasize recent nanoparticle-based strategies for efficient delivery of conventional and chemically-modified peptides nucleic acids.

Nanoparticles that deliver triplex-forming peptide nucleic acid molecules correct F508del CFTR in airway epithelium.

Cystic fibrosis (CF) is a lethal genetic disorder most commonly caused by the F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. It is not readily amenable to gene therapy because of its systemic nature and challenges including in vivo gene delivery and transient gene expression. Here we use triplex-forming peptide nucleic acids and donor DNA in biodegradable polymer nanoparticles to correct F508del. We confirm modification with sequencing and a functional chloride efflux assay. In vitro correction of chloride efflux occurs in up to 25% of human cells. Deep-sequencing reveals negligible off-target effects in partially homologous sites. Intranasal delivery of nanoparticles in CF mice produces changes in the nasal epithelium potential difference assay, consistent with corrected CFTR function. Also, gene correction is detected in the nasal and lung tissue. This work represents facile genome engineering in vivo with oligonucleotides using a nanoparticle system to achieve clinically relevant levels of gene editing without off-target effects.

Hybridization of G-quadruplex-forming peptide nucleic acids to guanine-rich DNA templates inhibits DNA polymerase η extension.

The guanine quadruplex (G-quadruplex) is a highly stable secondary structure that forms in G-rich repeats of DNA, which can interfere with DNA processes, including DNA replication and transcription. We showed previously that short guanine-rich peptide nucleic acids (PNAs) can form highly stable hybrid quadruplexes with DNA. We hypothesized that such structures would provide a stronger block to polymerase extension on G-rich templates than a native DNA homoquadruplex because of the greater thermodynamic stability of the PNA-DNA hybrid structures. To test this, we analyzed the DNA primer extension activity of polymerase η, a translesion polymerase implicated in synthesis past G-quadruplex blocks, on DNA templates containing guanine repeats. We observed a PNA concentration-dependent decrease in the level of polymerase η extension to the end of the template and an increase in the level of polymerase η inhibition at the sequence prior to the G-rich repeats. In contrast, the addition of a complementary C-rich PNA that hybridizes to the G-rich repeats by Watson-Crick base pairing led to a decrease in the level of polymerase inhibition and an increase in the level of full-length extension products. The G-quadruplex-forming PNA exhibited inhibition (IC50=16.2±3.3 nM) of polymerase η DNA synthesis on the G-rich templates stronger than that of the established G-quadruplex-stabilizing ligand BRACO-19 (IC50=42.5±4.8 nM). Our results indicate that homologous PNA targeting of G-rich sequences creates stable PNA-DNA heteroquadruplexes that inhibit polymerase η extension more effectively than a DNA homoquadruplex. The implications of these results for the potential development of homologous PNAs as therapeutics for halting proliferating cancer cells are discussed.

Modular evolution of DNA-binding preference of a Tbrain transcription factor provides a mechanism for modifying gene regulatory networks.

Gene regulatory networks (GRNs) describe the progression of transcriptional states that take a single-celled zygote to a multicellular organism. It is well documented that GRNs can evolve extensively through mutations to cis-regulatory modules (CRMs). Transcription factor proteins that bind these CRMs may also evolve to produce novelty. Coding changes are considered to be rarer, however, because transcription factors are multifunctional and hence are more constrained to evolve in ways that will not produce widespread detrimental effects. Recent technological advances have unearthed a surprising variation in DNA-binding abilities, such that individual transcription factors may recognize both a preferred primary motif and an additional secondary motif. This provides a source of modularity in function. Here, we demonstrate that orthologous transcription factors can also evolve a changed preference for a secondary binding motif, thereby offering an unexplored mechanism for GRN evolution. Using protein-binding microarray, surface plasmon resonance, and in vivo reporter assays, we demonstrate an important difference in DNA-binding preference between Tbrain protein orthologs in two species of echinoderms, the sea star, Patiria miniata, and the sea urchin, Strongylocentrotus purpuratus. Although both orthologs recognize the same primary motif, only the sea star Tbr also has a secondary binding motif. Our in vivo assays demonstrate that this difference may allow for greater evolutionary change in timing of regulatory control. This uncovers a layer of transcription factor binding divergence that could exist for many pairs of orthologs. We hypothesize that this divergence provides modularity that allows orthologous transcription factors to evolve novel roles in GRNs through modification of binding to secondary sites.

Strand invasion of DNA quadruplexes by PNA: comparison of homologous and complementary hybridization.

Molecular recognition of DNA quadruplex structures is envisioned to be a strategy for regulating gene expression at the transcriptional level and for in situ analysis of telomere structure and function. The recognition of DNA quadruplexes by peptide nucleic acid (PNA) oligomers is presented here, with a focus on comparing complementary, heteroduplex-forming and homologous, heteroquadruplex-forming PNAs. Surface plasmon resonance and optical spectroscopy experiments demonstrated that the efficacy of a recognition mode depended strongly on the target. Homologous PNA readily invades a quadruplex derived from the promoter regulatory region found upstream of the MYC proto-oncogene to form a heteroquadruplex at high potassium concentration mimicking the intracellular environment, whereas complementary PNA exhibits virtually no hybridization. In contrast, complementary PNA is superior to the homologous in hybridizing to a quadruplex modeled on the human telomere sequence. The results are discussed in terms of the different structural morphologies of the quadruplex targets and the implications for in vivo recognition of quadruplexes by PNAs.