Transcriptomic Analysis Reveals the Heterogeneous Role of Conducting Films Upon Electrical Stimulation.

Central nervous system (CNS) injuries and neurodegenerative diseases have markedly poor prognoses and can result in permanent dysfunction due to the general inability of CNS neurons to regenerate. Differentiation of transplanted stem cells has emerged as a therapeutic avenue to regenerate tissue architecture in damaged areas. Electrical stimulation is a promising approach for directing the differentiation outcomes and pattern of outgrowth of transplanted stem cells, however traditional inorganic bio-electrodes can induce adverse effects such as inflammation. This study demonstrates the implementation of two organic thin films, a polymer/reduced graphene oxide nanocomposite (P(rGO)) and PEDOT:PSS, that have favorable properties for implementation as conductive materials for electrical stimulation, as well as an inorganic indium tin oxide (ITO) conductive film. Transcriptomic analysis reveals that electrical stimulation improves neuronal differentiation of SH-SY5Y cells on all three films, with the greatest effect for P(rGO). Unique material- and electrical stimuli-mediated effects are observed, associated with differentiation, cell-substrate adhesion, and translation. The work demonstrates that P(rGO) and PEDOT:PSS are highly promising organic materials for the development of biocompatible, conductive scaffolds that will enhance electrically-aided stem cell therapeutics for CNS injuries and neurodegenerative diseases.

High-resolution visualisation of antisense oligonucleotide release from polymers in cells.

Antisense oligonucleotides (ASOs) are a well-established therapeutic modality based on RNA interference, but low cellular uptake, limited ability to direct ASO trafficking, and a range of intracellular barriers to successful activity compromise both gene silencing outcomes and clinical translations. Herein, we demonstrate that polymers can increase ASO internalisation via intracellular trafficking pathways that are distinct from lipid-based delivery reagents. For the first time, we spatially define internalisation and dissociation stages in the polymer-mediated cytosolic delivery of ASOs using Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS), which enables visualisation of ASO localisation at the organelle level. We find that polymer-ASO complexes are imported into cells, from which free ASO enters the cytosol following complex dissociation. This information enables a better understanding of the intracellular trafficking pathways of nucleic acid therapeutics and may be exploited for therapeutic delivery to enhance the effectiveness of nucleic acid therapeutics in the future.

Local therapy with combination TLR agonists stimulates systemic anti-tumor immunity and sensitizes tumors to immune checkpoint blockade.

Toll-like receptor (TLR) agonists are being developed as anti-cancer therapeutics due to their potent immunostimulatory properties. However, clinical trials testing TLR agonists as monotherapy have often failed to demonstrate significant improvement over standard of care. We hypothesized that the anti-cancer efficacy of TLR agonist immunotherapy could be improved by combinatorial approaches. To prevent increased toxicity, often seen with systemic combination therapies, we developed a hydrogel to deliver TLR agonist combinations at low doses, locally, during cancer debulking surgery. Using tumor models of WEHI 164 and bilateral M3-9-M sarcoma and CT26 colon carcinoma, we assessed the efficacy of pairwise combinations of poly(I:C), R848, and CpG in controlling local and distant tumor growth. We show that combination of the TLR3 agonist poly(I:C) and TLR7/8 agonist R848 drives anti-tumor immunity against local and distant tumors. In addition, combination of local poly(I:C) and R848 sensitized tumors to systemic immune checkpoint blockade, improving tumor control. Mechanistically, we demonstrate that local therapy with poly(I:C) and R848 recruits inflammatory monocytes to the tumor draining lymph nodes early in the anti-tumor response. Finally, we provide proof of concept for intraoperative delivery of poly(I:C) and R848 together via a surgically applicable biodegradable hydrogel.

Human genomic DNA is widely interspersed with i-motif structures.

DNA i-motif structures are formed in the nuclei of human cells and are believed to provide critical genomic regulation. While the existence, abundance, and distribution of i-motif structures in human cells has been demonstrated and studied by immunofluorescent staining, and more recently NMR and CUT&Tag, the abundance and distribution of such structures in human genomic DNA have remained unclear. Here we utilise high-affinity i-motif immunoprecipitation followed by sequencing to map i-motifs in the purified genomic DNA of human MCF7, U2OS and HEK293T cells. Validated by biolayer interferometry and circular dichroism spectroscopy, our approach aimed to identify DNA sequences capable of i-motif formation on a genome-wide scale, revealing that such sequences are widely distributed throughout the human genome and are common in genes upregulated in G0/G1 cell cycle phases. Our findings provide experimental evidence for the widespread formation of i-motif structures in human genomic DNA and a foundational resource for future studies of their genomic, structural, and molecular roles.

Preferential formation of Z-RNA over intercalated motifs in long noncoding RNA.

Secondary structure is a principal determinant of lncRNA function, predominantly regarding scaffold formation and interfaces with target molecules. Noncanonical secondary structures that form in nucleic acids have known roles in regulating gene expression and include G-quadruplexes (G4s), intercalated motifs (iMs), and R-loops (RLs). In this paper, we used the computational tools G4-iM Grinder and QmRLFS-finder to predict the formation of each of these structures throughout the lncRNA transcriptome in comparison to protein-coding transcripts. The importance of the predicted structures in lncRNAs in biological contexts was assessed by combining our results with publicly available lncRNA tissue expression data followed by pathway analysis. The formation of predicted G4 (pG4) and iM (piM) structures in select lncRNA sequences was confirmed in vitro using biophysical experiments under near-physiological conditions. We find that the majority of the tested pG4s form highly stable G4 structures, and identify many previously unreported G4s in biologically important lncRNAs. In contrast, none of the piM sequences are able to form iM structures, consistent with the idea that RNA is unable to form stable iMs. Unexpectedly, these C-rich sequences instead form Z-RNA structures, which have not been previously observed in regions containing cytosine repeats and represent an interesting and underexplored target for protein-RNA interactions. Our results highlight the prevalence and potential structure-associated functions of noncanonical secondary structures in lncRNAs, and show G4 and Z-RNA structure formation in many lncRNA sequences for the first time, furthering the understanding of the structure-function relationship in lncRNAs.

Docetaxel Inhibits Epithelial-Mesenchymal Transition in Human Mammary Cells.

Epithelial-mesenchymal transition (EMT) is a reversible and dynamic biological process in which epithelial cells acquire mesenchymal characteristics including enhanced stemness and migratory ability. EMT can facilitate cancer metastasis and is a known driver of cellular resistance to common chemotherapeutic drugs, such as docetaxel. Current chemotherapeutic practices such as docetaxel treatment can promote EMT and increase the chance of tumor recurrence and resistance, calling for new approaches in cancer treatment. Here we show that prolonged docetaxel treatment at a sub-IC50 concentration inhibits EMT in immortalized human mammary epithelial (HMLE) cells. Using immunofluorescence, flow cytometry, and bulk transcriptomic sequencing to assess EMT progression, we analyzed a range of cellular markers of EMT in docetaxel-treated cells and observed an upregulation of epithelial markers and downregulation of mesenchymal markers in the presence of docetaxel. This finding suggests that docetaxel may have clinical applications not only as a cytotoxic drug but also as an inhibitor of EMT-driven metastasis and multidrug resistance depending on the concentration of its use.

Elemental Mapping in a Preclinical Animal Model Reveals White Matter Copper Elevation in the Acute Phase of Central Nervous System Trauma.

Understanding the chemical events following trauma to the central nervous system could assist in identifying causative mechanisms and potential interventions to protect neural tissue. Here, we apply a partial optic nerve transection model of injury in rats and use synchrotron X-ray fluorescence microscopy (XFM) to perform elemental mapping of metals (K, Ca, Fe, Cu, Zn) and other related elements (P, S, Cl) in white matter tracts. The partial optic nerve injury model and spatial precision of microscopy allow us to obtain previously unattained resolution in mapping elemental changes in response to a primary injury and subsequent secondary effects. We observed significant elevation of Cu levels at multiple time points following the injury, both at the primary injury site and in neural tissue near the injury site vulnerable to secondary damage, as well as significant changes in Cl, K, P, S, and Ca. Our results suggest widespread metal dyshomeostasis in response to central nervous system trauma and that altered Cu homeostasis may be a specific secondary event in response to white matter injury. The findings highlight metal homeostasis as a potential point of intervention in limiting damage following nervous system injury.

G4-DNA formation and chromatin remodelling are interdependent in human cells.

DNA G-quadruplexes (G4s) have been identified as important biological targets for transcriptional, translational, and epigenetic regulation. The stabilisation of G4s with small molecule ligands has emerged as a technique to regulate gene expression and as a potential therapeutic approach for human diseases. Here, we demonstrate that ligand stabilisation of G4s causes altered chromatin accessibility dependent on the targeting specificity of the molecule. In particular, stabilisation of a target G4 using the highly specific GTC365 ligand resulted in differential accessibility of 61 genomic regions, while the broad-targeting G4 ligand, GQC-05, stabilised many G4s and induced a global shift towards increased accessibility of gene promoter regions. Interestingly, while we observed distinct effects of each ligand on RNA expression levels and the induction of DNA double-stranded breaks, both ligands modified DNA damage response pathways. Our work represents the dual possibility of G4-stabilising ligands for specific or global chromatin modulation via unique targeting characteristics.

A surgically optimized intraoperative poly(I:C)-releasing hydrogel prevents cancer recurrence.

Recurrences frequently occur following surgical removal of primary tumors. In many cancers, adjuvant therapies have limited efficacy. Surgery provides access to the tumor microenvironment, creating an opportunity for local therapy, in particular immunotherapy, which can induce local and systemic anti-cancer effects. Here, we develop a surgically optimized biodegradable hyaluronic acid-based hydrogel for sustained intraoperative delivery of Toll-like receptor 3 agonist poly(I:C) and demonstrate that it significantly reduces tumor recurrence after surgery in multiple mouse models. Mechanistically, poly(I:C) induces a transient interferon alpha (IFNα) response, reshaping the tumor/wound microenvironment by attracting inflammatory monocytes and depleting regulatory T cells. We demonstrate that a pre-existing IFN signature predicts response to the poly(I:C) hydrogel, which sensitizes tumors to immune checkpoint therapy. The safety, immunogenicity, and surgical feasibility are confirmed in a veterinary trial in canine soft tissue tumors. The surgically optimized poly(I:C)-loaded hydrogel provides a safe and effective approach to prevent cancer recurrence.

Millimeter waves alter DNA secondary structures and modulate the transcriptome in human fibroblasts.

As millimetre wave (MMW) frequencies of the electromagnetic spectrum are increasingly adopted in modern technologies such as mobile communications and networking, characterising the biological effects is critical in determining safe exposure levels. We study the exposure of primary human dermal fibroblasts to MMWs, finding MMWs trigger genomic and transcriptomic alterations. In particular, repeated 60 GHz, 2.6 mW cm-2, 46.8 J cm-2 d-1 MMW doses induce a unique physiological response after 2 and 4 days exposure. We show that high dose MMWs induce simultaneous non-thermal alterations to the transcriptome and DNA structural dynamics, including formation of G-quadruplex and i-motif secondary structures, but not DNA damage.

Intracellular Communication between Synthetic Macromolecules.

Non-viral delivery is an important strategy for selective and efficient gene therapy, immunization, and RNA interference, which overcomes problems of genotoxicity and inherent immunogenicity associated with viral vectors. Liposomes and polymers are compelling candidates as carriers for intracellular, non-viral delivery, but maximal efficiencies of around 1% have been reported for the most advanced non-viral carriers. Here, we develop a library of dendronized bottlebrush polymers with controlled defects, displaying a level of precision surpassed only by biological molecules like DNA, RNA, and proteins. We test concurrent and competitive delivery of DNA and show for the first time that, while intracellular communication is thought to be an exclusively biomolecular phenomenon, such communication between synthetic macromolecular complexes can also take place. Our findings challenge the assumption that delivery agents behave as bystanders that enable transfection by passive intracellular release of genetic cargo and improve upon coarse strategies in intracellular carrier design lacking control over polymer sequence, architecture, and composition, leading to a hit-or-miss outcome. Understanding the communication that takes place between macromolecules will help improve the design of non-viral delivery agents and facilitate translation of genome engineering, vaccines, and nucleic acid-based therapies.

Stability and context of intercalated motifs (i-motifs) for biological applications.

DNA is naturally dynamic and can self-assemble into alternative secondary structures including the intercalated motif (i-motif), a four-stranded structure formed in cytosine-rich DNA sequences. Until recently, i-motifs were thought to be unstable in physiological cellular environments. Studies demonstrating their existence in the human genome and role in gene regulation are now shining light on their biological relevance. Herein, we review the effects of epigenetic modifications on i-motif structure and stability, and biological factors that affect i-motif formation within cells. Furthermore, we highlight recent progress in targeting i-motifs with structure-specific ligands for biotechnology and therapeutic purposes.

The role of G-Quadruplex DNA in Paraspeckle formation in cancer.

Paraspeckles are RNA-protein structures within the nucleus of mammalian cells, capable of orchestrating various biochemical processes. An overexpression of the architectural component of paraspeckles, a long non-coding RNA called NEAT1 (Nuclear Enriched Abundant Transcript 1), has been linked to a variety of cancers and is often associated with poor patient prognosis. Thus, there is an accumulating interest in the role of paraspeckles in carcinogenesis, however there is a limited understanding of how NEAT1 expression is regulated. Here, we demonstrate that both nuclear G-quadruplex (G4) and paraspeckle formation are significantly increased in a human breast cancer cell line compared to non-tumorigenic breast cells. Moreover, we identified and characterized G4-forming sequences within the NEAT1 promoter and demonstrate stabilization of G4 DNA with a G4-stabilizing small molecule results in a significant alteration in both paraspeckle formation and NEAT1 expression. This G4-mediated alteration of NEAT1 at both the transcriptional and post-transcriptional levels was evident in U2OS osteosarcoma cells, MCF-7 breast adenocarcinoma and MDA-MB-231 triple negative breast cancer cells.

Regulation of Proteins to the Cytosol Using Delivery Systems with Engineered Polymer Architecture.

Intracellular protein delivery enables selective regulation of cellular metabolism, signaling, and development through introduction of defined protein quantities into the cell. Most applications require that the delivered protein has access to the cytosol, either for protein activity or as a gateway to other organelles such as the nucleus. The vast majority of delivery vehicles employ an endosomal pathway however, and efficient release of entrapped protein cargo from the endosome remains a challenge. Recent research has made significant advances toward efficient cytosolic delivery of proteins using polymers, but the influence of polymer architecture on protein delivery is yet to be investigated. Here, we developed a family of dendronized polymers that enable systematic alterations of charge density and structure. We demonstrate that while modulation of surface functionality has a significant effect on overall delivery efficiency, the endosomal release rate can be highly regulated by manipulating polymer architecture. Notably, we show that large, multivalent structures cause slower sustained release, while rigid spherical structures result in rapid burst release.

Modulating gene expression in breast cancer via DNA secondary structure and the CRISPR toolbox.

Breast cancer is the most commonly diagnosed malignancy in women, and while the survival prognosis of patients with early-stage, non-metastatic disease is ∼75%, recurrence poses a significant risk and advanced and/or metastatic breast cancer is incurable. A distinctive feature of advanced breast cancer is an unstable genome and altered gene expression patterns that result in disease heterogeneity. Transcription factors represent a unique therapeutic opportunity in breast cancer, since they are known regulators of gene expression, including gene expression involved in differentiation and cell death, which are themselves often mutated or dysregulated in cancer. While transcription factors have traditionally been viewed as 'undruggable', progress has been made in the development of small-molecule therapeutics to target relevant protein-protein, protein-DNA and enzymatic active sites, with varying levels of success. However, non-traditional approaches such as epigenetic editing, transcriptional control via CRISPR/dCas9 systems, and gene regulation through non-canonical nucleic acid secondary structures represent new directions yet to be fully explored. Here, we discuss these new approaches and current limitations in light of new therapeutic opportunities for breast cancers.

A dendronised polymer architecture breaks the conventional inverse relationship between porosity and mechanical properties of hydrogels.

We present a series of synthetic polymer hydrogels which break the traditional correlation between pore size and mechanical properties. The hydrogels are prepared from a dendronised polymer architecture based on a methacrylate copolymer to which poly(amido amine) dendrons are attached. Our approach will be useful in tailoring hydrogels for tissue engineering, controlled drug release, and flexible electronics.

The corona of a surface bubble promotes electrochemical reactions.

The evolution of gaseous products is a feature common to several electrochemical processes, often resulting in bubbles adhering to the electrode's surface. Adherent bubbles reduce the electrode active area, and are therefore generally treated as electrochemically inert entities. Here, we show that this general assumption does not hold for gas bubbles masking anodes operating in water. By means of imaging electrochemiluminescent systems, and by studying the anisotropy of polymer growth around bubbles, we demonstrate that gas cavities adhering to an electrode surface initiate the oxidation of water-soluble species more effectively than electrode areas free of bubbles. The corona of a bubble accumulates hydroxide anions, unbalanced by cations, a phenomenon which causes the oxidation of hydroxide ions to hydroxyl radicals to occur at potentials at least 0.7 V below redox tabled values. The downhill shift of the hydroxide oxidation at the corona of the bubble is likely to be a general mechanism involved in the initiation of heterogeneous electrochemical reactions in water, and could be harnessed in chemical synthesis.

DNA G-Quadruplex and i-Motif Structure Formation Is Interdependent in Human Cells.

Guanine- and cytosine-rich nucleic acid sequences have the potential to form secondary structures such as G-quadruplexes and i-motifs, respectively. We show that stabilization of G-quadruplexes using small molecules destabilizes the i-motifs, and vice versa, indicating these gene regulatory controllers are interdependent in human cells. This has important implications as these structures are predominately considered as isolated structural targets for therapy, but their interdependency highlights the interplay of both structures as an important gene regulatory switch.

A dendronized polymer variant that facilitates safe delivery of a calcium channel antagonist to the heart.

Therapeutic approaches for myocardial ischemia-reperfusion injury (MI) have been ineffective due to limited bioavailability and poor specificity. We have previously shown that a peptide that targets the α-interaction domain of the cardiac L-type calcium channel (AID-peptide) attenuates MI when tethered to transactivator of transcription sequence (TAT) or spherical nanoparticles. However some reservations remain regarding use of these delivery platforms due to the relationship with human immunodeficiency virus, off-target effects and toxicity. Here we investigate the use of linear dendronized polymers (denpols) to deliver AID-peptide as a potential MI therapy using in vitro, ex vivo and in vivo models. Optimized denpol-complexed AID-peptide facilitated in vitro cardiac uptake of AID-peptide, and reduced MI. Maximal in vivo cardiac uptake was achieved within the 2 h therapeutic time window for acute myocardial infarction. Importantly, optimized denpol-complexed AID-peptide was not toxic. This platform may represent an alternative therapeutic approach for the prevention of MI.

Surface Diffusion of Dendronized Polymers Correlates with Their Transfection Potential.

Successful intracellular delivery of therapeutics requires interactions at several liquid-solid interfaces, including cell surface, endosomal membranes, and-depending on the therapeutic-the nuclear membrane. Understanding the dynamics of polymer kinetics at the liquid-solid interface is fundamental for the design of polymers for such biomedical delivery applications. However, the effect of polymer architecture and charge density on polymer kinetics is not readily investigated using routine techniques, and the role of such parameters in the context of gene delivery remains unknown. We adopted a synthetic strategy which enabled the systematic manipulation of charge density, flexibility, and molecular weight using a dendronized linear polymeric architecture. High-speed atomic force microscopy (HS-AFM) was used as a label-free method to directly observe the polymers' dynamic properties, such as velocity, displacement, and diffusion, in physiologically relevant conditions. Importantly, we found that the physical parameters measured by HS-AFM relate to the transfection potential of the individual polymers and may be a valuable tool in screening structural polymer variants.