Broad-Spectrum Virus Trapping with Heparan Sulfate-Modified DNA Origami Shells.

Effective broadband antiviral platforms that can act on existing viruses and viruses yet to emerge are not available, creating a need to explore treatment strategies beyond the trodden paths. Here, we report virus-encapsulating DNA origami shells that achieve broadband virus trapping properties by exploiting avidity and a widespread background affinity of viruses to heparan sulfate proteoglycans (HSPG). With a calibrated density of heparin and heparan sulfate (HS) derivatives crafted to the interior of DNA origami shells, we could encapsulate adeno, adeno-associated, chikungunya, dengue, human papilloma, noro, polio, rubella, and SARS-CoV-2 viruses or virus-like particles, in one and the same HS-functionalized shell system. Additional virus-type-specific binders were not needed for the trapping. Depending on the relative dimensions of shell to virus particles, multiple virus particles may be trapped per shell, and multiple shells can cover the surface of clusters of virus particles. The steric occlusion provided by the heparan sulfate-coated DNA origami shells can prevent viruses from further interactions with receptors, possibly including those found on cell surfaces.

Programmable icosahedral shell system for virus trapping.

Broad-spectrum antiviral platforms that can decrease or inhibit viral infection would alleviate many threats to global public health. Nonetheless, effective technologies of this kind are still not available. Here, we describe a programmable icosahedral canvas for the self-assembly of icosahedral shells that have viral trapping and antiviral properties. Programmable triangular building blocks constructed from DNA assemble with high yield into various shell objects with user-defined geometries and apertures. We have created shells with molecular masses ranging from 43 to 925 MDa (8 to 180 subunits) and with internal cavity diameters of up to 280 nm. The shell interior can be functionalized with virus-specific moieties in a modular fashion. We demonstrate this virus-trapping concept by engulfing hepatitis B virus core particles and adeno-associated viruses. We demonstrate the inhibition of hepatitis B virus core interactions with surfaces in vitro and the neutralization of infectious adeno-associated viruses exposed to human cells.

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 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.

Macromolecular approach for targeted radioimmunotherapy in non-Hodgkin's lymphoma.

Polymers are an attractive anchoring platform for the synthesis of radioimmunoconjugates. They enable independent control over the amount of radioisotope loading and antibody attachment, which is pivotal in developing tailorable formulations for personalised medicine. Herein, we report the synthesis of p(HEMA-ran-GMA) for the conjugation of lutetium ions and rituximab as a functional platform for radioimmunotherapy. We demonstrate the suitability of this platform using non-Hodgkin's lymphoma cells.

Tumour suppression by targeted intravenous non-viral CRISPRa using dendritic polymers.

Aberrant gene expression is a hallmark of cancer. Although transcription is traditionally considered 'undruggable', the development of CRISPR-associated protein 9 (Cas9) systems offers enormous potential to rectify cancer-associated transcriptional abnormalities in malignant cells. However delivery of this technology presents a critical challenge to overcome in order to realize clinical translation for cancer therapy. In this article we demonstrate for the first time, a fully synthetic strategy to enable CRISPR-mediated activation (CRISPRa) of tumour suppressor genes in vivo using a targeted intravenous approach. We show this via highly efficient transcriptional activation of two model tumour suppressor genes, Mammary Serine Protease Inhibitor (MASPIN, SERPINB5) and cysteine-rich 61/connective tissue growth factor/nephroblastoma-overexpressed 6 (CCN6, WISP3), in a mouse model of breast cancer. In particular, we demonstrate that targeted intravenous delivery of can be achieved using a novel nanoscale dendritic macromolecular delivery agent, with negligible toxicity and long lasting therapeutic effects, outlining a targeted effective formulation with potential to treat aggressive malignancies.

A facile methodology using quantum dot multiplex labels for tracking co-transfection.

Advances in the field of genome engineering demand the development of efficient non-viral transfection agents capable of delivering multiple distinct nucleic acids efficiently to cells (co-transfection). However, current delivery methods result in lower co-transfection efficiency than single plasmid transfections, and the efficiency decreases further with increasing numbers of plasmids. The development of a high-throughput methodology is required for the validation of co-transfection platforms to facilitate independent tracking of not only the multiple DNA plasmids during transfection but also the localisation of transfection agents. This is pivotal to determine the bottlenecks in achieving high transfection efficiencies at various stages of the cell internalisation and plasmid expression process. Herein we demonstrate that this can be achieved using a facile methodology in which quantum dots (QDs) are used to label two different plasmid DNA assemblies that are delivered to cells simultaneously using a dendronised polymer system. Multispectral confocal imaging can be used to separate signals from each polyplex as well as the expressed fluorescent reporter proteins to determine whether co-transfection difficulties result from poor internalisation or the inability of DNA to escape from polyplexes. The results demonstrate the utility of this facile approach to label polyplexes without interfering with gene expression, and enable high-throughput screening of transfection reagents for achieving optimal co-transfection.

Intracellular speciation of gold nanorods alters the conformational dynamics of genomic DNA.

Gold nanorods are one of the most widely explored inorganic materials in nanomedicine for diagnostics, therapeutics and sensing1. It has been shown that gold nanorods are not cytotoxic and localize within cytoplasmic vesicles following endocytosis, with no nuclear localization2,3, but other studies have reported alterations in gene expression profiles in cells following exposure to gold nanorods, via unknown mechanisms4. In this work we describe a pathway that can contribute to this phenomenon. By mapping the intracellular chemical speciation process of gold nanorods, we show that the commonly used Au-thiol conjugation, which is important for maintaining the noble (inert) properties of gold nanostructures, is altered following endocytosis, resulting in the formation of Au(I)-thiolates that localize in the nucleus5. Furthermore, we show that nuclear localization of the gold species perturbs the dynamic microenvironment within the nucleus and triggers alteration of gene expression in human cells. We demonstrate this using quantitative visualization of ubiquitous DNA G-quadruplex structures, which are sensitive to ionic imbalances, as an indicator of the formation of structural alterations in genomic DNA.

Synthetically controlling dendrimer flexibility improves delivery of large plasmid DNA.

Tools for editing the genome and epigenome have revolutionised the field of molecular biology and represent a new frontier in targeted therapeutic intervention. Although efficiencies and specificities of genome editing technologies have improved with the development of TALEs and CRISPR platforms, intracellular delivery of these larger constructs still remains a challenge using existing delivery agents. Viral vectors, including lentiviruses and adeno-associated viruses, as well as some non-viral strategies, such as cationic polymers and liposomes, are limited by packaging capacity, poor delivery, toxicity, and immunogenicity. We report a highly controlled synthetic strategy to engineer a flexible dendritic polymer using click chemistry to overcome the aforementioned delivery challenges associated with genome engineering technologies. Using a systematic approach, we demonstrate that high transfection efficiencies and packaging capacity can be achieved using this non-viral delivery methodology to deliver zinc fingers, TALEs and CRISPR/dCas9 platforms.