RESUMEN
DNA origami enables the creation of complex 3D shapes from genetic material. Future uses could include the delivery of genetic instructions to cells, but nuclear import remains a major barrier to gene delivery due to the impermeability of the nuclear membrane. Here we realize active nuclear import of DNA origami objects in dividing and chemically arrested mammalian cells. We developed a custom DNA origami single-strand scaffold featuring a mammalian-cell expressible reporter gene (mCherry) and multiple Simian virus 40 (SV40) derived DNA nuclear targeting sequences (DTS). Inclusion of the DTS within DNA origami rescued gene expression in arrested cells, indicating that active transport into the nucleus occurs. Our work successfully adapts mechanisms known from viruses to promote the cellular expression of genetic instructions encoded within DNA origami objects.
Asunto(s)
Núcleo Celular , ADN , Animales , Transporte Activo de Núcleo Celular , ADN/metabolismo , Núcleo Celular/metabolismo , Genes Reporteros , Mamíferos/metabolismoRESUMEN
DNA origami may enable more versatile gene delivery applications through its ability to create custom nanoscale objects with specific targeting, cell-invading, and intracellular effector functionalities. Toward this goal here we describe the expression of genes folded in DNA origami objects delivered to mammalian cells. Genes readily express from custom-sequence single-strand scaffolds folded within DNA origami objects, provided that the objects can denature in the cell. We demonstrate enhanced gene expression efficiency by including and tuning multiple functional sequences and structures, including virus-inspired inverted-terminal repeat-like (ITR) hairpin motifs upstream or flanking the expression cassette. We describe gene-encoding DNA origami bricks that assemble into multimeric objects to enable stoichiometrically controlled co-delivery and expression of multiple genes in the same cells. Our work provides a framework for exploiting DNA origami for gene delivery applications.
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Nanoestructuras , Nanotecnología , Conformación de Ácido Nucleico , ADN/genética , ADN/química , Nanoestructuras/químicaRESUMEN
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.
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COVID-19 , Virus , Humanos , SARS-CoV-2 , Heparitina Sulfato/metabolismo , Proteoglicanos de Heparán Sulfato , ADNRESUMEN
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.
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ADN , Virus de la Hepatitis B , Nanopartículas , Microscopía Electrónica de Transmisión , Nanopartículas/química , Nanopartículas/ultraestructuraRESUMEN
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.
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Citosol/metabolismo , Polímeros/química , Ingeniería de Proteínas , Proteínas/metabolismo , Animales , Línea Celular , Citosol/química , Humanos , Ratones , Estructura Molecular , Polímeros/metabolismo , Proteínas/químicaRESUMEN
Breast cancer is the most commonly diagnosed malignancy in women, and while the survival prognosis of patients with early-stage, non-metastatic disease is â¼75%, 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.
RESUMEN
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.
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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 2â¯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.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/genética , Corazón/efectos de los fármacos , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Nanopartículas/química , Animales , Bloqueadores de los Canales de Calcio/química , Canales de Calcio Tipo L/efectos de los fármacos , Modelos Animales de Enfermedad , Cobayas , Corazón/fisiopatología , Humanos , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/patología , Miocitos Cardíacos/efectos de los fármacos , Péptidos/química , Péptidos/farmacología , Polímeros/química , Polímeros/farmacologíaRESUMEN
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.
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Inmunoconjugados/química , Linfoma no Hodgkin/radioterapia , Radioinmunoterapia , Antineoplásicos Inmunológicos/química , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéutico , Supervivencia Celular/efectos de los fármacos , Química Clic , Compuestos Epoxi/química , Humanos , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Lutecio/química , Metacrilatos/química , Polímeros/química , Rituximab/química , Rituximab/farmacología , Rituximab/uso terapéuticoRESUMEN
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.
RESUMEN
Protein delivery into cells is a potentially transformative tool for treating "undruggable" targets in diseases associated with protein deficiencies or mutations. The vast majority of these targets are accessed via the cytosol, a challenging prospect for proteins with therapeutic and diagnostic relevance. In this review we will present promising non-viral approaches for intracellular and ultimately cytosolic delivery of proteins using nanocarriers. We will also discuss the mechanistic properties that govern the efficacy of nanocarrier-mediated protein delivery, applications of nanomaterials, and key challenges and opportunities in the use of nanocarriers for intracellular protein delivery.
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Citosol/metabolismo , Portadores de Fármacos/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Endocitosis , Nanopartículas/metabolismo , Proteínas/metabolismoRESUMEN
The adsorption of serum proteins on the surface of nanoparticles (NPs) delivered into a biological environment has been known to alter NP surface properties and consequently their targeting efficiency. In this paper, we use random copolymer (p(HEMA- ran-GMA))-based NPs synthesized using 2-hydroxyethyl methacrylate (HEMA) and glycidyl methacrylate (GMA). We show that serum proteins bind to the NP and that functionalization with antibodies and peptides designed to facilitate NP passage across the blood-brain barrier (BBB) to bind specific cell types is ineffective. In particular, we use systematic in vitro and in vivo analyses to demonstrate that p(HEMA- ran-GMA) NPs functionalized with HIV-1 trans-activating transcriptor peptide (known to cross the BBB) and α neural/glial antigen 2 (NG2) (known for targeting oligodendrocyte precursor cells (OPCs)), individually and in combination, do not specifically target OPCs and are unable to cross the BBB, likely due to the serum protein binding to the NPs.
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Barrera Hematoencefálica/metabolismo , Nanopartículas/química , Nanopartículas/metabolismo , Animales , Transporte Biológico/fisiología , Compuestos Epoxi/química , Femenino , Masculino , Metacrilatos/química , Microscopía Confocal , Células Precursoras de Oligodendrocitos/metabolismo , Polímeros/química , RatasRESUMEN
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has revolutionized therapeutic gene editing by providing researchers with a new method to study and cure diseases previously considered untreatable. While the full range and power of CRISPR technology for therapeutics is being elucidated through in vitro studies, translation to in vivo studies is slow. To date there is no totally effective delivery strategy to carry CRISPR components to the target site in vivo. The complexity of in vivo delivery is furthered by the number of potential delivery methods, the different forms in which CRISPR can be delivered as a therapeutic, and the disease target and tissue type in question. There are major challenges and limitations to delivery strategies, and it is imperative that future directions are guided by well-conducted studies that consider the full effect these variables have on the eventual outcome. In this review we will discuss the advances of the latest in vivo CRISPR/Cas9 delivery strategies and highlight the challenges yet to be overcome.
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Edición Génica , Terapia Genética/métodos , Sistemas CRISPR-Cas , HumanosRESUMEN
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.
RESUMEN
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.
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Núcleo Celular/genética , ADN/química , G-Cuádruplex , Oro/metabolismo , Nanotubos , Compuestos de Sulfhidrilo/metabolismo , Núcleo Celular/metabolismo , ADN/genética , Endocitosis , Regulación de la Expresión Génica , Oro/análisis , Células HEK293 , Humanos , Células MCF-7 , Nanotubos/análisis , Nanotubos/ultraestructura , Compuestos de Sulfhidrilo/análisisRESUMEN
The potential impact of CRISPR/Cas9, TALE, and zinc finger technology is immense, both with respect to their use as tools for understanding the roles and functions of the genomic elements and epigenome modifications in an endogenous context and as new methods for treatment of diseases. Application of such technologies has drawn attention, however, to the prevailing lack of effective delivery methods. Promising viral and non-viral methods both currently fall short when the efficient delivery of large plasmids or multiple plasmids is required. Therefore, the use of TALE and CRISPR platforms has been severely limited in applications where selection methods to increase the relative proportion of treated cells are not applicable, and it represents a significant bottleneck in the further application of these tools as therapeutics.The protocol presented here describes the synthesis of a dendronized polymer as a highly efficient and nontoxic transfection agent. Furthermore, the optimization of the polymer as a co-transfection reagent for large and multiple plasmids in cell lines is described, in addition to general considerations for co-transfection experiments. Usage of this method has allowed for significantly improved large plasmid co-transfection efficiency over Lipofectamine 2000 in multiple cell lines, allowing an improved delivery of CRISPR/dCas9 and TALE systems.
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Química Clic/métodos , Dendrímeros/síntesis química , Transfección/métodos , Animales , Azidas/síntesis química , Azidas/química , Sistemas CRISPR-Cas , Línea Celular , Dendrímeros/química , Halogenación , Humanos , Transfección/economíaRESUMEN
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.