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RNA vaccines elicit protective immunity against SARS-CoV-2, but the use of mRNA as an antiviral immunotherapeutic is unexplored. Here, we investigate the activity of lipidoid nanoparticle (LNP)-formulated mRNA encoding human IFNλ1 (ETH47), which is a critical driver of innate immunity at mucosal surfaces protecting from viral infections. IFNλ1 mRNA administration promotes dose-dependent protein translation, induction of interferon-stimulated genes without relevant signs of unspecific immune stimulation, and dose-dependent inhibition of SARS-CoV-2 replication in vitro. Pulmonary administration of IFNλ1 mRNA in mice results in a potent reduction of virus load, virus-induced body weight loss and significantly increased survival. These data support the development of inhaled administration of IFNλ1 mRNA as a potential prophylactic option for individuals exposed to SARS-CoV-2 or at risk suffering from COVID-19. Based on the broad antiviral activity of IFNλ1 regardless of virus or variant, this approach might also be utilized for other respiratory viral infections or pandemic preparedness.
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Wireless Body Area Networks (WBANs) are an emerging industrial technology for monitoring physiological data. These networks employ medical wearable and implanted biomedical sensors aimed at improving quality of life by providing body-oriented services through a variety of industrial sensing gadgets. The sensors collect vital data from the body and forward this information to other nodes for further services using short-range wireless communication technology. In this paper, we provide a multi-aspect review of recent advancements made in this field pertaining to cross-domain security, privacy, and trust issues. The aim is to present an overall review of WBAN research and projects based on applications, devices, and communication architecture. We examine current issues and challenges with WBAN communications and technologies, with the aim of providing insights for a future vision of remote healthcare systems. We specifically address the potential and shortcomings of various Wireless Body Area Network (WBAN) architectures and communication schemes that are proposed to maintain security, privacy, and trust within digital healthcare systems. Although current solutions and schemes aim to provide some level of security, several serious challenges remain that need to be understood and addressed. Our aim is to suggest future research directions for establishing best practices in protecting healthcare data. This includes monitoring, access control, key management, and trust management. The distinguishing feature of this survey is the combination of our review with a critical perspective on the future of WBANs.
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Redes de Comunicación de Computadores , Calidad de Vida , Atención a la Salud , Privacidad , Encuestas y Cuestionarios , Tecnología InalámbricaRESUMEN
Plants and other organisms, but not insects or vertebrates, express the auxiliary respiratory enzyme alternative oxidase (AOX) that bypasses mitochondrial respiratory complexes III and/or IV when impaired. Persistent expression of AOX from Ciona intestinalis in mammalian models has previously been shown to be effective in alleviating some metabolic stresses produced by respiratory chain inhibition while exacerbating others. This implies that chronic AOX expression may modify or disrupt metabolic signaling processes necessary to orchestrate adaptive remodeling, suggesting that its potential therapeutic use may be confined to acute pathologies, where a single course of treatment would suffice. One possible route for administering AOX transiently is AOX-encoding nucleic acid constructs. Here we demonstrate that AOX-encoding chemically-modified RNA (cmRNA), sequence-optimized for expression in mammalian cells, was able to support AOX expression in immortalized mouse embryonic fibroblasts (iMEFs), human lung carcinoma cells (A549) and primary mouse pulmonary arterial smooth muscle cells (PASMCs). AOX protein was detectable as early as 3 h after transfection, had a half-life of ~4 days and was catalytically active, thus supporting respiration and protecting against respiratory inhibition. Our data demonstrate that AOX-encoding cmRNA optimized for use in mammalian cells represents a viable route to investigate and possibly treat mitochondrial respiratory disorders.
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Mitocondrias , ARN , Animales , Humanos , Ratones , Fibroblastos/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , ARN/metabolismo , Células A549 , TransfecciónRESUMEN
Extensive research in the past decade has brought mRNA closer to the clinical realization of its therapeutic potential. One common structural feature for all cellular messenger RNAs is a poly(A) tail, which can either be brought in cotranscriptionally via the DNA template (plasmid- or PCR-based) or added to the mRNA in a post-transcriptional enzymatic process. Plasmids containing poly(A) regions recombine in E. coli, resulting in extensive shortening of the poly(A) tail. Using a segmented poly(A) approach, we could significantly reduce recombination of plasmids in E. coli without any negative effect on mRNA half-life and protein expression. This effect was independent of the coding sequence. A segmented poly(A) tail is characterized in that it consists of at least two A-containing elements, each defined as a nucleotide sequence consisting of 40-60 adenosines, separated by a spacer element of different length. Furthermore, reducing the spacer length between the poly(A) segments resulted in higher translation efficiencies compared to homogeneous poly(A) tail and reduced recombination (depending upon the choice of spacer nucleotide). Our results demonstrate the superior potential of segmented poly(A) tails compared to the conventionally used homogeneous poly(A) tails with respect to recombination of the plasmids and the resulting mRNA performance (half-life and translational efficiency).
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Ingeniería Genética/métodos , Plásmidos/química , Poli A/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , Células A549 , Animales , Secuencia de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Células HEK293 , Semivida , Humanos , Plásmidos/metabolismo , Poli A/metabolismo , ARN Mensajero/metabolismo , Recombinación Genética , TransfecciónRESUMEN
Promising improvements in the field of transcript therapeutics have clearly enhanced the potential of mRNA as a new pillar for protein replacement therapies. Synthetic mRNAs are engineered to replace mutated mRNAs and to be immunologically inconspicuous and highly stable while maximizing protein expression. Approaches to deliver mRNA into the cellular cytoplasm safely and efficiently have been further developed so that two mRNA-based approaches replacing vascular endothelial growth factor (VEGF) and cystic fibrosis transmembrane conductance regulator (CFTR) have now made it into clinical trials. These studies bring mRNA therapeutics for protein replacement therapy closer to clinical realization. Herein, we provide an overview of preclinical and clinical developments of mRNA therapeutics for liver diseases.
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Sistemas de Liberación de Medicamentos , Hepatopatías/terapia , ARN Mensajero/genética , ARN Mensajero/uso terapéutico , Animales , ADN/genética , ADN/uso terapéutico , Terapia de Reemplazo Enzimático/métodos , Humanos , Lípidos/química , Ratones , Nanopartículas/química , Polímeros/químicaRESUMEN
The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform that combines simplicity, inexpensive manufacture, and favorable safety features in the context of human applications. However, efficient correction of hematopoietic stem and progenitor cells (HSPCs) with non-viral vector systems, including SB, demands further refinement of gene delivery techniques. We set out to improve SB gene transfer into hard-to-transfect human CD34+ cells by vectorizing the SB system components in the form of minicircles that are devoid of plasmid backbone sequences and are, therefore, significantly reduced in size. As compared to conventional plasmids, delivery of the SB transposon system as minicircle DNA is â¼20 times more efficient, and it is associated with up to a 50% reduction in cellular toxicity in human CD34+ cells. Moreover, providing the SB transposase in the form of synthetic mRNA enabled us to further increase the efficacy and biosafety of stable gene delivery into hematopoietic progenitors ex vivo. Genome-wide insertion site profiling revealed a close-to-random distribution of SB transposon integrants, which is characteristically different from gammaretroviral and lentiviral integrations in HSPCs. Transplantation of gene-marked CD34+ cells in immunodeficient mice resulted in long-term engraftment and hematopoietic reconstitution, which was most efficient when the SB transposase was supplied as mRNA and nucleofected cells were maintained for 4-8 days in culture before transplantation. Collectively, implementation of minicircle and mRNA technologies allowed us to further refine the SB transposon system in the context of HSPC gene delivery to ultimately meet clinical demands of an efficient and safe non-viral gene therapy protocol.
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Elementos Transponibles de ADN , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Células Madre Hematopoyéticas/metabolismo , Animales , Supervivencia Celular , Citometría de Flujo , Expresión Génica , Humanos , Ratones , Ratones Noqueados , Retroviridae/genética , Transfección , TransgenesRESUMEN
Recently, chemically modified mRNA (cmRNA) therapeutics have been the subject of extensive application-oriented research in both academia and industry as a safer alternative for gene and recombinant protein therapies. However, the lack of an efficient delivery system hinders widespread application. Here we used â¼100-nm lipoplexes and magnetic lipoplexes that can protect cmRNA from RNases and efficiently deliver it into muscle and fat tissues as well as to the endothelium of the carotid artery. Establishing magnetofection for ex vivo cmRNA delivery for the first time, we suggest this method for potential enhanced and targeted delivery of cmRNA. This study introduces optimal cmRNA complexes with high ex vivo efficiency as good candidates for further in vivo cmRNA delivery.
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Lípidos/química , Magnetismo/métodos , Nanopartículas de Magnetita/química , ARN Mensajero/administración & dosificación , ARN Mensajero/química , Transfección/métodos , Tejido Adiposo/metabolismo , Animales , Células Endoteliales/metabolismo , Liposomas/química , Ratones , Músculos/metabolismo , Células 3T3 NIH , ARN Mensajero/genética , Ovinos , PorcinosRESUMEN
The development of chemically modified mRNA holds great promise as a new class of biologic therapeutics. However, the intracellular delivery and endosomal escape of mRNA encapsulated in nanoparticles has not been systematically investigated. Here, we synthesized a diverse set of cationic polymers and lipids from a series of oligoalkylamines and subsequently characterized their mRNA delivery capability. Notably, a structure with an alternating alkyl chain length between amines showed the highest transfection efficiency, which was linked to a high buffering capacity in a narrow range of pHâ 6.2 to 6.5. Variation in only one methylene group resulted in enhanced mRNA delivery to both the murine liver as well as porcine lungs after systemic or aerosol administration, respectively. These findings reveal a novel fundamental structure-activity relationship for the delivery of mRNA that is independent of the class of mRNA carrier and define a promising new path of exploration in the field of mRNA therapeutics.
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Aminas/química , Lípidos/química , Polímeros/química , ARN Mensajero/genética , Animales , Cationes/química , Ratones , Células 3T3 NIH , Relación Estructura-Actividad , PorcinosRESUMEN
BACKGROUND: Cystic fibrosis (CF) is the most frequent lethal genetic disease in the Caucasian population. CF is caused by a defective gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP- and ATP-dependent Cl(-) channel and central regulatory protein in epithelia. CFTR influences the fluid composition of the mucus in the respiratory tract. The most common mutation inducing CF, ΔF508, impairs CFTR processing within the cell and thus prevents functional CFTR expression in the apical membrane. The present study aimed to investigate the functional restoration of CFTR in human CF airway epithelia after transfection with optimized wild-type (wt)CFTR-mRNA. METHODS: We used primary cultured human nasal epithelial (HNE) cells and the human bronchial epithelial cell line CFBE41o(-) that stably expresses ΔF508-CFTR and carried out transepithelial Ussing chamber measurements after transfection with optimized wtCFTR-mRNA. We confirmed the data obtained using immunofluorescence and protein biochemical approaches. RESULTS: Transfection of the CFBE41o(-) cells with wtCFTR-mRNA restored cAMP-induced CFTR currents similar to the values seen in control cells (16HBE14o(-)). Using immunofluorescence approaches, we demonstrated that a considerable amount of CFTR is located at the apical surface in the CF cells after transfection. Western blot analyses of wtCFTR-mRNA transfected CFBE41o(-) cells confirmed these findings. Furthermore, we demonstrated physiological relevance by using primary cultured HNE cells and showed an almost two-fold increase in the cAMP-stimulated CFTR current after transfection. CONCLUSIONS: From these data, we conclude that CFTR-mRNA transfection could comprise a novel alternative for gene therapy to restore impaired CFTR function.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Técnicas de Transferencia de Gen , ARN Mensajero , Animales , Línea Celular , Fibrosis Quística/terapia , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/metabolismo , Femenino , Expresión Génica , Terapia Genética , Humanos , Oocitos/metabolismo , Cultivo Primario de Células , Transfección/métodos , Xenopus laevisRESUMEN
The susceptibility of deep neural networks (DNNs) to adversarial intrusions, exemplified by adversarial examples, is well-documented. Conventional attacks implement unstructured, pixel-wise perturbations to mislead classifiers, which often results in a noticeable departure from natural samples and lacks human-perceptible interpretability. In this work, we present an adversarial attack strategy that implements fine-granularity, semantic-meaning-oriented structural perturbations. Our proposed methodology manipulates the semantic attributes of images through the use of disentangled latent codes. We engineer adversarial perturbations by manipulating either a single latent code or a combination thereof. To this end, we propose two unsupervised semantic manipulation strategies: one based on vector-disentangled representation and the other on feature map-disentangled representation, taking into consideration the complexity of the latent codes and the smoothness of the reconstructed images. Our empirical evaluations, conducted extensively on real-world image data, showcase the potency of our attacks, particularly against black-box classifiers. Furthermore, we establish the existence of a universal semantic adversarial example that is agnostic to specific images.
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PURPOSE: Targeted delivery of aerosols could not only improve efficacy of inhaled drugs but also reduce side effects resulting from their accumulation in healthy tissue. Here we investigated the impact of magnetized aerosols on model drug accumulation and transgene expression in magnetically targeted lung regions of unanesthetized mice. METHODS: Solutions containing superparamagnetic iron oxide nanoparticles (SPIONs) and model drugs (fluorescein or complexed plasmid DNA) were nebulized to unanesthetized mice under the influence of an external magnetic gradient directed to the lungs. Drug accumulation and transgene expression was subsequently measured at different time points. RESULTS: We could demonstrate 2-3 fold higher accumulation of the model drug fluorescein and specific transgene expression in lung regions of mice which had been exposed to an external magnetic gradient during nebulization compared to the control mice without any exposure to magnetic gradient. CONCLUSIONS: Magnetized aerosols present themselves as an efficient approach for targeted pulmonary delivery of drugs and gene therapeutic agents in order to treat localized diseases of the deeper airways.
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Aerosoles/química , Sistemas de Liberación de Medicamentos , Compuestos Férricos , Técnicas de Transferencia de Gen , Pulmón/metabolismo , Magnetismo , Nanopartículas del Metal , Animales , Femenino , Fluoresceína/farmacocinética , Fluoresceína/farmacología , Regulación de la Expresión Génica , Vectores Genéticos/genética , Ratones , Ratones Endogámicos BALB C , Plásmidos/genética , Transgenes/genéticaRESUMEN
PURPOSE: Cationic polymers have been intensively investigated for plasmid-DNA (pDNA), but few studies addressed their use for messenger-RNA (mRNA) delivery. We analyzed two types of polymers, linear polyethylenimine (l-PEI) and poly-N,N-dimethylaminoethylmethacrylate P(DMAEMA), to highlight specific requirements for the design of mRNA delivery reagents. The effect of PEGylation was investigated using P(DMAEMA-co-OEGMA) copolymer. METHODS: The influence of polymer structure on mRNA binding and particle formation was assessed in a side-by-side comparison with pDNA by methods such as agarose-retardation assay and scanning probe microscopy. Transfection studies were performed on bronchial epithelial cells. RESULTS: Binding of cationic polymers inversely correlated with type of nucleic acid. Whereas P(DMAEMA) bound strongly to pDNA, only weak mRNA binding was observed, which was vice versa for l-PEI. Both polymers resulted in self-assembled nanoparticles forming pDNA complexes of irregular round shape; mRNA particles were significantly smaller and more distinct. Surprisingly, PEGylation improved mRNA binding and transfection efficiency contrary to observations made with pDNA. Co-transfections with free polymer improved mRNA transfection. CONCLUSIONS: Gene delivery requires tailor-made design for each type of nucleic acid. PEGylation influenced mRNA-polymer binding efficiency and transfection and may provide a method of further improving mRNA delivery.
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Portadores de Fármacos/química , Nanopartículas/química , Polietilenglicoles/química , Polietileneimina/química , Ácidos Polimetacrílicos/química , ARN Mensajero , Transfección , Línea Celular , Portadores de Fármacos/síntesis química , Electroforesis en Gel de Agar , Células Epiteliales/metabolismo , Hemaglutininas Virales/química , Humanos , Luciferasas/genética , Metacrilatos/química , Microscopía de Fuerza Atómica , Polietilenglicoles/síntesis química , Ácidos Polimetacrílicos/síntesis química , ARN Mensajero/administración & dosificación , ARN Mensajero/genética , Propiedades de SuperficieRESUMEN
Currently, messenger RNA (mRNA)-based lipid nanoparticle formulations revolutionize the clinical field. Cationic polymer-based complexes (polyplexes) represent an alternative compound class for mRNA delivery. After establishing branched polyethylenimine with a succinylation degree of 10% (succPEI) as highly effective positive mRNA transfection standard, a diverse library of PEI-like peptides termed sequence-defined oligoaminoamides (OAAs) was screened for mRNA delivery. Notably, sequences, which had previously been identified as potent plasmid DNA (pDNA) or small-interfering RNA (siRNA) carriers, displayed only moderate mRNA transfection activity. A second round of screening combined the cationizable building block succinoyl tetraethylene pentamine and histidines for endosomal buffering, tyrosine tripeptides and various fatty acids for mRNA polyplex stabilization, as well as redox-sensitive units for programmed intracellular release. For the tested OAA carriers, balancing of extracellular stability, endosomal lytic activity, and intracellular release capability was found to be of utmost importance for optimum mRNA transfection efficiency. OAAs with T-shape topology containing two oleic acids as well-stabilizing fatty acids, attached via a dynamic bioreducible building block, displayed superior activity with up to 1000-fold increased transfection efficiency compared to their non-reducible analogs. In the absence of the dynamic linkage, incorporation of shorter less stabilizing fatty acids could only partly compensate for mRNA delivery. Highest GFP expression and the largest fraction of transfected cells (96%) could be detected for the bioreducible OAA with incorporated histidines and a dioleoyl motif, outperforming all other tested carriers as well as the positive control succPEI. The good in vitro performance of the dynamic lead structure was verified in vivo upon intratracheal administration of mRNA complexes in mice.
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Técnicas de Transferencia de Gen , Nanopartículas , Animales , Liposomas , Ratones , Plásmidos , Polietileneimina , ARN Mensajero , TransfecciónRESUMEN
Therapies to treat patients infected with human immunodeficiency virus (HIV) aim at preventing viral replication but fail to eliminate the virus. Although transplantation of allogeneic CCR5Δ32 homozygous stem cell grafts provided a cure for a few patients, this approach is not considered a general therapeutic strategy because of potential side effects. Conversely, gene editing to disrupt the C-C chemokine receptor type 5 (CCR5) locus, which encodes the major HIV coreceptor, has shown to confer resistance to CCR5-tropic HIV strains. Here, an engineered transcription activator-like effector nuclease (TALEN) that enables efficient CCR5 editing in hematopoietic cells is presented. After transferring TALEN-encoding mRNA into primary CD4+ T cells, up to 89% of CCR5 alleles are disrupted. Genotyping confirms the genetic stability of the CCR5-edited cells, and genome-wide off-target analyses established the absence of relevant mutagenic events. When challenging the edited T cells with CCR5-tropic HIV, protection in a dose-dependent manner is observed. Functional assessments reveal no significant differences between edited and control cells in terms of proliferation and their ability to secrete cytokines upon exogenous stimuli. In conclusion, a highly active and specific TALEN to disrupt CCR5 is successfully engineered, paving the way for its clinical application in hematopoietic stem cell grafts.
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Infecciones por VIH , VIH-1 , Receptores CCR5 , Nucleasas de los Efectores Tipo Activadores de la Transcripción , Resistencia a la Enfermedad , Infecciones por VIH/genética , Infecciones por VIH/prevención & control , VIH-1/genética , Humanos , Receptores CCR5/genética , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genética , Nucleasas de los Efectores Tipo Activadores de la Transcripción/farmacología , Efectores Tipo Activadores de la TranscripciónRESUMEN
Oligo(ethylene glycol) methyl ether methacrylates (OEGMA) of various chain lengths (i.e., 9, 23, or 45 EG units) and N,N-dimethylaminoethyl methacrylate (DMAEMA) were copolymerized by atom transfer radical polymerization (ATRP), yielding well-defined P(DMAEMA-co-OEGMA) copolymers with increasing OEGMA molar fractions (F(OEGMA)) but a comparable degree of polymerization (DP approximately 120). Increase of both F(OEGMA) and OEGMA chain lengths correlated inversely with gene vector size, morphology, and zeta potential. P(DMAEMA-co-OEGMA) copolymers prevented gene vector aggregation at high plasmid DNA (pDNA) concentrations in isotonic solution and did not induce cytotoxicity even at high concentrations. Transfection efficiency of the most efficient P(DMAEMA-co-OEGMA) copolymers was found to be >10-fold lower compared with branched polyethylenimine (PEI) 25 kDa. Although OEGMA copolymerization largely reduced gene vector binding with the cell surface, cellular internalization of the bound complexes was less affected. These observations suggest that inefficient endolysosomal escape limits transfection efficiency of P(DMAEMA-co-OEGMA) copolymer gene vectors. Despite this observation, optimized p(DMAEMA-co-OEGMA) gene vectors remained stable under conditions for in vivo application leading to 7-fold greater gene expression in the lungs compared with PEI. Tailor-made P(DMAEMA-co-OEGMA) copolymers are promising nonviral gene transfer agents that fulfill the requirements for successful in vivo gene delivery.
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Portadores de Fármacos/química , Vectores Genéticos/administración & dosificación , Vectores Genéticos/farmacología , Metacrilatos/química , Metilmetacrilato/química , Polímeros/química , Animales , Bronquios/citología , Bronquios/metabolismo , Supervivencia Celular , ADN/genética , ADN/metabolismo , Electroforesis en Gel de Agar , Células Epiteliales/metabolismo , Femenino , Citometría de Flujo , Técnicas de Transferencia de Gen , Humanos , Luciferasas/metabolismo , Ratones , Ratones Endogámicos BALB C , Polietileneimina , Polímeros/síntesis química , Polímeros/metabolismo , Alveolos Pulmonares/citología , Alveolos Pulmonares/metabolismo , TransfecciónRESUMEN
Genomic integration by the Streptomyces bacteriophage C31 integrase is a promising tool for non-viral gene therapy of various genetic disorders. We investigated the C31 integrase recombination activity in T cell derived cell lines, primary T lymphocytes and CD34(+) haematopoietic stem cells in comparison to mesenchymal stem cells and cell lines derived from lung-, liver- and cervix-tissue. In T cell lines, enhanced long-term expression above control was observed only with high amounts of integrase mRNA. Transfections of C31 integrase plasmids were not capable of mediating enhanced long-term transgene expression in T cell lines. In contrast, moderate to high efficiency could be detected in human mesenchymal stem cells, human lung, liver and cervix carcinoma cell lines. Up to 100-fold higher levels of recombination product was found in C31 integrase transfected A549 lung than Jurkat T cells. When the C31 integrase activity was normalized to the intracellular integrase mRNA levels, a 16-fold difference was found. As one possible inhibitor of the C31 integrase, we found 3- to 5-fold higher DAXX levels in Jurkat than in A549 cells, which could in addition to other yet unknown factors explain the observed discrepancy of C31 integrase activity.
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Bacteriófagos/enzimología , Integrasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/análisis , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Proteínas Co-Represoras , Expresión Génica , Genes Reporteros , Humanos , Células Jurkat , Chaperonas Moleculares , Proteínas Nucleares/análisis , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Recombinación Genética , Streptomyces/virología , Linfocitos T/metabolismo , TransfecciónRESUMEN
BACKGROUND: Supercoiled topology of transfected plasmid DNA (pDNA) is critical for transgene expression in mammalian cells. In the present study, we analysed transgene expression of transfected supercoiled pDNA concatemers. METHODS: Jurkat T cells were transfected with a supercoiled 4.7-kb monomeric and, in parallel, a 9.4-kb dimeric pEGFP plasmid concatemer using electroporation. The absolute amounts of pDNA delivered into the cytoplasm and the nucleus were quantified by quantitative real-time polymerase chain reaction. Further, the number and mean fluorescent intensity (MFI) of enhanced green fluorescent protein (EGFP) expressing cells and the relative amounts of TOTO-1 fluorescently-labeled pDNA associated with the cell, located in the cytoplasm, and in the nucleus, were analysed by flow cytometry. RESULTS: For both constructs, significantly higher amounts of pDNA were detected in the cytoplasm compared to the nucleus. Furthermore, from FACS analysis, we could infer the relative gene copy (E(gene)) and plasmid expression efficiency (E(plasmid)) by determining the ratio of the EGFP MFI of the transfected cells to TOTO-1 MFI per nucleus on the single cell level. E(gene) and E(plasmid) were significantly 1.6-and 3.5-fold higher for EGFP-dimer than for EGFP-monomer, although the transfection rates considering the number of transfected cells were significantly lower for EGFP-dimer than for EGFP-monomer. Together with hydrodynamic plasmid diameter measurements, these observations suggest that concatemer arrangement increases relative gene expression efficiency, whereas plasmid size is important for cell and nucleus entry after electroporation. CONCLUSIONS: We propose using preferably small supercoiled plasmid concatemers as the ideal plasmid vectors to maximize both transgene expression and the number of transfected target cells.
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ADN Concatenado/genética , ADN Superhelicoidal/genética , Regulación de la Expresión Génica , Plásmidos/genética , Transfección , Transgenes/genética , Southern Blotting , Compartimento Celular , Dimerización , Electroporación , Citometría de Flujo , Fluorescencia , Dosificación de Gen , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Células Jurkat , Luz , Dispersión de RadiaciónRESUMEN
We investigated in different human cell types nuclear positioning and transcriptional regulation of the functionally unrelated genes GASZ, CFTR, and CORTBP2, mapping to adjacent loci on human chromosome 7q31. When inactive, GASZ, CFTR, and CORTBP2 preferentially associated with the nuclear periphery and with perinuclear heterochromatin, whereas in their actively transcribed states the gene loci preferentially associated with euchromatin in the nuclear interior. Adjacent genes associated simultaneously with these distinct chromatin fractions localizing at different nuclear regions, in accordance with their individual transcriptional regulation. Although the nuclear localization of CFTR changed after altering its transcription levels, the transcriptional status of CFTR was not changed by driving this gene into a different nuclear environment. This implied that the transcriptional activity affected the nuclear positioning, and not vice versa. Together, the results show that small chromosomal subregions can display highly flexible nuclear organizations that are regulated at the level of individual genes in a transcription-dependent manner.
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Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Proteínas del Tejido Nervioso/genética , Transcripción Genética , Proteínas Portadoras/metabolismo , Línea Celular , Cromatina/metabolismo , Cromosomas Humanos Par 7 , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Eucromatina/metabolismo , Regulación de la Expresión Génica , Heterocromatina/metabolismo , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Proteínas de Transporte de Membrana , Microscopía Confocal , Proteínas del Tejido Nervioso/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Enhancing gene delivery and expression in alveolar epithelial cells could offer the opportunity for the treatment of acquired and inherited lung diseases. Here, we show that particle adsorption of human insulin (INS) is capable of increasing plasmid DNA (pDNA) delivery from polyethylenimine (PEI) nanoparticles specifically in alveolar epithelial cells. INS receptors were predominantly detected on alveolar but not on bronchial epithelial cells. INS was adsorbed on the surface of PEI gene vectors by spontaneous self-assembly resulting in ternary PEI-pDNA-INS nanoparticles. Surface adsorption was confirmed by particle size, surface charge, and fluorescence resonance energy transfer (FRET) measurements. INS adsorption significantly increased gene expression of PEI-pDNA nanoparticles up to 16-fold on alveolar epithelial cells but not on bronchial epithelial cells. This increased gene expression was INS receptor specific. Our results demonstrate that targeting INS receptor for gene delivery in alveolar epithelial cells represents a promising approach for enhanced gene delivery and expression.