RESUMO
Mutations in either mitochondrial DNA (mtDNA) or nuclear genes that encode mitochondrial proteins may lead to dysfunctional mitochondria, giving rise to mitochondrial diseases. Some mitochondrial myopathies, however, present without a known underlying cause. Interestingly, methylation of mtDNA has been associated with various clinical pathologies. The present study set out to assess whether mtDNA methylation could explain impaired mitochondrial function in patients diagnosed with myopathy without known underlying genetic mutations. Enhanced mtDNA methylation was indicated by pyrosequencing for muscle biopsies of 14 myopathy patients compared to four healthy controls, at selected cytosines in the Cytochrome B (CYTB) gene, but not within the displacement loop (D-loop) region. The mtDNA methylation patterns of the four healthy muscle biopsies were highly consistent and showed intriguing tissue-specific differences at particular cytosines with control skin fibroblasts cultured in vitro. Within individual myopathy patients, the overall mtDNA methylation pattern correlated well between muscle and skin fibroblasts. Despite this correlation, a pilot analysis of four myopathy and five healthy fibroblast samples did not reveal a disease-associated difference in mtDNA methylation. We did, however, detect increased expression of solute carrier family 25A26 (SLC25A26), encoding the importer of S-adenosylmethionine, together with enhanced mtDNA copy numbers in myopathy fibroblasts compared to healthy controls. To confirm that pyrosequencing indeed reflected DNA methylation and not bisulfite accessibility, mass spectrometry was employed. Although no myopathy-related differences in total amount of methylated cytosines were detected at this stage, a significant contribution of contaminating nuclear DNA (nDNA) was revealed, and steps to improve enrichment for mtDNA are reported. In conclusion, in this explorative study we show that analyzing the mitochondrial genome beyond its sequence opens novel avenues to identify potential molecular biomarkers assisting in the diagnosis of unexplained myopathies.
Assuntos
Epigenoma , Doenças Musculares , Sistemas de Transporte de Aminoácidos/genética , Proteínas de Ligação ao Cálcio/metabolismo , Citosina/metabolismo , Metilação de DNA , DNA Mitocondrial/metabolismo , Humanos , Mitocôndrias/metabolismo , Doenças Musculares/genética , Doenças Musculares/metabolismoRESUMO
Increasing evidence indicates that active DNA demethylation is involved in several processes in mammals, resulting in developmental stage-specificity and cell lineage-specificity. The recently discovered Ten-Eleven Translocation (TET) dioxygenases are accepted to be involved in DNA demethylation by initiating 5-mC oxidation. Aberrant DNA methylation profiles are associated with many diseases. For example in cancer, hypermethylation results in silencing of tumor suppressor genes. Such silenced genes can be re-expressed by epigenetic drugs, but this approach has genome-wide effects. In this study, fusions of designer DNA binding domains to TET dioxygenase family members (TET1, -2 or -3) were engineered to target epigenetically silenced genes (ICAM-1, EpCAM). The effects on targeted CpGs' methylation and on expression levels of the target genes were assessed. The results indicated demethylation of targeted CpG sites in both promoters for targeted TET2 and to a lesser extent for TET1, but not for TET3. Interestingly, we observed re-activation of transcription of ICAM-1. Thus, our work suggests that we provided a mechanism to induce targeted DNA demethylation, which facilitates re-activation of expression of the target genes. Furthermore, this Epigenetic Editing approach is a powerful tool to investigate functions of epigenetic writers and erasers and to elucidate consequences of epigenetic marks.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Dioxigenases/metabolismo , Epigênese Genética , Molécula 1 de Adesão Intercelular/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , Animais , Antígenos de Neoplasias/genética , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Metilação de DNA , Molécula de Adesão da Célula Epitelial , Células HEK293 , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Camundongos , Ativação Transcricional , Dedos de ZincoRESUMO
Activated endothelial cells play a pivotal role in the pathology of inflammatory diseases and present a rational target for therapeutic intervention by endothelial specific delivery of short interfering RNAs (siRNA). This study demonstrates the potential of the recently developed new generation of liposomes based on cationic amphiphile SAINT-C18 (1-methyl-4-(cis-9-dioleyl)methyl-pyridinium-chloride) for functional and selective delivery of siRNA into inflamed primary endothelial cells. To create specificity for inflamed endothelial cells, these so-called SAINT-O-Somes were harnessed with antibodies against vascular cell adhesion protein 1 (VCAM-1) or respectively E-selectin and tested in TNF-α activated primary endothelial cells from venous and aortic vascular beds. Both targeted SAINT-O-Somes carrying siRNA against the endothelial gene VE-cadherin specifically downregulated its target mRNA and protein without exerting cellular toxicity. SAINT-O-Somes formulated with siRNA formed small particles (106 nm) with a 71% siRNA encapsulation efficiency. SAINT-O-Somes were stable in the presence of serum at 37 °C, protected siRNA from degradation by serum RNases, and after i.v. injection displayed pharmacokinetic comparable to conventional long circulating liposomes. These anti-VCAM-1 and anti-E-selectin SAINT-O-Somes are thus a novel drug delivery system that can achieve specific and effective delivery of siRNA into inflamed primary endothelial cells and have physicochemical features that comply with in vivo application demands.
Assuntos
Selectina E/imunologia , Inflamação/metabolismo , RNA Interferente Pequeno/administração & dosagem , Molécula 1 de Adesão de Célula Vascular/imunologia , Animais , Anticorpos , Células Endoteliais , Células Endoteliais da Veia Umbilical Humana , Humanos , Lipossomos/química , Masculino , Camundongos , Tamanho da Partícula , RNA Interferente Pequeno/química , Fatores de Necrose Tumoral/metabolismoRESUMO
Increased insight in the role of endothelial cells in the pathophysiology of cancer, inflammatory and cardiovascular diseases, has drawn great interest in pharmacological interventions aiming at the endothelium in diseased sites. Their location in the body makes them suitable targets for therapeutic approaches based on targeted drug delivery. Functional heterogeneity of the microvascular bed in normal organ homeostasis has been appreciated for a long time, and more recent studies have revealed heterogeneity in endothelial reactivity to inflammatory stimuli as well. Upon stimulation, each organ displays a vascular bed specific pattern of cell adhesion molecules providing challenging opportunities to deliver drugs or small RNAs to organ specific (micro)vascular endothelial subsets. In this review we introduce general concepts of endothelial heterogeneity in relation to disease state and its consequences for targeted therapeutic interventions. Furthermore, we will describe novel approaches to interfere with endothelial cell engagement in disease with a main focus on siRNA therapeutics and currently used nonviral lipid and polymer-based siRNA delivery systems. The last part of this review addresses some technical issues that are essential in proving the concept of target mRNA knock down in a vascular bed specific manner, and the further development of effective endothelial cell specific drug delivery devices.
Assuntos
Endotélio Vascular/metabolismo , Microvasos/metabolismo , RNA Interferente Pequeno/administração & dosagem , Endotélio Vascular/citologia , Humanos , Microvasos/citologiaRESUMO
The epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein that is highly expressed on most carcinomas and therefore of potential use as a diagnostic and prognostic marker for a variety of carcinomas. Interestingly, EpCAM is explored as target in antibody-based therapies. Recently, EpCAM has been identified as an additional marker of cancer-initiating cells. In this review, we describe the controversial biological role of EpCAM with the focus on carcinogenesis: as an adhesion molecule, EpCAM mediates homophilic adhesion interactions, which in turn might prevent metastasis. On the other hand, EpCAM abrogates E-cadherin mediated cell-cell adhesion thereby promoting metastasis. Also, upon cleavage of EpCAM, the intracellular domain functions as a part of a transcriptional complex inducing c-myc and cyclin A and E. In line with these seemingly controversial roles, EpCAM overexpression has been associated with both decreased and increased survival of patients. Similarly, either induction or downregulation of EpCAM expression lowers the oncogenic potential depending on the cell type. As epigenetic dysregulation underlies aberrant EpCAM expression, we propose epigenetic editing as a novel approach to investigate the biological role of EpCAM, expanding the options for EpCAM as a therapeutic target in cancer.
Assuntos
Antígenos de Neoplasias/fisiologia , Moléculas de Adesão Celular/fisiologia , Neoplasias/metabolismo , Transformação Celular Neoplásica , Epigênese Genética , Molécula de Adesão da Célula Epitelial , HumanosRESUMO
The epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein that has been identified as a marker of cancer-initiating cells. EpCAM is highly expressed on most carcinomas, and transient silencing of EpCAM expression leads to reduced oncogenic potential. To silence the EpCAM gene in a persistent manner via targeted DNA methylation, a low activity mutant (C141S) of the CpG-specific DNA methyltransferase M.SssI was coupled to a triple-helix-forming oligonucleotide (TFO-C141S) specifically designed for the EpCAM gene. Reporter plasmids encoding the green fluorescent protein under control of different EpCAM promoter fragments were treated with the TFO-C141S conjugate to determine the specificity of targeted DNA methylation in the context of a functional EpCAM promoter. Treatment of the plasmids with TFO-C141S resulted in efficient and specific methylation of the targeted CpG located directly downstream of the triple helix forming site (TFS). No background DNA methylation was observed neither in a 700 bp region of the EpCAM promoter nor in a 400 bp region of the reporter gene downstream of the TFS. Methylation of the target CpG did not have a detectable effect on promoter activity. This study shows that the combination of a specific TFO and a reduced activity methyltransferase variant can be used to target DNA methylation to predetermined sites with high specificity, allowing determination of crucial CpGs for promoter activity.
Assuntos
Moléculas de Adesão Celular/metabolismo , Metilação de DNA/efeitos dos fármacos , Metilases de Modificação do DNA/metabolismo , DNA/efeitos dos fármacos , Células Epiteliais/metabolismo , Oligonucleotídeos/farmacologia , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , DNA/genética , Metilação de DNA/genética , Metilases de Modificação do DNA/química , Regulação para Baixo/efeitos dos fármacos , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Plasmídeos/genética , Regiões Promotoras Genéticas/genéticaRESUMO
The epithelial cell adhesion molecule (EpCAM) is expressed at high levels on the surface of most carcinoma cells. SiRNA silencing of EpCAM expression leads to reduced metastatic potential of tumor cells demonstrating its importance in oncogenesis and tumor progression. However, siRNA therapy requires either sequential delivery or integration into the host cell genome. Hence we set out to explore a more definite form to influence EpCAM gene expression. The mechanisms underlying the transcriptional activation of the EpCAM gene, both in normal epithelial tissue as well as in carcinogenesis, are poorly understood. We show that DNA methylation plays a crucial role in EpCAM expression, and moreover, active silencing of endogenous EpCAM via methylation of the EpCAM promoter results in a persistent downregulation of EpCAM expression. In a panel of carcinoma derived cell lines, bisulfite analyses showed a correlation between the methylation status of the EpCAM promoter and EpCAM expression. Treatment of EpCAM-negative cell lines with a demethylating agent induced EpCAM expression, both on mRNA and protein level, and caused upregulation of EpCAM expression in an EpCAM-positive cell line. After delivery of the DNA methyltransferase M.SssI into EpCAM-positive ovarian carcinoma cells, methylation of the EpCAM promoter resulted in silencing of EpCAM expression. SiRNA-mediated silencing remained for 4 days, after which EpCAM re-expression increased in time, while M.SssI-mediated downregulation of EpCAM maintained through successive cell divisions as the repression persisted for at least 17 days. This is the first study showing that active DNA methylation leads to sustained silencing of endogenous EpCAM expression.
Assuntos
Antígenos de Neoplasias/metabolismo , Carcinoma/metabolismo , Moléculas de Adesão Celular/metabolismo , Núcleo Celular/metabolismo , Metilação de DNA , Antígenos de Neoplasias/genética , Antineoplásicos/farmacologia , Azacitidina/farmacologia , Carcinoma/tratamento farmacológico , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , DNA-Citosina Metilases/metabolismo , Regulação para Baixo , Molécula de Adesão da Célula Epitelial , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica/efeitos dos fármacos , Humanos , Neoplasias Ovarianas/metabolismo , Regiões Promotoras Genéticas , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transcrição Gênica , Regulação para Cima/efeitos dos fármacosRESUMO
Microvascular endothelial cells play a pivotal role in the pathogenesis of sepsis-induced inflammatory responses and multiple organ failure. Therefore, they represent an important target for pharmacological intervention in the treatment of sepsis. Glucocorticosteroids were widely used in the treatment of sepsis but vast evidence to support their systemic use is lacking. The limited effects of glucocorticoids in the treatment of sepsis may be explained by differential effects of drug initiated NF-κB inhibition in different cell types and insufficient drug delivery in target cells. The current study aimed therefore to investigate the effects of an endothelial targeted delivery of dexamethasone in a mouse model of endotoxemia induced by two consecutive i.p. injections of lipopolysaccharide (LPS). To achieve endothelial cell specific delivery of dexamethasone, we modified SAINT-O-Somes, a new generation of liposomes that contain the cationic amphiphile SAINT-C18 (1-methyl-4-(cis-9-dioleyl) methyl-pyridinium chloride, with antibodies against vascular cell adhesion molecule-1 (VCAM-1). In LPS challenged mice, the systemic administration of free dexamethasone had negligible effects on the microvascular inflammatory endothelial responses. Dexamethasone-loaded anti-VCAM-1 SAINT-O-Somes specifically localized at VCAM-1 expressing endothelial cells in the microvasculature of inflamed organs. This was associated with a marginal attenuation of the expression of a few pro-inflammatory genes in kidney and liver, while no effects in the lung were observed. This study reveals that, although local accumulation of the targeted drug was achieved, endothelial targeted dexamethasone containing anti-VCAM-1 SAINT-O-Somes exhibited marginal effects on inflammatory endothelial cell activation in a model of endotoxemia. Studies with more potent drugs encapsulated into anti-VCAM-1 SAINT-O-Somes will in the future reveal whether this delivery system can be further developed for efficacious endothelial directed delivery of drugs in the treatment of sepsis.
Assuntos
Anticorpos/farmacologia , Dexametasona/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Endotélio Vascular/metabolismo , Endotoxemia , Molécula 1 de Adesão de Célula Vascular/metabolismo , Animais , Modelos Animais de Doenças , Endotélio Vascular/patologia , Endotoxemia/induzido quimicamente , Endotoxemia/tratamento farmacológico , Endotoxemia/metabolismo , Endotoxemia/patologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Lipopolissacarídeos/toxicidade , Masculino , CamundongosRESUMO
Like the nucleus, mitochondria contain their own DNA and recent reports provide accumulating evidence that also the mitochondrial DNA (mtDNA) is subjective to DNA methylation. This evidence includes the demonstration of mitochondria-localised DNA methyltransferases and demethylases, and the detection of mtDNA methylation as well as hydroxymethylation. Importantly, differential mtDNA methylation has been linked to aging and diseases, including cancer and diabetes. However, functionality of mtDNA methylation has not been demonstrated. Therefore, we targeted DNA methylating enzymes (modifying cytosine in the CpG or GpC context) to the mtDNA. Unexpectedly, mtDNA gene expression remained unchanged upon induction of CpG mtDNA methylation, whereas induction of C-methylation in the GpC context decreased mtDNA gene expression. Intriguingly, in the latter case, the three mtDNA promoters were differentially affected in each cell line, while cellular function seemed undisturbed. In conclusion, this is the first study which directly addresses the potential functionality of mtDNA methylation. Giving the important role of mitochondria in health and disease, unravelling the impact of mtDNA methylation adds to our understanding of the role of mitochondria in physiological and pathophysiological processes.
Assuntos
Metilação de DNA , DNA Mitocondrial/química , Proteínas Mitocondriais/genética , Linhagem Celular , DNA Mitocondrial/genética , Sequência Rica em GC , Regulação da Expressão Gênica , Células HCT116 , Células HEK293 , HumanosRESUMO
Histone modifications reflect gene activity, but the relationship between cause and consequence of transcriptional control is heavily debated. Recent developments in rewriting local histone codes of endogenous genes elucidated instructiveness of certain marks in regulating gene expression. Maintenance of such repressive epigenome editing is controversial, while stable reactivation is still largely unexplored. Here we demonstrate sustained gene re-expression using two types of engineered DNA-binding domains fused to a H3K4 methyltransferase. Local induction of H3K4me3 is sufficient to allow re-expression of silenced target genes in various cell types. Maintenance of the re-expression is achieved, but strongly depends on the chromatin microenvironment (that is, DNA methylation status). We further identify H3K79me to be essential in allowing stable gene re-expression, confirming its role in epigenetic crosstalk for stable reactivation. Our approach uncovers potent epigenetic modifications to be directly written onto genomic loci to stably activate any given gene.
Assuntos
Metilação de DNA/genética , Inativação Gênica , Histonas/genética , Ativação Transcricional , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Engenharia Genética/métodos , Histonas/metabolismo , Humanos , Metiltransferases/genética , Metiltransferases/metabolismo , Domínios Proteicos/genéticaRESUMO
Dendritic cell (DC)-based vaccination is an appealing strategy to boost graft-versus-tumor immunity after allogeneic stem cell transplantation (allo-SCT), and thereby prevent or counteract tumor recurrence. By exploiting minor histocompatibility antigens (MiHA) presented on hematopoietic cells, donor CD8 T-cell immunity can be selectively targeted to patient's hematological tumor cells without the risk of inducing graft-versus-host disease. Previously, we demonstrated that silencing RNA (siRNA) of programmed death-ligand 1 (PD-L1) and PD-L2 on DCs markedly augments the expansion and function of MiHA-specific CD8 T cells. However, previously applied methods based on electroporation or lipid nanoparticles were either incompatible with target antigen mRNA delivery or required complex manufacturing compliant to Good Manufacturing Practice. Here, we investigated whether transfection using lipoplexes composed of PD-L1 and PD-L2 siRNAs plus SAINT-18:DOPE (ie, SAINT-RED) is an effective and feasible clinical-grade method in DC vaccine manufacturing. We observed that a single siRNA/SAINT-RED transfection resulted in efficient and long-term knockdown of the PD-1 ligands without affecting DC maturation or viability. Furthermore, we demonstrated that SAINT-RED can be heat sterilized without loss of function, facilitating its use in aseptic DC vaccine production. Finally, we showed that the established transfection method can be combined with target antigen mRNA or peptide loading to efficiently stimulate MiHA-specific T-cell expansion and cytokine production. Together, these findings indicate that the developed PD-L siRNA/SAINT-RED transfection protocol in combination with MiHA mRNA or peptide loading can be applied in the generation of clinical-grade DC vaccines to boost antitumor immunity after allo-SCT.
Assuntos
Antígeno B7-H1/antagonistas & inibidores , Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Transplante de Células-Tronco Hematopoéticas , Neoplasias/terapia , Proteína 2 Ligante de Morte Celular Programada 1/antagonistas & inibidores , RNA Interferente Pequeno/genética , Transfecção/métodos , Antígeno B7-H1/genética , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Células Clonais , Citocinas/metabolismo , Efeito Enxerto vs Tumor/genética , Humanos , Antígenos de Histocompatibilidade Menor/imunologia , Proteína 2 Ligante de Morte Celular Programada 1/genética , Compostos de Piridínio/química , Transplante HomólogoRESUMO
Together with mesangial cells, glomerular endothelial cells and the basement membrane, podocytes constitute the glomerular filtration barrier (GFB) of the kidney. Podocytes play a pivotal role in the progression of various kidney-related diseases such as glomerular sclerosis and glomerulonephritis that finally lead to chronic end-stage renal disease. During podocytopathies, the slit-diaphragm connecting the adjacent podocytes are detached leading to severe loss of proteins in the urine. The pathophysiology of podocytopathies makes podocytes a potential and challenging target for nanomedicine development, though there is a lack of known molecular targets for cell selective drug delivery. To identify VCAM-1 as a cell-surface receptor that is suitable for binding and internalization of nanomedicine carrier systems by podocytes, we investigated its expression in the immortalized podocyte cell lines AB8/13 and MPC-5, and in primary podocytes. Gene and protein expression analyses revealed that VCAM-1 expression is increased by podocytes upon TNFα-activation for up to 24 h. This was paralleled by anti-VCAM-1 antibody binding to the TNFα-activated cells, which can be employed as a ligand to facilitate the uptake of nanocarriers under inflammatory conditions. Hence, we next explored the possibilities of using VCAM-1 as a cell-surface receptor to deliver the potent immunosuppressant rapamycin to TNFα-activated podocytes using the lipid-based nanocarrier system Saint-O-Somes. Anti-VCAM-1-rapamycin-SAINT-O-Somes more effectively inhibited the cell migration of AB8/13 cells than free rapamycin and non-targeted rapamycin-SAINT-O-Somes indicating the potential of VCAM-1 targeted drug delivery to podocytes.
Assuntos
Lipídeos , Nanoconjugados , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Sirolimo/administração & dosagem , Molécula 1 de Adesão de Célula Vascular/metabolismo , Animais , Anticorpos Monoclonais , Diferenciação Celular , Linhagem Celular , Expressão Gênica , Humanos , Imunossupressores/administração & dosagem , Inflamação/metabolismo , Masculino , Camundongos , Podócitos/citologia , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
Interference with acute and chronic inflammatory processes by means of delivery of siRNAs into microvascular endothelial cells at a site of inflammation demands specific, non-toxic and effective siRNA delivery system. In the current work we describe the design and characterization of siRNA carriers based on cationic pyridinium-derived lipid 1-methyl-4-(cis-9-dioleyl)methyl-pyridinium-chloride) (SAINT-C18) and the transfection enhancer protamine, complexed with siRNA/carrier DNA or siRNA only. These carriers, called SAINT-liposome-polycation-DNA (S-LPD) and SAINT-liposome-polycation (S-LP), have a high efficiency of siRNA encapsulation, low cellular toxicity, and superior efficacy of gene downregulation in endothelial cells in vitro as compared to DOTAP-LPD. Incorporation of 10 mol% PEG and anti-E-selectin antibody in these formulations resulted in selective siRNA delivery into activated endothelial cells. Furthermore, we showed that the physicochemical characteristics of S-LPD and S-LP, including size-stability and maintenance of the siRNA integrity in the presence of serum at 37 °C, comply with requirements for in vivo application.
Assuntos
Portadores de Fármacos/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Inflamação/tratamento farmacológico , Lipossomos/farmacologia , Poliaminas/farmacologia , Compostos de Piridínio/farmacologia , RNA Interferente Pequeno/farmacologia , Células Cultivadas , Química Farmacêutica/métodos , DNA/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Selectina E/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inflamação/metabolismo , Lipídeos/farmacologia , Tamanho da Partícula , Polieletrólitos , Transfecção/métodosRESUMO
EGP-2, also known as Ep-CAM, is expressed at high levels on the surface of most carcinomas and is therefore considered an attractive target for anticancer strategies. To explore the mechanisms regulating the expression of EGP-2, sequences 3.4 kb upstream of the transcription start site were isolated and assayed for their ability to control the expression of the EGP-2 cDNA, the green fluorescent protein, the luciferase reporter gene and the thymidine kinase and cytosine deaminase suicide genes. Expression of these chimeric constructs as assessed in a range of different cell lines was restricted to cell lines expressing EGP-2. In addition, only cells expressing EGP-2 were sensitive for gancyclovir after being transiently transfected with EGP-2 promoter-driven thymidine kinase. Deletion analyses defined 687 bp upstream as the basic proximal promoter region, which could confer epithelial-specific expression to the GFP reporter gene in vitro. As these EGP-2 sequences can confer promoter activity to reporter and suicide genes in an EGP-2 restricted manner, they may be useful for gene therapy of EGP-2 expressing carcinomas.
Assuntos
Antígenos de Neoplasias/genética , Moléculas de Adesão Celular/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Neoplasias/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/metabolismo , Animais , Antígenos de Neoplasias/metabolismo , Antivirais/farmacologia , Sequência de Bases , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Citosina Desaminase/metabolismo , Molécula de Adesão da Célula Epitelial , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Ganciclovir/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Luciferases/metabolismo , Camundongos , Dados de Sequência Molecular , Neoplasias/genética , Neoplasias/patologia , Deleção de Sequência , Timidina Quinase/metabolismo , Sítio de Iniciação de Transcrição , TransfecçãoRESUMO
In recent years much research in RNA nanotechnology has been directed to develop an efficient and clinically suitable delivery system for short interfering RNA (siRNA). The current study describes the in vivo siRNA delivery using PEGylated antibody-targeted SAINT-based-lipoplexes (referred to as antibody-SAINTPEGarg/PEG2%), which showed superior siRNA delivery capacity and effective down-regulation of VE-cadherin gene expression in vitro in inflammation-activated primary endothelial cells of different vascular origins. PEGylation of antibody-SAINTPEGarg resulted in more desirable pharmacokinetic behavior than that of non-PEGylated antibody-SAINTPEGarg. To create specificity for inflammation-activated endothelial cells, antibodies against vascular cell adhesion molecule-1 (VCAM-1) were employed. In TNFα-challenged mice, these intravenously administered anti-VCAM-1-SAINTPEGarg/PEG2% homed to VCAM-1 protein expressing vasculature. Confocal laser scanning microscopy revealed that anti-VCAM-1-SAINTPEGarg/PEG2% co-localized with endothelial cells in lung postcapillary venules. Furthermore, they did not exert any liver and kidney toxicity. Yet, lack of in vivo gene silencing as assessed in whole lung and in laser microdissected lung microvascular segments indicates that in vivo internalization and/or intracellular trafficking of the delivery system and its cargo in the target cells are not sufficient, and needs further attention, emphasizing the essence of evaluating siRNA delivery systems in an appropriate in vivo animal model at an early stage in their development.
Assuntos
Anticorpos/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Endotélio Vascular/metabolismo , Inflamação/metabolismo , Lipídeos/química , Pulmão/irrigação sanguínea , Polietilenoglicóis/química , Compostos de Piridínio/química , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transfecção/métodos , Molécula 1 de Adesão de Célula Vascular/metabolismo , Animais , Anticorpos/química , Antígenos CD/genética , Caderinas/genética , Modelos Animais de Doenças , Endotélio Vascular/imunologia , Regulação da Expressão Gênica , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/prevenção & controle , Masculino , Camundongos Endogâmicos C57BL , Microscopia Confocal , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Fatores de Tempo , Distribuição Tecidual , Fator de Necrose Tumoral alfa , Molécula 1 de Adesão de Célula Vascular/imunologia , Vênulas/metabolismoRESUMO
We have previously shown that the combination of MIDGE-Th1 DNA vectors with the cationic lipid SAINT-18 increases the immune response to the encoded antigen in mice. Here, we report on experiments to further optimize and characterize this approach. We evaluated different formulations of MIDGE-Th1 vectors with SAINT-18 by assessing their influence on the transfection efficiency in cell culture and on the immune response in mice. We found that high amounts of SAINT-18 in formulations with a w/w ratio MIDGE Th1/SAINT-18 of 1:4.8 are beneficial for cell transfection in vitro. In contrast, the formulation of HBsAg-encoding MIDGE-Th1 DNA vectors with the lowest amount of SAINT-18 (w/w ratio MIDGE Th1/SAINT-18 of 1:0.5) resulted in the highest serum IgG1 and IgG2a levels after intradermal immunization of mice. Consequently, latter formulation was selected for a comparative biodistribution study in rats. Following intradermal administration of both naked and formulated MIDGE-Th1 DNA, the vectors localized primarily at the site of injection. Vector DNA levels decreased substantially over the two months duration of the study. When administered in combination with SAINT-18, the vectors were found in significantly higher amounts in draining lymph nodes in comparison to administration of naked MIDGE-Th1 DNA. We propose that the high immune responses induced by MIDGE-Th1/SAINT-18 lipoplexes are mediated by enhanced transfection of cells in vivo, resulting in stronger antigen expression and presentation. Importantly, the combination of MIDGE-Th1 vectors with SAINT-18 was well tolerated in mice and rats and is expected to be safe in human clinical applications.
Assuntos
Vetores Genéticos/química , Compostos de Piridínio/química , Vacinas de DNA/imunologia , Animais , Anticorpos Antivirais/sangue , Cátions , Feminino , Vetores Genéticos/farmacocinética , Antígenos de Superfície da Hepatite B/imunologia , Imunoglobulina G/sangue , Linfonodos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Compostos de Piridínio/farmacocinética , Ratos , Ratos Wistar , Células Th1/imunologia , Distribuição Tecidual , Transfecção , Vacinas de DNA/farmacocinéticaRESUMO
Currently marketed vaccines against hepatitis B virus (HBV) based on the small (S) hepatitis B surface antigen (HBsAg) fail to induce a protective immune response in about 10% of vaccinees. DNA vaccination and the inclusion of PreS1 and PreS2 domains of HBsAg have been reported to represent feasible strategies to improve the efficacy of HBV vaccines. Here, we evaluated the immunogenicity of SAINT-18-formulated MIDGE-Th1 vectors encoding the S or the large (L) protein of HBsAg in mice and pigs. In both animal models, vectors encoding the secretion-competent S protein induced stronger humoral responses than vectors encoding the L protein, which was shown to be retained mainly intracellularly despite the presence of a heterologous secretion signal. In pigs, SAINT-18-formulated MIDGE-Th1 vectors encoding the S protein elicited an immune response of the same magnitude as the licensed protein vaccine Engerix-B, with S protein-specific antibody levels significantly higher than those considered protective in humans, and lasting for at least six months after the third immunization. Thus, our results provide not only the proof of concept for the SAINT-18-formulated MIDGE-Th1 vector approach but also confirm that with a cationic-lipid formulation, a DNA vaccine at a relatively low dose can elicit an immune response similar to a human dose of an aluminum hydroxide-adjuvanted protein vaccine in large animals.
Assuntos
Vetores Genéticos/química , Antígenos de Superfície da Hepatite B/imunologia , Compostos de Piridínio/química , Células Th1/imunologia , Vacinas de DNA/imunologia , Animais , Células CHO , Cátions , Cricetulus , Feminino , Vacinas contra Hepatite B/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , SuínosRESUMO
The pivotal role of endothelial cells in the pathology of inflammatory diseases raised interest in the development of short interfering RNA (siRNA) delivery devices for selective pharmacological intervention in the inflamed endothelium. The current study demonstrates endothelial specific delivery of siRNAs and downregulation of inflammatory genes in activated endothelium in vivo by applying a novel type of targeted liposomes based on the cationic amphiphile SAINT-C18 (1-methyl-4-(cis-9-dioleyl)methyl-pyridinium-chloride). To create specificity for inflamed endothelial cells, these so-called SAINT-O-Somes were harnessed with antibodies against vascular cell adhesion protein 1 (VCAM-1). In TNFα challenged mice, intravenously administered anti-VCAM-1 SAINT-O-Somes exerted long circulation times and homed to VCAM-1 expressing endothelial cells in inflamed organs. The formulations were devoid of liver and kidney toxicity. Using anti-VCAM-1 SAINT-O-Somes we successfully delivered siRNA to knock down VE-cadherin mRNA in inflamed renal microvasculature, as demonstrated by using laser microdissection of different microvascular beds prior to analysis of gene expression. Using the same strategy, we demonstrated local attenuation of endothelial inflammatory response towards lipopolysaccharide in kidneys of mice treated with anti-VCAM-1 SAINT-O-Somes containing NFκB p65 specific siRNA. This study is the first demonstration of a novel, endothelial specific carrier that is suitable for selective in vivo delivery of siRNAs into inflamed microvascular segments and interference with disease associated endothelial activation.
Assuntos
Anticorpos/administração & dosagem , Antígenos CD/genética , Caderinas/genética , Compostos de Piridínio/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Fator de Transcrição RelA/genética , Molécula 1 de Adesão de Célula Vascular/imunologia , Animais , Encéfalo/metabolismo , Células Cultivadas , Regulação para Baixo , Endotélio Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Inflamação/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Lipossomos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Pulmão/metabolismo , Masculino , Camundongos , Miocárdio/metabolismo , Compostos de Piridínio/farmacocinética , RNA Interferente Pequeno/farmacocinética , Distribuição Tecidual , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
The endothelium represents an attractive therapeutic target due to its pivotal role in many diseases including chronic inflammation and cancer. Small interfering RNAs (siRNAs) specifically interfere with the expression of target genes and are considered an important new class of therapeutics. However, due to their size and charge, siRNAs do not spontaneously enter unperturbed endothelial cells (EC). To overcome this problem, we developed novel lipoplexes for siRNA delivery that are based on the cationic amphiphilic lipid SAINT-C18. Antibodies recognizing disease induced cell adhesion molecules were employed to create cell specificity resulting in so-called antibody-SAINTargs. To improve particle stability, antibody-SAINTargs were further optimized for EC-specific siRNA-mediated gene silencing by addition of polyethylene glycol (PEG). Although PEGylated antibody-SAINTargs maintained specificity, they lost their siRNA delivery capacity. Coupling of antibodies to the distal end of PEG (so-called antibody-SAINTPEGargs), resulted in anti-E-selectin- and anti-vascular cell adhesion molecule (VCAM)-1-SAINTPEGarg that preserved their antigen recognition and their capability to specifically deliver siRNA into inflammation-activated primary endothelial cells. The enhanced uptake of siRNA by antibody-SAINTPEGargs was followed by improved silencing of the target gene VE-cadherin, demonstrating that antibody-SAINTPEGargs were capable of functionally delivering siRNA into primary endothelial cells originating from different vascular beds. In conclusion, the newly developed, physicochemically stable, and EC-specific siRNA carrying antibody-SAINTPEGargs selectively down-regulate target genes in primary endothelial cells that are generally difficult to transfect.
Assuntos
Selectina E/química , Células Endoteliais/patologia , Endotélio Vascular/patologia , Inflamação/patologia , Lipídeos/química , Polietilenoglicóis/química , Compostos de Piridínio/química , RNA Interferente Pequeno/administração & dosagem , Molécula 1 de Adesão de Célula Vascular/química , Capilares/citologia , Células Cultivadas , Sistemas de Liberação de Medicamentos , Eletroquímica , Citometria de Fluxo , Expressão Gênica , Técnicas de Transferência de Genes , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Tamanho da Partícula , Reação em Cadeia da Polimerase em Tempo Real , TransfecçãoRESUMO
BACKGROUND: Podocytes are uniquely structured cells that are critical to the kidney filtration barrier. Their anatomic location on the outer side of the glomerular capillaries expose podocytes to large quantities of both plasma and urinary components and thus are reachable for drug delivery. Recent years have made clear that interference with podocyte-specific disease pathways can modulate glomerular function and influence severity and progression of glomerular disease. METHODOLOGY/PRINCIPAL FINDINGS: Here, we describe studies that show efficient transport of proteins into the mammalian cells mouse 3T3 fibroblasts and podocytes, utilizing an approach termed profection. We are using synthetic lipid structures that allow the safe packing of proteins or antibodies resulting in the subsequent delivery of protein into the cell. The uptake of lipid coated protein is facilitated by the intrinsic characteristic of cells such as podocytes to engulf particles that are physiologically retained in the extracellular matrix. Profection of the restriction enzyme MunI in 3T3 mouse fibroblasts caused an increase in DNA degradation. Moreover, purified proteins such as beta-galactosidase and the large GTPase dynamin could be profected into podocytes using two different profection reagents with the success rate of 95-100%. The delivered beta-galactosidase enzyme was properly folded and able to cleave its substrate X-gal in podocytes. Diseased podocytes are also potential recipients of protein cargo as we also delivered fluorophore labeled IgG into puromycin treated podocytes. We are currently optimizing our protocol for in vivo profection. CONCLUSIONS: Protein transfer is developing as an exciting tool to study and target highly differentiated cells such as podocytes.