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A major limiting factor for systemically delivered gene therapies is the lack of novel tissue specific AAV (Adeno-associated virus) derived vectors. Bispecific antibodies can be used to redirect AAVs to specific target receptors. Here, we demonstrate that the insertion of a short linear epitope "2E3" derived from human proprotein-convertase subtilisin/kexin type 9 (PCSK9) into different surface loops of the VP capsid proteins can be used for AAV de-targeting from its natural receptor(s), combined with a bispecific antibody-mediated retargeting. We chose to target a set of distinct disease relevant membrane proteins-fibroblast activation protein (FAP), which is upregulated on activated fibroblasts within the tumor stroma and in fibrotic tissues, as well as programmed death-ligand 1 (PD-L1), which is strongly upregulated in many cancers. Upon incubation with a bispecific antibody recognizing the 2E3 epitope and FAP or PD-L1, the bispecific antibody/rAAV complex was able to selectively transduce receptor positive cells. In summary, we developed a novel, rationally designed vector retargeting platform that can target AAVs to a new set of cellular receptors in a modular fashion. This versatile platform may serve as a valuable tool to investigate the role of disease relevant cell types and basis for novel gene therapy approaches.
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Anticorpos Biespecíficos/imunologia , Proteínas do Capsídeo/imunologia , Capsídeo/imunologia , Dependovirus/genética , Endopeptidases/imunologia , Epitopos/imunologia , Vetores Genéticos/administração & dosagem , Proteínas de Membrana/imunologia , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/imunologia , Pró-Proteína Convertase 9/metabolismo , Transdução GenéticaRESUMO
Nintedanib, a tyrosine kinase inhibitor approved for the treatment of idiopathic pulmonary fibrosis, has anti-fibrotic, anti-inflammatory, and anti-angiogenic activity. We explored the impact of nintedanib on microvascular architecture in a pulmonary fibrosis model. Lung fibrosis was induced in C57Bl/6 mice by intratracheal bleomycin (0.5 mg/kg). Nintedanib was started after the onset of lung pathology (50 mg/kg twice daily, orally). Micro-computed tomography was performed via volumetric assessment. Static lung compliance and forced vital capacity were determined by invasive measurements. Mice were subjected to bronchoalveolar lavage and histologic analyses, or perfused with a casting resin. Microvascular corrosion casts were imaged by scanning electron microscopy and synchrotron radiation tomographic microscopy, and quantified morphometrically. Bleomycin administration resulted in a significant increase in higher-density areas in the lungs detected by micro-computed tomography, which was significantly attenuated by nintedanib. Nintedanib significantly reduced lung fibrosis and vascular proliferation, normalized the distorted microvascular architecture, and was associated with a trend toward improvement in lung function and inflammation. Nintedanib resulted in a prominent improvement in pulmonary microvascular architecture, which outperformed the effect of nintedanib on lung function and inflammation. These findings uncover a potential new mode of action of nintedanib that may contribute to its efficacy in idiopathic pulmonary fibrosis.
Assuntos
Fibrose Pulmonar Idiopática/tratamento farmacológico , Indóis/uso terapêutico , Microvasos/ultraestrutura , Animais , Bleomicina , Proliferação de Células/efeitos dos fármacos , Colágeno/metabolismo , Modelos Animais de Doenças , Fibrose Pulmonar Idiopática/diagnóstico por imagem , Fibrose Pulmonar Idiopática/patologia , Fibrose Pulmonar Idiopática/fisiopatologia , Imageamento Tridimensional , Camundongos Endogâmicos C57BL , Microvasos/diagnóstico por imagem , Microvasos/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Pneumonia/complicações , Pneumonia/diagnóstico por imagem , Pneumonia/patologia , Pneumonia/fisiopatologia , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/patologia , Alvéolos Pulmonares/ultraestrutura , Testes de Função Respiratória , Microtomografia por Raio-XRESUMO
Cytotoxicity of transgenes carried by adeno-associated virus (AAV) vectors might be desired, for instance, in oncolytic virotherapy or occur unexpectedly in exploratory research when studying sparsely characterized genes. To date, most AAV-based studies use constitutively active promoters (e.g., the CMV promoter) to drive transgene expression, which often hampers efficient AAV production due to cytotoxic, antiproliferative, or unknown transgene effects interfering with producer cell performance. Therefore, we explored artificial riboswitches as novel tools to control transgene expression during AAV production in mammalian cells. Our results demonstrate that the guanine-responsive GuaM8HDV aptazyme efficiently attenuates transgene expression and associated detrimental effects, thereby boosting AAV vector yields up to 23-fold after a single addition of guanine. Importantly, riboswitch-harboring vectors preserved their ability to express functional transgene at high levels in the absence of ligand, as demonstrated in a mouse model of AAV-TGFß1-induced pulmonary fibrosis. Thus, our study provides the first application-ready biotechnological system-based on aptazymes, which should enable high viral vector yields largely independent of the transgene used. Moreover, the RNA-intrinsic, small-molecule regulatable mode of action of riboswitches provides key advantages over conventional transcription factor-based regulatory systems. Therefore, such riboswitch vectors might be ultimately applied to temporally control therapeutic transgene expression in vivo.
Assuntos
Dependovirus/genética , Vetores Genéticos/genética , Riboswitch , Transgenes , Replicação Viral , Animais , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Ordem dos Genes , Genes Reporter , Guanina/metabolismo , Guanina/farmacologia , Células HEK293 , Humanos , Ligantes , Camundongos , Transdução Genética , Replicação Viral/efeitos dos fármacosRESUMO
Viral vectors have been applied successfully to generate disease-related animal models and to functionally characterize target genes in vivo. However, broader application is still limited by complex vector production, biosafety requirements, and vector-mediated immunogenic responses, possibly interfering with disease-relevant pathways. Here, we describe adeno-associated virus (AAV) variant 6.2 as an ideal vector for lung delivery in mice, overcoming most of the aforementioned limitations. In a proof-of-concept study using AAV6.2 vectors expressing IL-13 and transforming growth factor-ß1 (TGF-ß1), we were able to induce hallmarks of severe asthma and pulmonary fibrosis, respectively. Phenotypic characterization and deep sequencing analysis of the AAV-IL-13 asthma model revealed a characteristic disease signature. Furthermore, suitability of the model for compound testing was also demonstrated by pharmacological intervention studies using an anti-IL-13 antibody and dexamethasone. Similarly, the AAV-TGF-ß1 fibrosis model showed several disease-like pathophenotypes monitored by micro-computed tomography imaging and lung function measurement. Most importantly, analyses using stuffer control vectors demonstrated that in contrast to a common adenovirus-5 vector, AAV6.2 vectors did not induce any measurable inflammation and therefore carry a lower risk of altering relevant readouts. In conclusion, we propose AAV6.2 as an ideal vector system for the functional characterization of target genes in the context of pulmonary diseases in mice.
Assuntos
Asma/imunologia , Dependovirus/genética , Fibrose Pulmonar Idiopática/imunologia , Animais , Asma/genética , Asma/metabolismo , Modelos Animais de Doenças , Feminino , Vetores Genéticos , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/metabolismo , Interleucina-13/biossíntese , Interleucina-13/genética , Camundongos Endogâmicos BALB C , Transdução Genética , Fator de Crescimento Transformador beta1/biossíntese , Fator de Crescimento Transformador beta1/genéticaRESUMO
Compressed sensing is an image reconstruction technique to achieve high-quality results from limited amount of data. In order to achieve this, it utilizes prior knowledge about the samples that shall be reconstructed. Focusing on image reconstruction in nanotomography, this work proposes enhancements by including additional problem-specific knowledge. In more detail, we propose further classes of algebraic inequalities that are added to the compressed sensing model. The first consists in a valid upper bound on the pixel brightness. It only exploits general information about the projections and is thus applicable to a broad range of reconstruction problems. The second class is applicable whenever the sample material is of roughly homogeneous composition. The model favors a constant density and penalizes deviations from it. The resulting mathematical optimization models are algorithmically tractable and can be solved to global optimality by state-of-the-art available implementations of interior point methods. In order to evaluate the novel models, obtained results are compared to existing image reconstruction methods, tested on simulated and experimental data sets. The experimental data comprise one 360° electron tomography tilt series of a macroporous zeolite particle and one absorption contrast nano X-ray computed tomography (nano-CT) data set of a copper microlattice structure. The enriched models are optimized quickly and show improved reconstruction quality, outperforming the existing models. Promisingly, our approach yields superior reconstruction results, particularly when only a small number of tilt angles is available.
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BACKGROUND AND PURPOSE: Toll-like receptors 4 (TLR4) and TLR7/TLR8 play an important role in mediating the inflammatory effects of bacterial and viral pathogens. Interleukin-1 receptor-associated kinase 4 (IRAK4) is an important regulator of signalling by toll-like receptor (TLR) and hence is a potential therapeutic target in diseases characterized by increased lung inflammatory signalling. EXPERIMENTAL APPROACH: We used an established murine model of acute lung inflammation, and studied human lung tissue ex vivo, to investigate the effects of inhibiting IRAK4 on lung inflammatory pathways. KEY RESULTS: We show that TLR4 stimulation produces an inflammatory response characterized by neutrophil influx and tumour necrosis factor-α (TNF-α) production in murine lungs and that these responses are markedly reduced in IRAK4 kinase-dead mice. In addition, we characterize a novel selective IRAK4 inhibitor, BI1543673, and show that this compound can reduce lipopolysaccharide (LPS)-induced airway inflammation in wild-type mice. Additionally, BI1543673 reduced inflammatory responses to both TLR4 and TLR7/8 stimulation in human lung tissue studied ex vivo. CONCLUSION AND IMPLICATIONS: These data demonstrate a key role for IRAK4 signalling in lung inflammation and suggest that IRAK4 inhibition has potential utility to treat lung diseases characterized by inflammatory responses driven through TLR4 and TLR7/8.
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Hepatocellular carcinoma (HCC) and solid cancers with liver metastases are indications with high unmet medical need. Interleukin-12 (IL-12) is a proinflammatory cytokine with substantial anti-tumor properties, but its therapeutic potential has not been realized due to severe toxicity. Here, we show that orthotopic liver tumors in mice can be treated by targeting hepatocytes via systemic delivery of adeno-associated virus (AAV) vectors carrying the murine IL-12 gene. Controlled cytokine production was achieved in vivo by using the tetracycline-inducible K19 riboswitch. AAV-mediated expression of IL-12 led to STAT4 phosphorylation, interferon-γ (IFNγ) production, infiltration of T cells and, ultimately, tumor regression. By detailed analyses of efficacy and tolerability in healthy and tumor-bearing animals, we could define a safe and efficacious vector dose. As a potential clinical candidate, we characterized vectors carrying the human IL-12 (huIL-12) gene. In mice, bioactive human IL-12 was expressed in a vector dose-dependent manner and could be induced by tetracycline, suggesting tissue-specific AAV vectors with riboswitch-controlled expression of highly potent proinflammatory cytokines as an attractive approach for vector-based cancer immunotherapy.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Riboswitch , Camundongos , Humanos , Animais , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/patologia , Terapia Genética , Interleucina-12/genética , Interleucina-12/metabolismo , Tetraciclina/farmacologiaRESUMO
Enhancers are important cis-regulatory elements controlling cell-type specific expression patterns of genes. Furthermore, combinations of enhancers and minimal promoters are utilized to construct small, artificial promoters for gene delivery vectors. Large-scale functional screening methodology to construct genomic maps of enhancer activities has been successfully established in cultured cell lines, however, not yet applied to terminally differentiated cells and tissues in a living animal. Here, we transposed the Self-Transcribing Active Regulatory Region Sequencing (STARR-seq) technique to the mouse brain using adeno-associated-viruses (AAV) for the delivery of a highly complex screening library tiling entire genomic regions and covering in total 3 Mb of the mouse genome. We identified 483 sequences with enhancer activity, including sequences that were not predicted by DNA accessibility or histone marks. Characterizing the expression patterns of fluorescent reporters controlled by nine candidate sequences, we observed differential expression patterns also in sparse cell types. Together, our study provides an entry point for the unbiased study of enhancer activities in organisms during health and disease.
Assuntos
Elementos Facilitadores Genéticos , Genômica , Animais , Camundongos , Genômica/métodos , Mapeamento Cromossômico/métodos , Regiões Promotoras Genéticas , EncéfaloRESUMO
Adeno-associated virus (AAV) vector applications are often limited by capsid-directed humoral immune responses, mainly through neutralizing antibodies (NAbs), which are present throughout the human population due to natural AAV infections. Currently, antibody levels are often quantified via ELISA-based protocols or by cellular NAb assays and less frequently by in vivo NAb assays in mice. These methods need optimization for each serotype and are often not applicable to AAV variants with poor in vitro transduction. To tackle these limitations, we have established Meso Scale Discovery (MSD)-based assays for the quantification of binding antibodies (BAbs) and NAbs against the three most commonly used AAV serotypes, AAV2, AAV8, and AAV9. Both assays detect anti-AAV-IgG1-3 with high sensitivity and consistency as shown in a screen of sera from 40 healthy human donors. Subsequently, BAb and NAb titers were determined for identification of seronegative animals in a non-human primate (NHP) cohort. Moreover, the MSD-based BAb assay protocol was extended to a panel of 14 different AAV serotypes. In summary, our platform allows a rapid and quantitative assessment of the immunological properties of any natural or engineered AAV variant irrespective of transduction efficiency and enables high-throughput screens.
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We have previously established a novel mouse model of lung fibrosis based on Adeno-associated virus (AAV)-mediated pulmonary overexpression of TGFß1. Here, we provide an in-depth characterization of phenotypic and transcriptomic changes (mRNA and miRNA) in a head-to-head comparison with Bleomycin-induced lung injury over a 4-week disease course. The analyses delineate the temporal state of model-specific and commonly altered pathways, thereby providing detailed insights into the processes underlying disease development. They further guide appropriate model selection as well as interventional study design. Overall, Bleomycin-induced fibrosis resembles a biphasic process of acute inflammation and subsequent transition into fibrosis (with partial resolution), whereas the TGFß1-driven model is characterized by pronounced and persistent fibrosis with concomitant inflammation and an equally complex disease phenotype as observed upon Bleomycin instillation. Finally, based on an integrative approach combining lung function data, mRNA/miRNA profiles, their correlation and miRNA target predictions, we identify putative drug targets and miRNAs to be explored as therapeutic candidates for fibrotic diseases. Taken together, we provide a comprehensive analysis and rich data resource based on RNA-sequencing, along with a strategy for transcriptome-phenotype coupling. The results will be of value for TGFß research, drug discovery and biomarker identification in progressive fibrosing interstitial lung diseases.
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MicroRNAs , Fibrose Pulmonar , Animais , Bleomicina/efeitos adversos , Bleomicina/metabolismo , Dependovirus/genética , Modelos Animais de Doenças , Fibrose , Perfilação da Expressão Gênica , Inflamação/patologia , Pulmão/patologia , Camundongos , MicroRNAs/metabolismo , Fenótipo , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/genética , Fibrose Pulmonar/metabolismo , RNA Mensageiro/metabolismoRESUMO
Aging is accompanied by disrupted information flow, resulting from accumulation of molecular mistakes. These mistakes ultimately give rise to debilitating disorders including skeletal muscle wasting, or sarcopenia. To derive a global metric of growing 'disorderliness' of aging muscle, we employed a statistical physics approach to estimate the state parameter, entropy, as a function of genes associated with hallmarks of aging. Escalating network entropy reached an inflection point at old age, while structural and functional alterations progressed into oldest-old age. To probe the potential for restoration of molecular 'order' and reversal of the sarcopenic phenotype, we systemically overexpressed the longevity protein, Klotho, via AAV. Klotho overexpression modulated genes representing all hallmarks of aging in old and oldest-old mice, but pathway enrichment revealed directions of changes were, for many genes, age-dependent. Functional improvements were also age-dependent. Klotho improved strength in old mice, but failed to induce benefits beyond the entropic tipping point.
Assuntos
Envelhecimento/metabolismo , Glucuronidase/metabolismo , Músculo Esquelético/metabolismo , Sarcopenia/metabolismo , Fatores Etários , Envelhecimento/genética , Envelhecimento/patologia , Animais , Dependovirus/genética , Dependovirus/metabolismo , Feminino , Regulação da Expressão Gênica , Terapia Genética , Vetores Genéticos , Glucuronidase/genética , Células HEK293 , Humanos , Proteínas Klotho , Masculino , Camundongos Endogâmicos C57BL , Força Muscular , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Recuperação de Função Fisiológica , Sarcopenia/genética , Sarcopenia/fisiopatologia , Sarcopenia/terapia , TranscriptomaRESUMO
Gene therapies using adeno-associated viruses (AAVs) are among the most promising strategies to treat or even cure hereditary and acquired retinal diseases. However, the development of new efficient AAV vectors is slow and costly, largely because of the lack of suitable non-clinical models. By faithfully recreating structure and function of human tissues, human induced pluripotent stem cell (iPSC)-derived retinal organoids could become an essential part of the test cascade addressing translational aspects. Organ-on-chip (OoC) technology further provides the capability to recapitulate microphysiological tissue environments as well as a precise control over structural and temporal parameters. By employing our recently developed retina on chip that merges organoid and OoC technology, we analyzed the efficacy, kinetics, and cell tropism of seven first- and second-generation AAV vectors. The presented data demonstrate the potential of iPSC-based OoC models as the next generation of screening platforms for future gene therapeutic studies.
Assuntos
Dependovirus/genética , Vetores Genéticos/genética , Células-Tronco Pluripotentes Induzidas/citologia , Dispositivos Lab-On-A-Chip , Organoides/metabolismo , Retina/metabolismo , Transdução Genética , Biomarcadores , Técnicas de Cultura de Células , Técnicas de Cultura de Células em Três Dimensões , Diferenciação Celular , Imunofluorescência , Expressão Gênica , Genes Reporter , Terapia Genética , Humanos , Organoides/citologia , Retina/citologia , TransgenesRESUMO
Synthetic riboswitches mediating ligand-dependent RNA cleavage or splicing-modulation represent elegant tools to control gene expression in various applications, including next-generation gene therapy. However, due to the limited understanding of context-dependent structure-function relationships, the identification of functional riboswitches requires large-scale-screening of aptamer-effector-domain designs, which is hampered by the lack of suitable cellular high-throughput methods. Here we describe a fast and broadly applicable method to functionally screen complex riboswitch libraries (~1.8 × 104 constructs) by cDNA-amplicon-sequencing in transiently transfected and stimulated human cells. The self-barcoding nature of each construct enables quantification of differential mRNA levels without additional pre-selection or cDNA-manipulation steps. We apply this method to engineer tetracycline- and guanine-responsive ON- and OFF-switches based on hammerhead, hepatitis-delta-virus and Twister ribozymes as well as U1-snRNP polyadenylation-dependent RNA devices. In summary, our method enables fast and efficient high-throughput riboswitch identification, thereby overcoming a major hurdle in the development cascade for therapeutically applicable gene switches.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Riboswitch/genética , Biologia Computacional/métodos , Código de Barras de DNA Taxonômico , DNA Complementar , Regulação da Expressão Gênica/efeitos dos fármacos , Guanina/farmacologia , Células HEK293 , Vírus Delta da Hepatite/genética , Humanos , RNA Catalítico/genética , Ribonucleoproteína Nuclear Pequena U1/genética , Riboswitch/efeitos dos fármacos , Biologia Sintética/métodos , Tetraciclina/farmacologiaRESUMO
Adeno-associated viral (AAV) vector-mediated gene therapy holds great potential for future medical applications. However, to facilitate safer and broader applicability and to enable patient-centric care, therapeutic protein expression should be controllable, ideally by an orally administered drug. The use of protein-based systems is considered rather undesirable, due to potential immunogenicity and the limited coding space of AAV. Ligand-dependent riboswitches, in contrast, are small and characterized by an attractive mode-of-action based on mRNA-self-cleavage, independent of coexpressed foreign protein. While a promising approach, switches available to date have only shown moderate potency in animals. In particular, ON-switches that induce transgene expression upon ligand administration so far have achieved rather disappointing results. Here we present the utilization of the previously described tetracycline-dependent ribozyme K19 for controlling AAV-mediated transgene expression in mice. Using this tool switch, we provide first proof for the feasibility of clinically desired key features, including multiorgan functionality, potent regulation (up to 15-fold induction), reversibility, and the possibility to fine-tune and repeatedly induce expression. The systematic assessment of ligand and reporter protein plasma levels further enabled the characterization of pharmacokinetic-pharmacodynamic relationships. Thus, our results strongly support future efforts to develop engineered riboswitches for applications in clinical gene therapy.
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Antibacterianos/farmacologia , Dependovirus/genética , Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/metabolismo , RNA Catalítico/metabolismo , Regiões 3' não Traduzidas , Animais , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Linhagem Celular , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Fígado/metabolismo , Pulmão/metabolismo , Camundongos , RNA Catalítico/genética , Tetraciclina/farmacologiaRESUMO
BACKGROUND: Acetyl-CoA carboxylases (ACC) 1 and 2 are central enzymes in lipid metabolism. To further investigate their relevance for the development of obesity and type 2 diabetes, expression of both ACC isoforms was analyzed in obese fa/fa Zucker fatty and Zucker diabetic fatty rats at different ages in comparison to Zucker lean controls. METHODS: ACC1 and ACC2 transcript levels were measured by quantitative real-time polymerase chain reaction in metabolically relevant tissues of Zucker fatty, Zucker diabetic fatty and Zucker lean control animals. Quantitative real-time polymerase chain reaction was also applied to measure ACC tissue distribution in human tissues. For confirmation on a protein level, quantitative mass spectrometry was used. RESULTS: Disease-related transcriptional changes of both ACC isoforms were observed in various tissues of Zucker fatty and Zucker diabetic fatty rats including liver, pancreas and muscle. Changes were most prominent in oxidative tissues of diabetic rats, where ACC2 was significantly increased and ACC1 was reduced compared with Zucker lean control animals. A comparison of the overall tissue distribution of both ACC isoforms in humans and rats surprisingly revealed strong differences. While in rats ACC1 was mainly expressed in lipogenic and ACC2 in oxidative tissues, ACC2 was predominant in oxidative and lipogenic tissues in humans. CONCLUSION: Our data support a potential role for both ACC isoforms in the development of obesity and diabetes in rats. However, the finding of fundamental species differences in ACC1 and ACC2 tissue expression might be indicative for different functions of both isoforms in humans and rats and raises the question to which degree these models are predictive for the physiology and pathophysiology of lipid metabolism in humans.
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Acetil-CoA Carboxilase/metabolismo , Diabetes Mellitus/enzimologia , Regulação Enzimológica da Expressão Gênica , Obesidade/enzimologia , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/isolamento & purificação , Envelhecimento , Análise de Variância , Animais , Glicemia/análise , Peso Corporal , Dieta , Jejum/metabolismo , Ácidos Graxos não Esterificados/sangue , Feminino , Humanos , Técnicas de Diluição do Indicador , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Masculino , Especificidade de Órgãos , Fragmentos de Peptídeos/análise , Proteômica/métodos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Ratos Zucker , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da Espécie , Espectrometria de Massas em Tandem , Triglicerídeos/sangueRESUMO
Fas (APO-1/CD95) is the prototypic death receptor, and the molecular mechanisms of Fas-induced apoptosis are comparably well understood. Here, we show that Fas activates NFkappaB via a pathway involving RIP, FADD, and caspase-8. Remarkably, the enzymatic activity of the latter was dispensable for Fas-induced NFkappaB signaling pointing to a scaffolding-related function of caspase-8 in nonapoptotic Fas signaling. NFkappaB was activated by overexpressed FLIPL and FLIPS in a cell type-specific manner. However, in the context of Fas signaling both isoforms blocked FasL-induced NFkappaB activation. Moreover, down-regulation of both endogenous FLIP isoforms or of endogenous FLIPL alone was sufficient to enhance FasL-induced expression of the NFkappaB target gene IL8. As NFkappaB signaling is inhibited during apoptosis, FasL-induced NFkappaB activation was most prominent in cells that were protected by Bcl2 expression or caspase inhibitors and expressed no or minute amounts of FLIP. Thus, protection against Fas-induced apoptosis in a FLIP-independent manner converted a proapoptotic Fas signal into an inflammatory NFkappaB-related response.
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Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Caspases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , NF-kappa B/metabolismo , Proteínas/metabolismo , Receptor fas/metabolismo , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Caspase 8 , Proteína de Domínio de Morte Associada a Fas , Regulação da Expressão Gênica/fisiologia , Humanos , Proteínas I-kappa B/metabolismo , Inibidor de NF-kappaB alfa , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Proteína Serina-Treonina Quinases de Interação com Receptores , Regulação para CimaRESUMO
Adeno-associated virus (AAV) vectors currently represent the most attractive platform for viral gene therapy and are also valuable research tools to study gene function or establish disease models. Consequently, many academic labs, core facilities, and biotech/pharma companies meanwhile produce AAVs for research and early clinical development. Whereas fast, universal protocols for vector purification (downstream processing) are available, AAV production using adherent HEK-293 cells still requires time-consuming passaging and extensive culture expansion before transfection. Moreover, most scalable culture platforms require special equipment or extensive method development. To tackle these limitations in upstream processing, this study evaluated frozen high-density cell stocks as a ready-to-seed source of producer cells, and further investigated the multilayered CELLdisc culture system for upscaling. The results demonstrate equal AAV productivity using frozen cell stock-derived cultures compared to conventionally cultured cells, as well as scalability using CELLdiscs. Thus, by directly seeding freshly thawed cells into CELLdiscs, AAV production can be easily upscaled and efficiently standardized to low-passage, high-viability cells in a timely flexible manner, potentially dismissing time-consuming routine cell culture work. In conjunction with a further optimized iodixanol protocol, this process enabled supply to a large-animal study with two high-yield AAV2 capsid variant batches (0.6-1.2 × 1015 vector genomes) in as little as 4 weeks.
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Dependovirus/genética , Vetores Genéticos , Biotecnologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/isolamento & purificação , Técnicas de Cultura de Células , Dependovirus/crescimento & desenvolvimento , Dependovirus/isolamento & purificação , Terapia Genética/métodos , Células HEK293 , Humanos , TransfecçãoRESUMO
FasL and gamma interferon (IFN-gamma) are produced by activated T cells and NK cells and synergistically induce apoptosis. Although both cytokines can also elicit proinflammatory responses, a possible cross talk of these ligands with respect to nonapoptotic signaling has been poorly addressed. Here, we show that IFN-gamma sensitizes KB cells for apoptosis induction by facilitating death-inducing signaling complex (DISC)-mediated caspase 8 processing. Moreover, after protection against death receptor-induced apoptosis by caspase inhibition or Bcl2 overexpression, IFN-gamma also sensitized for Fas- and TRAIL death receptor-mediated NF-kappaB activation leading to synergistic upregulation of a variety of proinflammatory genes. In contrast, Fas-mediated activation of JNK, p38, and p42/44 occurred essentially independent from IFN-gamma sensitization, indicating that the apoptosis- and NF-kappaB-related FasL-IFN-gamma cross talk was not due to a simple global enhancement of Fas signaling. Overexpression of FLIP(L) and FLIP(S) inhibited Fas- as well as TRAIL-mediated NF-kappaB activation and apoptosis induction in IFN-gamma-primed cells suggesting that both responses are coregulated at the level of the DISC.
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Caspases/metabolismo , Interferon gama/fisiologia , Glicoproteínas de Membrana/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/fisiologia , Apoptose/fisiologia , Caspase 8 , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte , Proteína Ligante Fas , Humanos , Células KB , Glicoproteínas de Membrana/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNFRESUMO
Extracellular matrix (ECM) composition and stiffness are major driving forces for the development and persistence of fibrotic diseases. Lysyl oxidase (LOX) and LOX-like (LOXL) proteins play crucial roles in ECM remodeling due to their collagen crosslinking and intracellular functions. Here, we systematically investigated LOX/L expression in primary fibroblasts and epithelial cells under fibrotic conditions, Bleomycin (BLM) induced lung fibrosis and in human IPF tissue. Basal expression of all LOX/L family members was detected in epithelial cells and at higher levels in fibroblasts. Various pro-fibrotic stimuli broadly induced LOX/L expression in fibroblasts, whereas specific induction of LOXL2 and partially LOX was observed in epithelial cells. Immunohistochemical analysis of lung tissue from 14 IPF patients and healthy donors revealed strong induction of LOX and LOXL2 in bronchial and alveolar epithelium as well as fibroblastic foci. Using siRNA experiments we observed that LOXL2 and LOXL3 were crucial for fibroblast-to-myofibroblast transition (FMT). As FMT could only be reconstituted with an enzymatically active LOXL2 variant, we conclude that LOXL2 enzymatic function is crucial for fibroblast transdifferentiation. In summary, our study provides a comprehensive analysis of the LOX/L family in fibrotic lung disease and indicates prominent roles for LOXL2/3 in fibroblast activation and LOX/LOXL2 in IPF.
Assuntos
Aminoácido Oxirredutases/metabolismo , Bleomicina/efeitos adversos , Proteína-Lisina 6-Oxidase/metabolismo , Fibrose Pulmonar/enzimologia , Animais , Linhagem Celular , Transdiferenciação Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Masculino , Camundongos , Família Multigênica , Miofibroblastos/citologia , Miofibroblastos/metabolismo , Fibrose Pulmonar/induzido quimicamenteRESUMO
Current literature on pulmonary fibrosis induced in animal models highlights the need of an accurate, reliable and reproducible histological quantitative analysis. One of the major limits of histological scoring concerns the fact that it is observer-dependent and consequently subject to variability, which may preclude comparative studies between different laboratories. To achieve a reliable and observer-independent quantification of lung fibrosis we developed an automated software histological image analysis performed from digital image of entire lung sections. This automated analysis was compared to standard evaluation methods with regard to its validation as an end-point measure of fibrosis. Lung fibrosis was induced in mice by intratracheal administration of bleomycin (BLM) at 0.25, 0.5, 0.75 and 1 mg/kg. A detailed characterization of BLM-induced fibrosis was performed 14 days after BLM administration using lung function testing, micro-computed tomography and Ashcroft scoring analysis. Quantification of fibrosis by automated analysis was assessed based on pulmonary tissue density measured from thousands of micro-tiles processed from digital images of entire lung sections. Prior to analysis, large bronchi and vessels were manually excluded from the original images. Measurement of fibrosis has been expressed by two indexes: the mean pulmonary tissue density and the high pulmonary tissue density frequency. We showed that tissue density indexes gave access to a very accurate and reliable quantification of morphological changes induced by BLM even for the lowest concentration used (0.25 mg/kg). A reconstructed 2D-image of the entire lung section at high resolution (3.6 µm/pixel) has been performed from tissue density values allowing the visualization of their distribution throughout fibrotic and non-fibrotic regions. A significant correlation (p<0.0001) was found between automated analysis and the above standard evaluation methods. This correlation establishes automated analysis as a novel end-point measure of BLM-induced lung fibrosis in mice, which will be very valuable for future preclinical drug explorations.