Pharmacological and pre-clinical safety profile of rSIV.F/HN, a hybrid lentiviral vector for cystic fibrosis gene therapy.

Rationale And Objective: Cystic fibrosis (CF) is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene. CFTR modulators offer significant improvements, but approximately 10% of patients remain nonresponsive or are intolerant. This study provides an analysis of rSIV.

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a critical regulator of inflammatory signalling through toll-like receptors 4 and 7/8 in murine and human lungs.

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.

Improving reconstructions in nanotomography for homogeneous materials mathematical optimization.

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.

Riboswitch-controlled IL-12 gene therapy reduces hepatocellular cancer in mice.

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.

An unbiased AAV-STARR-seq screen revealing the enhancer activity map of genomic regions in the mouse brain in vivo.

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.

Time and phenotype-dependent transcriptome analysis in AAV-TGFβ1 and Bleomycin-induced lung fibrosis models.

We have previously established a novel mouse model of lung fibrosis based on Adeno-associated virus (AAV)-mediated pulmonary overexpression of TGFβ1. 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.

MSD-based assays facilitate a rapid and quantitative serostatus profiling for the presence of anti-AAV antibodies.

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 NAb assays in mice. These methods need optimization for each serotype and are often not applicable to AAV variants with poor transduction.

Human stem cell-based retina on chip as new translational model for validation of AAV retinal gene therapy vectors.

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.

The biphasic and age-dependent impact of klotho on hallmarks of aging and skeletal muscle function.

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.

A Small-Molecule-Responsive Riboswitch Enables Conditional Induction of Viral Vector-Mediated Gene Expression in Mice.

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.

High-throughput identification of synthetic riboswitches by barcode-free amplicon-sequencing in human cells.

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.

Standardized, Scalable, and Timely Flexible Adeno-Associated Virus Vector Production Using Frozen High-Density HEK-293 Cell Stocks and CELLdiscs.

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.

Effects of nintedanib on the microvascular architecture in a lung fibrosis model.

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.

Comparative analysis of lysyl oxidase (like) family members in pulmonary fibrosis.

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.

Quantification of Pulmonary Fibrosis in a Bleomycin Mouse Model Using Automated Histological Image Analysis.

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.

Riboswitch-mediated Attenuation of Transgene Cytotoxicity Increases Adeno-associated Virus Vector Yields in HEK-293 Cells.

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.

Modeling Pulmonary Disease Pathways Using Recombinant Adeno-Associated Virus 6.2.

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.

Gene delivery to adipose tissue using transcriptionally targeted rAAV8 vectors.

In recent years, the increasing prevalence of obesity and obesity-related co-morbidities fostered intensive research in the field of adipose tissue biology. To further unravel molecular mechanisms of adipose tissue function, genetic tools enabling functional studies in vitro and in vivo are essential. While the use of transgenic animals is well established, attempts using viral and non-viral vectors to genetically modify adipocytes in vivo are rare.

Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain.

Recombinant Adeno-associated virus vectors (rAAV) are widely used for gene delivery and multiple naturally occurring serotypes have been harnessed to target cells in different tissues and organs including the brain. Here, we provide a detailed and quantitative analysis of the transduction profiles of rAAV vectors based on six of the most commonly used serotypes (AAV1, AAV2, AAV5, AAV6, AAV8, AAV9) that allows systematic comparison and selection of the optimal vector for a specific application. In our studies we observed marked differences among serotypes in the efficiency to transduce three different brain regions namely the striatum, hippocampus and neocortex of the mouse.

Off-target analysis of control siRNA molecules reveals important differences in the cytokine profile and inflammation response of human fibroblasts.

The use of RNA interference for the manipulation of gene expression has seen great applications from basic science to clinical investigations. However, limited selectivity and the induction of off-target effects by double stranded RNA molecules have been analyzed and discussed since the discovery of this gene expression regulation mechanism. In this study, the specificity of 13 commercially available control siRNA molecules is addressed by the analysis of gene expression profiles in 2 human cell lines HT1080 and HaCaT and in the mouse cell line 3T3-L1.

Acetyl-CoA carboxylases 1 and 2 show distinct expression patterns in rats and humans and alterations in obesity and diabetes.

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.

Death receptor-induced signaling pathways are differentially regulated by gamma interferon upstream of caspase 8 processing.

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.

NFkappaB activation by Fas is mediated through FADD, caspase-8, and RIP and is inhibited by FLIP.

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.