Therapeutic protein transduction of mammalian cells and mice by nucleic acid-free lentiviral nanoparticles

The straightforward production and dose-controlled administration of protein therapeutics remain major challenges for the biopharmaceutical manufacturing and gene therapy communities. Transgenes linked to HIV-1-derived vpr and pol-based protease cleavage (PC) sequences were co-produced as chimeric fusion proteins in a lentivirus production setting, encapsidated and processed to fusion peptide-free native protein in pseudotyped lentivirions for intracellular delivery and therapeutic action in target cells. Devoid of viral genome sequences, protein-transducing nanoparticles (PTNs) enabled transient and dose-dependent delivery of therapeutic proteins at functional quantities into a variety of mammalian cells in the absence of host chromosome modifications. PTNs delivering Manihot esculenta linamarase into rodent or human, tumor cell lines and spheroids mediated hydrolysis of the innocuous natural prodrug linamarin to cyanide and resulted in efficient cell killing. Following linamarin injection into nude mice, linamarase-transducing nanoparticles impacted solid tumor development through the bystander effect of cyanid

Therapeutic protein transduction of mammalian cells and mice by nucleic acid-free lentiviral nanoparticles

The straightforward production and dose-controlled administration of protein therapeutics remain major challenges for the biopharmaceutical manufacturing and gene therapy communities. Transgenes linked to HIV-1-derived vpr and pol-based protease cleavage (PC) sequences were co-produced as chimeric fusion proteins in a lentivirus production setting, encapsidated and processed to fusion peptide-free native protein in pseudotyped lentivirions for intracellular delivery and therapeutic action in target cells. Devoid of viral genome sequences, protein-transducing nanoparticles (PTNs) enabled transient and dose-dependent delivery of therapeutic proteins at functional quantities into a variety of mammalian cells in the absence of host chromosome modifications. PTNs delivering Manihot esculenta linamarase into rodent or human, tumor cell lines and spheroids mediated hydrolysis of the innocuous natural prodrug linamarin to cyanide and resulted in efficient cell killing. Following linamarin injection into nude mice, linamarase-transducing nanoparticles impacted solid tumor development through the bystander effect of cyanide

Conditional human VEGF‐mediated vascularization in chicken embryos using a novel temperature‐inducible gene regulation (TIGR) system

Advanced heterologous transcription control systems for adjusting desired transgene expression are essential for gene function assignments, drug discovery, manufacturing of difficult to produce protein pharmaceuticals and precise dosing of gene‐based therapeutic interventions. Conversion of the Streptomyces albus heat shock response regulator (RheA) into an artificial eukaryotic transcription factor resulted in a vertebrate thermosensor (CTA; cold‐inducible transactivator), which is able to adjust transcription initiation from chimeric target promoters (PCTA) in a low‐temperature‐ inducible manner. Evaluation of the temperature‐dependent CTA-PCTA interaction using a tailored ELISA‐like cell‐free assay correlated increased affinity of CTA for PCTA with temperature downshift. The temperature‐inducible gene regulation (TIGR) system enabled tight repression in the chicken bursal B‐cell line DT40 at 41°C as well as precise titration of model product proteins up to maximum expression at or below 37°C. Implantation of microencapsulated DT40 cells engineered for TIGR‐controlled expression of the human vascular endothelial growth factor A (hVEGF121) provided low‐temperature‐induced VEGF‐mediated vascularization in chicken embryo

Streptomyces‐derived quorum‐sensing systems engineered for adjustable transgene expression in mammalian cells and mice

Prokaryotic transcriptional regulatory elements have been adopted for controlled expression of cloned genes in mammalian cells and animals, the cornerstone for gene‐function correlations, drug discovery, biopharmaceutical manufacturing as well as advanced gene therapy and tissue engineering. Many prokaryotes have evolved specific molecular communication systems known as quorum‐sensing to coordinate population‐wide responses to physiological and/or physicochemical signals. A generic bacterial quorum‐sensing system is based on a diffusible signal molecule that prevents binding of a repressor to corresponding operator sites thus resulting in derepression of a target regulon. In Streptomyces, a family of butyrolactones and their corresponding receptor proteins, serve as quorum‐sensing systems that control morphological development and antibiotic biosynthesis. Fusion of the Streptomyces coelicolor quorum‐sensing receptor (ScbR) to a eukaryotic transactivation domain (VP16) created a mammalian transactivator (SCA) which binds and adjusts transcription from chimeric promoters containing an SCA‐specific operator module (PSPA). Expression of erythropoietin or the human secreted alkaline phosphatase (SEAP) by this quorum‐sensor‐regulated gene expression system (QuoRex) could be fine‐tuned by non‐toxic butyrolactones in a variety of mammalian cells including human primary and mouse embryonic stem cells. Following intraperitoneal implantation of microencapsulated Chinese hamster ovary cells transgenic for QuoRex‐controlled SEAP expression into mice, the serum levels of this model glycoprotein could be adjusted to desired concentrations using different butyrolactone dosing regime

Occurrence of Anti-Drug Antibodies against Interferon-Beta and Natalizumab in Multiple Sclerosis: A Collaborative Cohort Analysis

Immunogenicity of biopharmaceutical products in multiple sclerosis is a frequent side effect which has a multifactorial etiology. Here we study associations between anti-drug antibody (ADA) occurrence and demographic and clinical factors. Retrospective data from routine ADA test laboratories in Sweden, Denmark, Austria and Germany (Dusseldorf group) and from one research study in Germany (Munich group) were gathered to build a collaborative multi-cohort dataset within the framework of the ABIRISK project. A subset of 5638 interferon-beta (IFN beta)-treated and 3440 natalizumab-treated patients having data on at least the first two years of treatment were eligible for interval-censored time-to-event analysis. In multivariate Cox regression, IFN beta-1a subcutaneous and IFN beta-1b subcutaneous treated patients were at higher risk of ADA occurrence compared to IFN beta-1a intramuscular-treated patients (pooled HR = 6.4, 95% CI 4.9-8.4 and pooled HR = 8.7, 95% CI 6.6-11.4 respectively). Patients older than 50 years at start of IFN beta therapy developed ADA more frequently than adult patients younger than 30 (pooled HR = 1.8, 95% CI 1.4-2.3). Men developed ADA more frequently than women (pooled HR = 1.3, 95% CI 1.1-1.6). Interestingly we observed that in Sweden and Germany, patients who started IFN beta in April were at higher risk of developing ADA (HR = 1.6, 95% CI 1.1-2.4 and HR = 2.4, 95% CI 1.5-3.9 respectively). This result is not confirmed in the other cohorts and warrants further investigations. Concerning natalizumab, patients older than 45 years had a higher ADA rate (pooled HR = 1.4, 95% CI 1.0-1.8) and women developed ADA more frequently than men (pooled HR = 1.4, 95% CI 1.0-2.0). We confirmed previously reported differences in immunogenicity of the different types of IFN beta. Differences in ADA occurrence by sex and age are reported here for the first time. These findings should be further investigated taking into account other exposures and biomarkers

Occurrence of Anti-Drug Antibodies against Interferon-Beta and Natalizumab in Multiple Sclerosis: A Collaborative Cohort Analysis

Immunogenicity of biopharmaceutical products in multiple sclerosis is a frequent side effect which has a multifactorial etiology. Here we study associations between anti-drug antibody (ADA) occurrence and demographic and clinical factors. Retrospective data from routine ADA test laboratories in Sweden, Denmark, Austria and Germany (Dusseldorf group) and from one research study in Germany (Munich group) were gathered to build a collaborative multi-cohort dataset within the framework of the ABIRISK project. A subset of 5638 interferon-beta (IFN beta)-treated and 3440 natalizumab-treated patients having data on at least the first two years of treatment were eligible for interval-censored time-to-event analysis. In multivariate Cox regression, IFN beta-1a subcutaneous and IFN beta-1b subcutaneous treated patients were at higher risk of ADA occurrence compared to IFN beta-1a intramuscular-treated patients (pooled HR = 6.4, 95% CI 4.9-8.4 and pooled HR = 8.7, 95% CI 6.6-11.4 respectively). Patients older than 50 years at start of IFN beta therapy developed ADA more frequently than adult patients younger than 30 (pooled HR = 1.8, 95% CI 1.4-2.3). Men developed ADA more frequently than women (pooled HR = 1.3, 95% CI 1.1-1.6). Interestingly we observed that in Sweden and Germany, patients who started IFN beta in April were at higher risk of developing ADA (HR = 1.6, 95% CI 1.1-2.4 and HR = 2.4, 95% CI 1.5-3.9 respectively). This result is not confirmed in the other cohorts and warrants further investigations. Concerning natalizumab, patients older than 45 years had a higher ADA rate (pooled HR = 1.4, 95% CI 1.0-1.8) and women developed ADA more frequently than men (pooled HR = 1.4, 95% CI 1.0-2.0). We confirmed previously reported differences in immunogenicity of the different types of IFN beta. Differences in ADA occurrence by sex and age are reported here for the first time. These findings should be further investigated taking into account other exposures and biomarkers

Development of the Low Frequency Telescope Focal Plane Detector Modules for LiteBIRD

LiteBIRD is a JAXA-led strategic large-class satellite mission designed to measure the polarization of the cosmic microwave background and Galactic foregrounds from 34 to 448 GHz across the entire sky from L2 in the late 2020s. The scientific payload includes three telescopes which are called the low-, mid-, and high-frequency telescopes each with their own receiver that covers a portion of the mission's frequency range. The low frequency telescope will map synchrotron radiation from the Galactic foreground and the cosmic microwave background. We discuss the design, fabrication, and characterization of the low-frequency focal plane modules for low-frequency telescope, which has a total bandwidth ranging from 34 to 161 GHz. There will be a total of 4 different pixel types with 8 overlapping bands to cover the full frequency range. These modules are housed in a single low-frequency focal plane unit which provides thermal isolation, mechanical support, and radiative baffling for the detectors. The module design implements multi-chroic lenslet-coupled sinuous antenna arrays coupled to transition edge sensor bolometers read out with frequency-domain mulitplexing. While this technology has strong heritage in ground-based cosmic microwave background experiments, the broad frequency coverage, low optical loading conditions, and the high cosmic ray background of the space environment require further development of this technology to be suitable for LiteBIRD. In these proceedings, we discuss the optical and bolometeric characterization of a triplexing prototype pixel with bands centered on 78, 100, and 140 GHz.Comment: SPIE Astronomical Telescope + Instrumentation (AS22

A MAFG-lncRNA axis links systemic nutrient abundance to hepatic glucose metabolism

Obesity and type 2 diabetes mellitus are global emergencies and long noncoding RNAs (lncRNAs) are regulatory transcripts with elusive functions in metabolism. Here we show that a high fraction of lncRNAs, but not protein-coding mRNAs, are repressed during diet-induced obesity (DIO) and refeeding, whilst nutrient deprivation induced lncRNAs in mouse liver. Similarly, lncRNAs are lost in diabetic humans. LncRNA promoter analyses, global cistrome and gain-of-function analyses confirm that increased MAFG signaling during DIO curbs lncRNA expression. Silencing Mafg in mouse hepatocytes and obese mice elicits a fasting-like gene expression profile, improves glucose metabolism, de-represses lncRNAs and impairs mammalian target of rapamycin (mTOR) activation. We find that obesity-repressed LincIRS2 is controlled by MAFG and observe that genetic and RNAi-mediated LincIRS2 loss causes elevated blood glucose, insulin resistance and aberrant glucose output in lean mice. Taken together, we identify a MAFG-lncRNA axis controlling hepatic glucose metabolism in health and metabolic disease