Generation and GMP scale-up of human CAR-T cells using non-viral transposons for B cell malignances.

Most CAR-T therapies rely on genetic T cell engineering with integrating viral vectors that, although effective, are associated with prohibitive costs. Here we have generated TranspoCART19 cells, a fully functional 4-1BB second-generation CAR-T cell product targeting CD19, fused to a truncated version of the human EGFR (hEGFRt) as reporter gene and safety switch, based on the transposon delivery system. Our manufacturing protocol allowed generation of TranspoCART19 cells under GMP conditions, showing similar and antitumoral efficacy than conventional CAR-T cells generated with lentiviral vectors.

DNA Meets Protein─Development, Characterization, and Application of Aptamers against Peanut Allergen.

Allergen detection methods support food labeling and quality assessment at the allergen component level of allergen preparations used for allergy diagnosis and immunotherapy (AIT). Commonly applied enzyme-linked immunosorbent assay (ELISA) requires animal antibodies but potentially shows batch variations. We developed synthetic aptamers as alternative binders in allergen detection to meet the replacement, reduction, and refinement (3R) principle on animal protection in science.

Unraveling the dynamics of hepatitis C virus adaptive mutations and their impact on antiviral responses in primary human hepatocytes.

Unlabelled: Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture-adapted HCV population (p100pop) which exhibits increased replicative capacity in various liver cell lines, this study investigated virus and host determinants that underlie enhanced viral fitness.

Hepatitis C virus cell culture adaptive mutations enhance cell culture propagation by multiple mechanisms but boost antiviral responses in primary human hepatocytes.

Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture adapted HCV population (p100pop) which exhibits increased replicative capacity in various liver cell lines, this study investigated virus and host determinants which underlie enhanced viral fitness.

Spatiotemporal analysis of SARS-CoV-2 infection reveals an expansive wave of monocyte-derived macrophages associated with vascular damage and virus clearance in hamster lungs.

We present the first study of the 3D kinetics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the early host response in a large lung volume using a combination of tissue imaging and transcriptomics. This approach allowed us to make a number of important findings: Spatially restricted antiviral response is shown, including the formation of monocytic macrophage clusters and upregulation of the major histocompatibility complex II in infected epithelial cells. The monocyte-derived macrophages are linked to SARS-CoV-2 clearance, and the appearance of these cells is associated with post-infection endothelial damage; thus, we shed light on the role of these cells in infected tissue.

Transposon Insertions into Nucleolar DNA by an Engineered Transposase Localized in the Nucleolus.

Transposons are nature's gene delivery vehicles that can be harnessed for experimental and therapeutic purposes. The (SB) transposon shows efficient transposition and long-term transgene expression in human cells, and is currently under clinical development for gene therapy. SB transposition occurs into the human genome in a random manner, which carries a risk of potential genotoxic effects associated with transposon integration.

Erratum: Anti-CD117 CAR T cells incorporating a safety switch eradicate human acute myeloid leukemia and hematopoietic stem cells.

[This corrects the article DOI: 10.1016/j.omto.

Anti-CD117 CAR T cells incorporating a safety switch eradicate human acute myeloid leukemia and hematopoietic stem cells.

Discrimination between hematopoietic stem cells and leukemic stem cells remains a major challenge for acute myeloid leukemia immunotherapy. CAR T cells specific for the CD117 antigen can deplete malignant and healthy hematopoietic stem cells before consolidation with allogeneic hematopoietic stem cell transplantation in absence of cytotoxic conditioning. Here we exploit non-viral technology to achieve early termination of CAR T cell activity to prevent incoming graft rejection.

Passer, a highly active transposon from a fish genome, as a potential new robust genetic manipulation tool.

The discovery of new, active DNA transposons can expand the range of genetic tools and provide more options for genomic manipulation. In this study, a bioinformatics analysis suggested that Passer (PS) transposons, which are members of the pogo superfamily, show signs of recent and current activity in animals and may be active in some species. Cell-based transposition assays revealed that the native PS transposases from Gasterosteus aculeatus and Danio rerio displayed very high activity in human cells relative to the Sleeping Beauty transposon.

Horizontal Transfer and Evolutionary Profiles of Two DD34E Transposons ( and ) in Vertebrates.

Both ZeBrafish (), a recently identified DNA transposon in the zebrafish genome, and , a reconstructed transposon originally discovered in several fish species, are known to exhibit high transposition activity in vertebrate cells. Although a similar structural organization was observed for and transposons, the evolutionary profiles of their homologs in various species remain unknown. In the present study, we compared their taxonomic ranges, structural arrangements, sequence identities, evolution dynamics, and horizontal transfer occurrences in vertebrates.

Second hepatitis C virus transmission by blood components since introduction of mandatory NAT screening in Germany.

Background: Viral safety of blood products in Germany has improved significantly over the last two decades. We describe the second documented transfusion-transmitted (TT) episode for the hepatitis C virus (HCV) in Germany since mandatory nucleic acid amplification techniques (NAT) screening was introduced in 1999.

Study Design And Methods: When a repeat donor who had tested negative for anti-HCV tested positive for HCV RNA by NAT in a minipool (MP) of eight, a look-back procedure was initiated.

Molecular and Functional Characterization of BDNF-Overexpressing Human Retinal Pigment Epithelial Cells Established by Transposon-Mediated Gene Transfer.

More and more patients suffer from multifactorial neurodegenerative diseases, such as age-related macular degeneration (AMD). However, their pathological mechanisms are still poorly understood, which complicates the development of effective therapies. To improve treatment of multifactorial diseases, cell-based gene therapy can be used to increase the expression of therapeutic factors.

Influenza A virus infection instructs hematopoiesis to megakaryocyte-lineage output.

Respiratory tract infections are among the deadliest communicable diseases worldwide. Severe cases of viral lung infections are often associated with a cytokine storm and alternating platelet numbers. We report that hematopoietic stem and progenitor cells (HSPCs) sense a non-systemic influenza A virus (IAV) infection via inflammatory cytokines.

Vaccine-associated enhanced respiratory pathology in COVID-19 hamsters after T2-biased immunization.

Vaccine-associated enhanced respiratory disease (VAERD) is a severe complication for some respiratory infections. To investigate the potential for VAERD induction in coronavirus disease 2019 (COVID-19), we evaluate two vaccine leads utilizing a severe hamster infection model: a T helper type 1 (T1)-biased measles vaccine-derived candidate and a T2-biased alum-adjuvanted, non-stabilized spike protein. The measles virus (MeV)-derived vaccine protects the animals, but the protein lead induces VAERD, which can be alleviated by dexamethasone treatment.

Generation of CAR-T Cells with Sleeping Beauty Transposon Gene Transfer.

Human T lymphocytes that transgenically express a chimeric antigen receptor (CAR) have proven efficacy and safety in gene- and cell-based immunotherapy of certain hematological cancers. Appropriate gene vectors and methods of genetic engineering are required for therapeutic cell products to be biologically potent and their manufacturing to be economically viable. Transposon-based gene transfer satisfies these needs, and is currently being evaluated in clinical trials.

In Vitro Insertional Mutagenesis Screen Identifies Novel Genes Driving Breast Cancer Metastasis.

Unlabelled: Metastasis, a complex, multistep process, is responsible for the overwhelming majority of cancer-related deaths. Despite its devastating consequences, it is not possible to effectively treat cancer that has spread to vital organs, the mechanisms leading to metastasis are still poorly understood, and the catalog of metastasis promoting genes is still incomprehensive. To identify new driver genes of metastasis development, we performed an in vitro Sleeping Beauty transposon-based forward genetic screen in nonmetastatic SKBR3 human breast cancer cells.

Engineered Sleeping Beauty transposase redirects transposon integration away from genes.

The Sleeping Beauty (SB) transposon system is a popular tool for genome engineering, but random integration into the genome carries a certain genotoxic risk in therapeutic applications. Here we investigate the role of amino acids H187, P247 and K248 in target site selection of the SB transposase. Structural modeling implicates these three amino acids located in positions analogous to amino acids with established functions in target site selection in retroviral integrases and transposases.

Genome Sequences of West Nile Virus Reference Materials.

We report the sequences of two West Nile virus (WNV) strains (lineages 1 and 2) developed by the Paul-Ehrlich-Institut as reference materials. The materials are calibrated against the 1 World Health Organization WNV RNA International Standard and are intended for use in nucleic acid technology assays supporting transfusion safety.

The Flagellin:Allergen Fusion Protein rFlaA:Betv1 Induces a MyD88- and MAPK-Dependent Activation of Glucose Metabolism in Macrophages.

TLR5 ligand flagellin-containing fusion proteins are potential vaccine candidates for many diseases. A recombinant fusion protein of flagellin A and the major birch pollen allergen Bet v 1 (rFlaA:Betv1) modulates immune responses in vitro and in vivo. We studied the effects of rFlaA:Betv1 on bone marrow-derived macrophages (BMDMs).

Persistence of infectious SARS-CoV-2 particles for up to 37 days in patients with mild COVID-19.

Background: People suffering from COVID-19 are typically considered non-infectious 14 days after diagnosis if symptoms have disappeared for at least 48 h. We describe three patients who independently acquired their infection. These three patients experienced mild COVID-19 and completely recovered symptomatically within 10 days, but remained PCR-positive in deep pharyngeal samples for at least 38 days.

Genome-wide mapping of binding sites of the transposase-derived SETMAR protein in the human genome.

Throughout evolution, DNA transposons provide a recurrent supply of genetic information to give rise to novel gene functions by fusion of their transposase domain to various domains of host-encoded proteins. One of these "domesticated", transposase-derived factors is SETMAR/Metnase which is a naturally occurring fusion protein that consists of a histone-lysine methyltransferase domain and an transposase. To elucidate the biological role of SETMAR, it is crucial to identify genomic targets to which SETMAR specifically binds and link these sites to the regulation of gene expression.