Engineering a bacterial toxin deaminase from the DYW-family into a novel cytosine base editor for plants and mammalian cells.

Base editors are precise editing tools that employ deaminases to modify target DNA bases. The DYW-family of cytosine deaminases is structurally and phylogenetically distinct and might be harnessed for genome editing tools. We report a novel CRISPR/Cas9-cytosine base editor using SsdA, a DYW-like deaminase and bacterial toxin.

Alpharetroviral Vector-Mediated Gene Therapy for IL7RA-Deficient Severe Combined Immunodeficiency.

Severe combined immunodeficiency (SCID) encompasses rare primary immunodeficiency disorders characterized by deficient T-cell development, which leads to a severely compromised immune system and susceptibility to life-threatening infections. Among SCID subtypes, IL7RA-SCID is caused by mutations in the interleukin 7 receptor alpha chain (IL7RA) and represents a significant subset of patients with limited treatment options. This study investigated the efficacy of a self-inactivating (SIN) alpharetroviral vector (ARV) engineered to deliver a codon-optimized cDNA to restore T-cell development in -knockout mice.

HYKYHT: A versatile, affordable, and open-source 3D-printed liquid aspiration system.

This paper aims to provide the details for making affordable single and multichannel liquid aspirators for wet labs. A liquid aspirator is a basic laboratory device that can cost several hundred Euros. We present a < €25 3D print solution that performs equally well in daily lab routines and is compatible with various vacuum sources, including an aquarium pump or household vacuum cleaner.

A tetravalent bispecific antibody outperforms the combination of its parental antibodies and neutralizes diverse SARS-CoV-2 variants.

The devastating impact of COVID-19 on global health shows the need to increase our pandemic preparedness. Recombinant therapeutic antibodies were successfully used to treat and protect at-risk patients from COVID-19. However, the currently circulating Omicron subvariants of SARS-CoV-2 are largely resistant to therapeutic antibodies, and novel approaches to generate broadly neutralizing antibodies are urgently needed.

Capsid-modified adeno-associated virus vectors as novel vaccine platform for cancer immunotherapy.

Immunotherapy has significantly improved treatment outcomes in various cancer entities. To enhance immunogenicity and efficacy, and to further broaden its applicability, co-administration of anti-tumor vaccines is considered as a promising strategy. Here, we introduce adeno-associated virus (AAV) vectors, widely used for gene therapy, as a potent cancer vaccine platform.

Generation of hiPSC-derived low threshold mechanoreceptors containing axonal termini resembling bulbous sensory nerve endings and expressing Piezo1 and Piezo2.

Somatosensory low threshold mechanoreceptors (LTMRs) sense innocuous mechanical forces, largely through specialized axon termini termed sensory nerve endings, where the mechanotransduction process initiates upon activation of mechanotransducers. In humans, a subset of sensory nerve endings is enlarged, forming bulb-like expansions, termed bulbous nerve endings. There is no in vitro human model to study these neuronal endings.

Predicting genotoxicity of viral vectors for stem cell gene therapy using gene expression-based machine learning.

Hematopoietic stem cell gene therapy is emerging as a promising therapeutic strategy for many diseases of the blood and immune system. However, several individuals who underwent gene therapy in different trials developed hematological malignancies caused by insertional mutagenesis. Preclinical assessment of vector safety remains challenging because there are few reliable assays to screen for potential insertional mutagenesis effects in vitro.

An Improved Lentiviral Fluorescent Genetic Barcoding Approach Distinguishes Hematopoietic Stem Cell Properties in Multiplexed Experiments.

Hematopoietic stem cells (HSCs) represent a rare cell population of particular interest for biomedical research and regenerative medicine. Various marker combinations enable the isolation of HSCs, but fail to reach purity in transplantation assays. To reduce animal consumption, we developed a multiplexing system based on lentiviral fluorescent genetic barcoding (FGB) to enable the parallel characterization of multiple HSC samples within single animals.

Conditionally immortalised leukaemia initiating cells co-expressing Hoxa9/Meis1 demonstrate microenvironmental adaptation properties ex vivo while maintaining myelomonocytic memory.

Regulation of haematopoietic stem cell fate through conditional gene expression could improve understanding of healthy haematopoietic and leukaemia initiating cell (LIC) biology. We established conditionally immortalised myeloid progenitor cell lines co-expressing constitutive Hoxa9.EGFP and inducible Meis1.

Multiple Genes Surrounding , a Common Retroviral Insertion Site, Can Influence Hematopoiesis Individually or in Concert.

Retroviral insertional mutagenesis (RIM) is both a relevant risk in gene therapy and a powerful tool for identifying genes that enhance the competitiveness of repopulating hematopoietic stem and progenitor cells (HSPCs). However, focusing only on the gene closest to the retroviral vector insertion site (RVIS) may underestimate the effects of RIM, as dysregulation of distal and/or multiple genes by a single insertion event was reported in several studies. As a proof of concept, we examined the common insertion site (CIS) , which revealed seven genes located within ±150 kb from the RVIS for our study.

Competitive sgRNA Screen Identifies p38 MAPK as a Druggable Target to Improve HSPC Engraftment.

Previous gene therapy trials for X-linked chronic granulomatous disease (X-CGD) lacked long-term engraftment of corrected hematopoietic stem and progenitor cells (HSPCs). Chronic inflammation and high levels of interleukin-1 beta (IL1B) might have caused aberrant cell cycling in X-CGD HSPCs with a concurrent loss of their long-term repopulating potential. Thus, we performed a targeted CRISPR-Cas9-based sgRNA screen to identify candidate genes that counteract the decreased repopulating capacity of HSPCs during gene therapy.

Inducible Forward Programming of Human Pluripotent Stem Cells to Hemato-endothelial Progenitor Cells with Hematopoietic Progenitor Potential.

Induced pluripotent stem cells (iPSCs) offer a promising platform to model early embryonic developmental processes, to create disease models that can be evaluated by drug screens as well as proof-of-concept experiments for regenerative medicine. However, generation of iPSC-derived hemato-endothelial and hematopoietic progenitor cells for these applications is challenging due to variable and limited cell numbers, which necessitates enormous up-scaling or development of demanding protocols. Here, we unravel the function of key transcriptional regulators SCL, LMO2, GATA2, and ETV2 (SLGE) on early hemato-endothelial specification and establish a fully inducible and stepwise hemato-endothelial forward programming system based on SLGE-regulated overexpression.

Cytokine Selection of MSC Clones with Different Functionality.

Mesenchymal stromal cells (MSCs) are used in many clinical applications. However, ex vivo expansion is required to reach clinically relevant cell numbers, which might lead to selection of clones with different characteristics. To follow clonal selection, we transduced MSC progenitors in umbilical cord pieces (UCPs) with vectors encoding fluorescent proteins and genetic barcodes.

Comparison of Tetracycline-regulated Promoters in Lentiviral-based Vectors in Murine Transplantation Studies.

Tetracycline-regulated systems with efficient temporal and dose regulation of transgene expression are useful for development of new physiologic/pathophysiologic experimental models and gene therapy approaches. Lentiviral vectors with improved tetracycline-regulated promoters help to overcome the existing limitations such as basal activity in the drug absence, poor inducibility or unstable transgene expression. To compare conventional and improved tetracycline-regulated promoters in lentiviral based vectors in vivo, we investigated doxycycline-regulated gene transfer/expression levels in a long-term murine transplantation model and demonstrated that the lentiviral vector with the improved T11 promoter exhibited more efficient inducibility and higher gene transfer level.

Genetic reporter analysis reveals an expandable reservoir of OCT4+ cells in adult skin.

The transcription factor Oct4 (Pou5f1) is a critical regulator of pluripotency in embryonic and induced pluripotent stem cells. Therefore, Oct4 expression might identify somatic stem cell populations with inherent multipotent potential or a propensity for facilitated reprogramming. However, analysis of Oct4 expression is confounded by Oct4 pseudogenes or non-pluripotency-related isoforms.

Dose response and clonal variability of lentiviral tetracycline-regulated vectors in murine hematopoietic cells.

Tetracycline-regulated integrating vectors allow pharmacologically controlled genetic modification of murine and human hematopoietic stem cells and provide the opportunity for time- and dose-controlled reversible transgene expression in hematopoietic stem cells in vitro and in vivo. However, the background activity of tetracycline-regulated promoters (tetPs) in the absence of induction or vector integration in the vicinity of proto-oncogenes can diminish the advantages of the system. Here we investigated the effect of lentiviral transduction rate on tetP background activity, vector copy number (VCN), and clonal variability as a consequence of vector integration.

All-in-One inducible lentiviral vector systems based on drug controlled FLP recombinase.

Site specific recombinases are frequently used as gene switches in transgenic animals where recombination is induced by drug treatment or by tissue specific recombinase expression. Alternatively, lentiviral gene transfer can be utilized for the genetic modification of a wide variety of cell types, albeit systems for tight control of transcriptional activity are scarce. Here, we combined lentiviral gene transfer and the development of a tightly drug-controlled FLP recombinase for the construction of "All-in-One" inducible gene expression systems.