Dissociating sensory and cognitive contributions to visual persistence II. Photoreceptor integration of flash pairs as a function of interflash interval, intensity, integration site, and candidate code

Patient material from rare diseases such as very early-onset inflammatory bowel disease (VEO-IBD) is often limited. The use of patient-derived induced pluripotent stem cells (iPSCs) for disease modeling is a promising approach to investigate disease pathomechanisms and therapeutic strategies. We successfully developed VEO-IBD patient-derived iPSC lines harboring a mutation in the IL-10 receptor β-chain (IL-10RB) associated with defective IL-10 signaling. To characterize the disease phenotype, healthy control and VEO-IBD iPSCs were differentiated into macrophages. IL-10 stimulation induced characteristic signal transducer and activator of transcription 3 (STAT3) and suppressor of cytokine signaling 3 (SOCS3) downstream signaling and anti-inflammatory regulation of lipopolysaccharide (LPS)-mediated cytokine secretion in healthy control iPSC-derived macrophages. In contrast, IL-10 stimulation of macrophages derived from patient iPSCs did not result in STAT3 phosphorylation and subsequent SOCS3 expression, recapitulating the phenotype of cells from patients with IL-10RB deficiency. In line with this, LPS-induced cytokine secretion (e.g., IL-6 and tumor necrosis factor-α (TNF-α)) could not be downregulated by exogenous IL-10 stimulation in VEO-IBD iPSC-derived macrophages. Correction of the IL-10RB defect via lentiviral gene therapy or genome editing in the adeno-associated virus integration site 1 (AAVS1) safe harbor locus led to reconstitution of the anti-inflammatory response. Corrected cells showed IL-10RB expression, IL-10-inducible phosphorylation of STAT3, and subsequent SOCS3 expression. Furthermore, LPS-mediated TNF-α secretion could be modulated by IL-10 stimulation in gene-edited VEO-IBD iPSC-derived macrophages. Our established disease models provide the opportunity to identify and validate new curative molecular therapies and to investigate phenotypes and consequences of additional individual IL-10 signaling pathway-dependent VEO-IBD mutations.

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Long-term lymphoid progenitors independently sustain naïve T and NK cell production in humans

Nature Communications ◽

10.1038/s41467-021-21834-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Natalia Izotova ◽

Christine Rivat ◽

Cristina Baricordi ◽

Elena Blanco ◽

Danilo Pellin ◽

...

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Nk Cell ◽

Integration Site ◽

Hematopoietic Stem ◽

Cell Production ◽

Lymphoid Progenitors ◽

Clinical Gene Therapy ◽

Cancer Immunotherapies

AbstractOur mathematical model of integration site data in clinical gene therapy supported the existence of long-term lymphoid progenitors capable of surviving independently from hematopoietic stem cells. To date, no experimental setting has been available to validate this prediction. We here report evidence of a population of lymphoid progenitors capable of independently maintaining T and NK cell production for 15 years in humans. The gene therapy patients of this study lack vector-positive myeloid/B cells indicating absence of engineered stem cells but retain gene marking in both T and NK. Decades after treatment, we can still detect and analyse transduced naïve T cells whose production is likely maintained by a population of long-term lymphoid progenitors. By tracking insertional clonal markers overtime, we suggest that these progenitors can support both T and NK cell production. Identification of these long-term lymphoid progenitors could be utilised for the development of next generation gene- and cancer-immunotherapies.

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Loss of Wnt16 Leads to Skeletal Deformities and Downregulation of Bone Developmental Pathway in Zebrafish

International Journal of Molecular Sciences ◽

10.3390/ijms22136673 ◽

2021 ◽

Vol 22 (13) ◽

pp. 6673

Author(s):

Xiaochao Qu ◽

Mei Liao ◽

Weiwei Liu ◽

Yisheng Cai ◽

Qiaorong Yi ◽

...

Keyword(s):

Gene Knockout ◽

Integration Site ◽

Skeletal Development ◽

Rna Seq ◽

Developmental Pathway ◽

Mineral Density ◽

Zebrafish Model ◽

Protein Protein Interaction ◽

Ct Analysis

Wingless-type MMTV integration site family, member 16 (wnt16), is a wnt ligand that participates in the regulation of vertebrate skeletal development. Studies have shown that wnt16 can regulate bone metabolism, but its molecular mechanism remains largely undefined. We obtained the wnt16-/- zebrafish model using the CRISPR-Cas9-mediated gene knockout screen with 11 bp deletion in wnt16, which led to the premature termination of amino acid translation and significantly reduced wnt16 expression, thus obtaining the wnt16-/- zebrafish model. The expression of wnt16 in bone-related parts was detected via in situ hybridization. The head, spine, and tail exhibited significant deformities, and the bone mineral density and trabecular bone decreased in wnt16-/- using light microscopy and micro-CT analysis. RNA sequencing was performed to explore the differentially expressed genes (DEGs). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that the down-regulated DEGs are mainly concentrated in mTOR, FoxO, and VEGF pathways. Protein–protein interaction (PPI) network analysis was performed with the detected DEGs. Eight down-regulated DEGs including akt1, bnip4, ptena, vegfaa, twsg1b, prkab1a, prkab1b, and pla2g4f.2 were validated by qRT-PCR and the results were consistent with the RNA-seq data. Overall, our work provides key insights into the influence of wnt16 gene on skeletal development.

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WGS of Commensal Neisseria Reveals Acquisition of a New Ribosomal Protection Protein (MsrD) as a Possible Explanation for High Level Azithromycin Resistance in Belgium

Pathogens ◽

10.3390/pathogens10030384 ◽

2021 ◽

Vol 10 (3) ◽

pp. 384

Author(s):

Tessa de Block ◽

Jolein Gyonne Elise Laumen ◽

Christophe Van Dijck ◽

Said Abdellati ◽

Irith De Baetselier ◽

...

Keyword(s):

Integration Site ◽

Bacterial Species ◽

Resistance Mechanism ◽

Health Concern ◽

Sex With Men ◽

High Level ◽

Ribosomal Protection Protein ◽

Commensal Neisseria ◽

Level Resistance ◽

Ribosomal Protection

In this study, we characterized all oropharyngeal and anorectal isolates of Neisseria spp. in a cohort of men who have sex with men. This resulted in a panel of pathogenic Neisseria (N. gonorrhoeae [n = 5] and N. meningitidis [n = 5]) and nonpathogenic Neisseria (N. subflava [n = 11], N. mucosa [n = 3] and N. oralis [n = 2]). A high proportion of strains in this panel were resistant to azithromycin (18/26) and ceftriaxone (3/26). Whole genome sequencing (WGS) of these strains identified numerous mutations that are known to confer reduced susceptibility to azithromycin and ceftriaxone in N. gonorrhoeae. The presence or absence of these known mutations did not explain the high level resistance to azithromycin (>256 mg/L) in the nonpathogenic isolates (8/16). After screening for antimicrobial resistance (AMR) genes, we found a ribosomal protection protein, Msr(D), in these highly azithromycin resistant nonpathogenic strains. The complete integration site originated from Streptococcus pneumoniae and is associated with high level resistance to azithromycin in many other bacterial species. This novel AMR resistance mechanism to azithromycin in nonpathogenic Neisseria could be a public health concern if it were to be transmitted to pathogenic Neisseria. This study demonstrates the utility of WGS-based surveillance of nonpathogenic Neisseria.

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Genome-wide integration site detection using Cas9 enriched amplification-free long-range sequencing

Nucleic Acids Research ◽

10.1093/nar/gkaa1152 ◽

2020 ◽

Author(s):

Joost van Haasteren ◽

Altar M Munis ◽

Deborah R Gill ◽

Stephen C Hyde

Keyword(s):

Long Range ◽

Insertional Mutagenesis ◽

Lentiviral Vector ◽

Integration Site ◽

Low Cost ◽

Safety Evaluation ◽

Read Length ◽

Chromosomal Dna ◽

Wide Range

Abstract The gene and cell therapy fields are advancing rapidly, with a potential to treat and cure a wide range of diseases, and lentivirus-based gene transfer agents are the vector of choice for many investigators. Early cases of insertional mutagenesis caused by gammaretroviral vectors highlighted that integration site (IS) analysis was a major safety and quality control checkpoint for lentiviral applications. The methods established to detect lentiviral integrations using next-generation sequencing (NGS) are limited by short read length, inadvertent PCR bias, low yield, or lengthy protocols. Here, we describe a new method to sequence IS using Amplification-free Integration Site sequencing (AFIS-Seq). AFIS-Seq is based on amplification-free, Cas9-mediated enrichment of high-molecular-weight chromosomal DNA suitable for long-range Nanopore MinION sequencing. This accessible and low-cost approach generates long reads enabling IS mapping with high certainty within a single day. We demonstrate proof-of-concept by mapping IS of lentiviral vectors in a variety of cell models and report up to 1600-fold enrichment of the signal. This method can be further extended to sequencing of Cas9-mediated integration of genes and to in vivo analysis of IS. AFIS-Seq uses long-read sequencing to facilitate safety evaluation of preclinical lentiviral vector gene therapies by providing IS analysis with improved confidence.

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New Quinoxaline Derivatives as Dual Pim-1/2 Kinase Inhibitors: Design, Synthesis and Biological Evaluation

Molecules ◽

10.3390/molecules26040867 ◽

2021 ◽

Vol 26 (4) ◽

pp. 867

Author(s):

Bruno Oyallon ◽

Marie Brachet-Botineau ◽

Cédric Logé ◽

Thomas Robert ◽

Stéphane Bach ◽

...

Keyword(s):

Colorectal Carcinoma ◽

Cancer Progression ◽

Kinase Inhibitors ◽

Integration Site ◽

Leukemia Virus ◽

Biological Evaluation ◽

Design Synthesis ◽

Proviral Integration Site ◽

Quinoxaline Derivatives

Proviral integration site for Moloney murine leukemia virus (Pim)-1/2 kinase overexpression has been identified in a variety of hematologic (e.g., multiple myeloma or acute myeloid leukemia (AML)) and solid (e.g., colorectal carcinoma) tumors, playing a key role in cancer progression, metastasis, and drug resistance, and is linked to poor prognosis. These kinases are thus considered interesting targets in oncology. We report herein the design, synthesis, structure–activity relationships (SAR) and in vitro evaluations of new quinoxaline derivatives, acting as dual Pim1/2 inhibitors. Two lead compounds (5c and 5e) were then identified, as potent submicromolar Pim-1 and Pim-2 inhibitors. These molecules were also able to inhibit the growth of the two human cell lines, MV4-11 (AML) and HCT-116 (colorectal carcinoma), expressing high endogenous levels of Pim-1/2 kinases.

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From cytopenia to leukemia: the role of Gfi1 and Gfi1b in blood formation

Blood ◽

10.1182/blood-2015-06-655043 ◽

2015 ◽

Vol 126 (24) ◽

pp. 2561-2569 ◽

Cited By ~ 51

Author(s):

Tarik Möröy ◽

Lothar Vassen ◽

Brian Wilkes ◽

Cyrus Khandanpour

Keyword(s):

Zinc Finger ◽

Target Genes ◽

Integration Site ◽

Wide Spectrum ◽

Human Leukemia ◽

Specific Expression ◽

Hematopoietic Stem ◽

Proviral Integration Site ◽

Cell Type Specific Expression

AbstractThe DNA-binding zinc finger transcription factors Gfi1 and Gfi1b were discovered more than 20 years ago and are recognized today as major regulators of both early hematopoiesis and hematopoietic stem cells. Both proteins function as transcriptional repressors by recruiting histone-modifying enzymes to promoters and enhancers of target genes. The establishment of Gfi1 and Gfi1b reporter mice made it possible to visualize their cell type–specific expression and to understand their function in hematopoietic lineages. We now know that Gfi1 is primarily important in myeloid and lymphoid differentiation, whereas Gfi1b is crucial for the generation of red blood cells and platelets. Several rare hematologic diseases are associated with acquired or inheritable mutations in the GFI1 and GFI1B genes. Certain patients with severe congenital neutropenia carry mutations in the GFI1 gene that lead to the disruption of the C-terminal zinc finger domains. Other mutations have been found in the GFI1B gene in families with inherited bleeding disorders. In addition, the Gfi1 locus is frequently found to be a proviral integration site in retrovirus-induced lymphomagenesis, and new, emerging data suggest a role of Gfi1 in human leukemia and lymphoma, underlining the role of both factors not only in normal hematopoiesis, but also in a wide spectrum of human blood diseases.

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