Search bioRxiv⌕ Search

Biology subjects

Corre, G.

Publications and source records attributed to Corre, G..

7 recordsLinked to original sources
bioRxiv2026

CRISPR-Mediated Linearization of IDLV Donor Enables Precise Targeted Integration in Human Hematopoietic Stem Cells

Ex vivo genome editing of human hematopoietic stem and progenitor cells (HSPCs) requires targeted integration strategies that support large therapeutic DNA payloads while preserving stem cell fitness. Although CRISPR/Cas9-mediated homology-directed repair using AAV donors is effective, it is constrained by limited cargo capacity and adverse effects on long-term HSPCs function. Integrase-defective lentiviral vectors (IDLVs) offer an alternative donor platform, yet their precise and controlled genomic integration remains inefficient. Here, we describe TILV (Targeted Integration of Lentiviral Vector), a CRISPR-assisted knock-in strategy that exploits Cas9-mediated linearization of episomal IDLV DNA to expose a single homology arm and engage homology-mediated end-joining repair pathways. TILV enables precise, directional and seamless integration of transgenes in multiple loci, enabling constitutive or physiological expression. Using single-cell clonal analyses and targeted long-read sequencing, we define the molecular features of TILV-mediated integration and demonstrate preferential use of CRISPR-linearized episomal substrates. TILV supports accurate insertion of large therapeutic transgenes, without compromising HSPC viability or multilineage potential. We further show that transient modulation of DNA repair pathway, in combination with extended homology arms, enhances integration efficiency and junctional precision. Importantly, optimized TILV enables targeted integration in phenotypically defined long-term HSPCs, highlighting its potential for scalable and durable gene therapy.

molecular biology↗
bioRxiv2025

Advanced pipeline for CRISPR/Cas9 offtargets detection in Guide-seq and related integration-based assays

We developed GENETHOFF, a flexible single-command versatile Snakemake workflow designed for the comprehensive analysis of CRISPR/Cas9 related OFF-targets genomic positions from GUIDE-Seq derived protocols. It efficiently processes multiplexed libraries from different organisms, PCR orientations, and Cas nucleases with varying PAM specificities in a single run, all based on a simple user-specified datasheet containing sample metadata.

bioinformatics↗
bioRxiv2024

Rescue of lysosomal acid lipase deficiency in mice by rAAV8 liver gene transfer.

Lysosomal acid lipase deficiency (LAL-D) is an autosomal recessive disorder caused by mutations in the LIPA gene, which results in lipid accumulation leading to multi-organ failure. If left untreated, the severe form of LAL-D results in premature death within the first year of life due to failure to thrive and hepatic insufficiency. Enzyme replacement therapy is the only available supportive treatment consisting in weekly systemic injections of recombinant LAL protein. Here, we characterized a novel Lipa-/- mouse model and developed a curative gene therapy treatment based on the in vivo administration of recombinant (r)AAV8 vector encoding the human LIPA transgene under the control of a hepatocyte-specific promoter. We defined the minimal rAAV8 dose required to rescue disease lethality and to correct cholesterol and triglyceride accumulation in multiple organs and blood. Finally, using liver transcriptomic and biochemical analysis, we showed mitochondrial impairment in Lipa-/- mice and its recovery by gene therapy. Overall, our in vivo gene therapy strategy achieves a stable long-term LAL expression sufficient to correct the disease phenotype in the Lipa-/-mouse model and offers a new therapeutic option for LAL-D patients. One Sentence SummaryWeve developed a liver-targeted gene therapy using recombinant AAV8 to effectively cure Lysosomal acid lipase deficiency by correcting lipid accumulation and by normalizing gene expression pattern and mitochondrial function in Lipa-/- mouse model.

molecular biology↗
bioRxiv2020

Cholesterol metabolism is a potential therapeutic target in Duchenne Muscular Dystrophy

BackgroundDuchenne Muscular Dystrophy (DMD) is a lethal muscle disease detected in approximately 1:5000 male births. DMD is caused by mutations in the DMD gene, encoding a critical protein that link the cytoskeleton and the extracellular matrix in skeletal and cardiac muscles. The primary consequence of the disrupted link between the extracellular matrix and the myofiber actin cytoskeleton is thought to involve sarcolemma destabilization, perturbation of Ca+2 homeostasis, activation of proteases, mitochondrial damage and tissue degeneration. A recently emphasized secondary aspect of the dystrophic process is a progressive metabolic change of the dystrophic tissue; however, the mechanism and nature of the metabolic dysregulation is yet poorly understood. In this study, we characterized a molecular mechanism of metabolic perturbation in DMD. MethodsWe sequenced plasma miRNA in a DMD cohort, comprising of 54 DMD patients treated or not by glucocorticoid, compared to 27 healthy controls, in three age groups. We developed an original approach for the biological interpretation of miRNA dysregulation, and produced a novel hypothesis concerning metabolic perturbation in DMD. We then used the mdx mouse model for DMD for the investigation of this hypothesis. ResultsWe identified 96 dysregulated miRNAs, of which 74 were up- and 22 down-regulated in DMD. We confirmed the dysregulation in DMD of Dystro-miRs, Cardio-miRs and a large number of the DLK1-DIO3 miRNAs. We also identified numerous dysregulated miRNAs, yet unreported in DMD. Bioinformatics analysis of both target and host genes for dysregulated miRNAs predicted that lipid metabolism might be a critical metabolic perturbation in DMD. Investigation of skeletal muscles of the mdx mouse uncovered dysregulation of transcription factors of cholesterol and fatty acid metabolism (SREBP1 and SREBP2), perturbation of the mevalonate pathway, and accumulation of cholesterol. Elevated cholesterol level was also found in muscle biopsies of DMD patients. Treatment of mdx mice with Simvastatin, a cholesterol-reducing agent, normalized these perturbations and partially restored the dystrophic parameters. ConclusionThis investigation supports that cholesterol metabolism and the mevalonate pathway are potential therapeutic targets in DMD.

genetics↗
bioRxiv2020

Selective silencing rather than targeted activation of gene expression underlies fate choice in human hematopoietic stem cells.

When human cord blood derived CD34+ cells are induced to differentiate in vitro, they undergo rapid and dynamic morphological and molecular transformations that are critical for fate commitment. Using ATAC-seq and single-cell RNA sequencing, we detected two phases in this process. In the first phase, we observed a rapid and global chromatin opening that makes most of the gene promoters in the genome accessible, followed by widespread upregulation of gene transcription and a concomitant increase in the cell-to-cell variability of gene expression. The second phase is marked by a slow chromatin closure and a subsequent overall downregulation of gene transcription and emergence of coherent expression profiles corresponding to distinct cell subpopulations. These observations are consistent with a model based on the spontaneous probabilistic organization of the cellular process of fate commitment.

cell biology↗
bioRxiv2017

Integrated time-lapse and single-cell transcription studies highlight the variable and dynamic nature of human hematopoietic cell fate commitment.

Individual cells take lineage commitment decisions in a way that is not necessarily uniform. We address this issue by characterizing transcriptional changes in cord blood derived CD34+ cells at the single-cell level and integrating data with cell division history and morphological changes determined by time-lapse microscopy. We show, that major transcriptional changes leading to a multilineage-primed gene expression state occur very rapidly during the first cell cycle. One of the two stable lineage-primed patterns emerges gradually in each cell with variable timing. Some cells reach a stable morphology and molecular phenotype by the end of the first cell cycle and transmit it clonally. Others fluctuate between the two phenotypes over several cell cycles. Our analysis highlights the dynamic nature and variable timing of cell fate commitment in hematopoietic cells, links the gene expression pattern to cell morphology and identifies a new category of cells with fluctuating phenotypic characteristics, demonstrating the complexity of the fate decision process, away from a simple binary switch between two options as it is usually envisioned.

cell biology↗