Biology subjects

Doyon, Y.

Publications and source records attributed to Doyon, Y..

9 recordsLinked to original sources

bioRxiv2025

Autophagy disruption primes CAR-T cell metabolism for sustained rejection of ovarian tumors

T-cell based immunotherapies such as chimeric antigen receptor T (CAR-T) cell therapy face substantial hurdles when confronting solid tumors such as ovarian cancer, where metabolic constraints in the tumor microenvironment limit T cell infiltration and function. In particular, T cells exposed to nutrient deprivation and hypoxia upregulate autophagy, a lysosomal degradation pathway that negatively regulates effector responses. Here, we used CRISPR-Cas9 to target a folate receptor alpha (FR) CAR expression cassette into the locus of the essential autophagy gene ATG5, thereby generating autophagy-deficient CAR-T cells in a single editing step. Targeted metabolite profiling revealed that deletion of ATG5 induced widespread metabolic reprogramming characterized by increased glucose and amino acid uptake. Functionally, ATG5-knockout CAR-T cells maintained high cytolytic activity when assayed in patient-derived ascites in vitro, and exhibited superior and long-lasting tumor control against ovarian tumors in vivo. Taken together, our results suggest that deletion of ATG5 metabolically primes CAR-T cells for enhanced cytotoxicity in immune-suppressive conditions, thereby improving the therapeutic potential of FR CAR-T cells for ovarian cancer immunotherapy.

immunology↗

bioRxiv2025

NTBC dosing and outcomes in hereditary tyrosinemia type 1: insights from a representative human model and 99 patients

Hereditary tyrosinemia type 1 (HT1) is a rare and severe metabolic liver disorder caused by fumarylacetoacetate hydrolase (FAH) deficiency. The optimal dose and long-term effects of the only available treatment, nitisinone (NTBC), remain unclear due to the absence of clinical trial data. Here, we generated a representative human in vitro model of HT1 using iPSC-derived hepatocytes, which faithfully recapitulated key disease features. We investigated the mechanisms of FAH deficiency-induced hepatocellular injury and evaluated the effects of NTBC treatment. We confirmed treatment efficacy and identified 50 {micro}mol/L as the minimal effective NTBC concentration to prevent cellular damage. This protective dose was subsequently validated in a large cohort of 99 HT1 patients, providing compelling evidence for establishing minimal therapeutic NTBC levels. Notably, approximately 10% of disease-associated genes, many implicated in hepatocellular carcinoma, remained dysregulated despite treatment, raising concerns that NTBC may not fully eliminate long-term oncogenic risk.

genetics↗

bioRxiv2023

In vivo dissection of the mouse tyrosine catabolic pathway with CRISPR-Cas9 identifies modifier genes affecting hereditary tyrosinemia type 1

Hereditary tyrosinemia type 1 is an autosomal recessive disorder caused by mutations (pathogenic variants) in fumarylacetoacetate hydrolase, an enzyme involved in tyrosine degradation. Its loss results in the accumulation of toxic metabolites that mainly affect the liver and kidneys and can lead to severe liver disease and liver cancer. Tyrosinemia type 1 has a global prevalence of approximately 1 in 100,000 births but can reach up to 1 in 1,500 births in some regions of Quebec, Canada. Mutating functionally related modifier genes (i.e., genes that, when mutated, affect the phenotypic impacts of mutations in other genes) is an emerging strategy for treating human genetic diseases. In vivo somatic genome editing in animal models of these diseases is a powerful means to identify modifier genes and fuel treatment development. In this study, we demonstrate that mutating additional enzymes in the tyrosine catabolic pathway through liver-specific genome editing can relieve or worsen the phenotypic severity of a murine model of tyrosinemia type 1. Neonatal gene delivery using recombinant adeno-associated viral vectors expressing Staphylococcus aureus Cas9 under the control of a liver-specific promoter led to efficient gene disruption and metabolic rewiring of the pathway, with systemic effects that were distinct from the phenotypes observed in whole-body knockout models. Our work illustrates the value of using in vivo genome editing in model organisms to study the direct effects of combining pathological mutations with modifier gene mutations in isogenic settings.

genetics↗

bioRxiv2023

MTOR as a selectable genomic harbor for CRISPR-engineered CAR-T cell therapy

Chimeric antigen receptors (CARs) reprogram T cells to recognize and target cancer cells. Despite remarkable responses observed with CAR-T cell therapy in patients with hematological malignancies, CAR-T cell engineering still relies mostly on randomly integrating vectors, limiting the possibilities of fine-tuning T cell function. Here, we designed a CRISPR-based marker-free selection strategy to simultaneously target a therapeutic transgene and a gain-of-function mutation to the MTOR locus to enrich cells resistant to rapamycin, a clinically used immunosuppressant. We readily engineered rapamycin-resistant (RapaR) CAR-T cells by targeting CAR expression cassettes to the MTOR locus. Using in vitro cytotoxicity assays, and a humanized mouse model of acute lymphoblastic leukemia, we show that RapaR-CAR-T cells can efficiently target CD19+ leukemia cells in presence of immunosuppressing doses of rapamycin. Furthermore, our strategy allows multiplexed targeting of rapamycin-regulated immunoreceptors complexes (DARICs) to the MTOR and TRAC loci to pharmacologically control CAR-T cells activity. We foresee that our approach could both facilitate the enrichment of CRISPR-engineered CAR-T cells ex vivo and in vivo while improving tumor eradication.

cancer biology↗

bioRxiv2022

Accessory-cell-free differentiation of hematopoietic stem and progenitor cells into mature red blood cells

The culture and ex vivo engineering of red blood cells (RBCs) can help characterize genetic variants, model diseases, and may eventually spur the development of applications in transfusion medicine. In the last decade, improvements to the in vitro production of RBCs have enabled efficient erythroid progenitor proliferation and high enucleation levels from several sources of hematopoietic stem and progenitor cells (HSPCs). Despite these advances, there remains a need for refining the terminal step of in vitro human erythropoiesis -- i.e., the terminal maturation of reticulocytes into erythrocytes -- so that it can occur without feeder or accessory cells and animal components. Here, we describe the near-complete erythroid differentiation of cultured RBCs (cRBCs) from adult HSPCs in accessory-cell-free and animal-component-free conditions. The approach improves post-enucleation cell integrity and cell survival, and enables subsequent storage of cRBCs for up to 42 days in classical nutritive solution conditions, without any specialized equipment. We foresee that these improvements will facilitate the characterization of RBCs derived from gene-edited HSPCs. KEY POINTSO_LIErythroid progenitors were differentiated into fully mature RBCs in a medium free of accessory cells C_LIO_LICultured RBCs can be stored up to 42 days in a standard nutritive solution C_LI

cell biology↗

bioRxiv2021

Marker-free coselection for successive rounds of prime editing in human cells

Prime editing enables the introduction of precise point mutations, small insertions, or short deletions without requiring donor DNA templates. However, efficiency remains a key challenge in a broad range of human cell types. In this work, we designed a robust coselection strategy through coediting of the ubiquitous and essential sodium/potassium pump (Na+/K+ ATPase). We readily engineered highly modified pools of cells and clones with homozygous modifications for functional studies with minimal pegRNA optimization. This process revealed that nicking the non-edited strand stimulates multiallelic editing but often generates tandem duplications and large deletions at the target site, an outcome dictated by the relative orientation of the protospacer adjacent motifs. Our approach streamlines the production of cell lines with multiple genetic modifications to create cellular models for biological research and lays the foundation for the development of cell-type specific coselection strategies.

molecular biology↗

bioRxiv2021

Recurrent chromosomal translocations in sarcomas create a mega-complex that mislocalizes NuA4/TIP60 to Polycomb target loci

Chromosomal translocations frequently promote carcinogenesis by producing gain-of-function fusion proteins. Recent studies have identified highly recurrent chromosomal translocations in patients with Endometrial Stromal Sarcomas (ESS) and Ossifying FibroMyxoid Tumors (OFMT) leading to an in-frame fusion of PHF1 (PCL1) to six different subunits of the NuA4/TIP60 complex. While NuA4/TIP60 is a co-activator that acetylates chromatin and loads the H2A.Z histone variant, PHF1 is part of the Polycomb repressive complex 2 (PRC2) linked to transcriptional repression of key developmental genes through methylation of histone H3 on lysine 27. In this study, we characterize the fusion protein produced by the EPC1-PHF1 translocation. The chimeric protein assembles a mega-complex harboring both NuA4/TIP60 and PRC2 activities and leads to mislocalization of chromatin marks in the genome, in particular over an entire topologically- associating domain including part of the HOXD cluster. This is linked to aberrant gene expression, most notably increased expression of PRC2 target genes. Furthermore, we show that JAZF1, implicated with a PRC2 component in the most frequent translocation in ESS, JAZF1-SUZ12, is a potent transcription activator that physically associates with NuA4/TIP60, its fusion creating similar outcomes as EPC1-PHF1. Importantly, the specific increased expression of PRC2 targets/HOX genes was also confirmed with ESS patient samples. Altogether, these results indicate that most chromosomal translocations linked to these sarcomas employ the same molecular oncogenic mechanism through a physical merge of NuA4/TIP60 and PRC2 complexes leading to mislocalization of histone marks and aberrant polycomb target gene expression.

molecular biology↗

bioRxiv2018

Versatile in vivo genome editing with the prototypical Streptococcus thermophilus CRISPR1-Cas9 system

Targeting definite genomic locations using CRISPR-Cas systems requires a set of enzymes with unique protospacer adjacent motif (PAM) compatibilities. To expand this repertoire, we engineered nucleases, cytosine base editors, and adenine base editors from the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9) system. We found that St1Cas9 strain variants enable targeting to five distinct A-rich PAMs and provide structural basis for their specificities. The small size of this ortholog enables expression of the holoenzyme from a single adeno-associated viral vector for in vivo editing applications. Delivery of St1Cas9 to the neonatal liver efficiently rewired metabolic pathways, leading to phenotypic rescue in a mouse model of hereditary tyrosinemia. These robust enzymes expand and complement current editing platforms available for tailoring mammalian genomes.

molecular biology↗

bioRxiv2017

A marker-free co-selection strategy for high efficiency human genome engineering

Targeted genome editing using engineered nucleases facilitates the creation of bona fide cellular models for biological research and may be applied to human cell-based therapies. Broadly applicable and versatile methods for increasing the levels of gene editing in cell populations remain highly desirable due to the variable efficiency between distinct genomic loci and cell types. Harnessing the multiplexing capabilities of CRISPR-Cas9 and Cpf1 systems, we designed a simple and robust co-selection strategy for enriching cells harboring either nuclease-driven non-homologous end joining (NHEJ) or homology-directed repair (HDR) events. Selection for dominant alleles of the endogenous sodium-potassium pump (Na+,K+-ATPase) that render cells resistant to ouabain is used to enrich for custom modifications at another unlinked locus of interest, effectively increasing the recovery of engineered cells. The process was readily adaptable to transformed and primary cells, including hematopoietic stem and progenitor cells (HSPCs). The use of universal CRISPR reagents and a commercially available small molecule inhibitor streamlines the incorporation of marker-free genetic changes in human cells.

molecular biology↗