Biology subjects

French, D. L.

Publications and source records attributed to French, D. L..

8 recordsLinked to original sources

bioRxiv2025

CRISPR-mediated transcriptional activation as a mutation-independent therapeutic strategy for SYNGAP1-related intellectual disability

Synaptic Ras GTPase-activating protein (SynGAP) regulates synaptic strength and neuronal signaling, with essential roles in cortical development and synaptic plasticity. Heterozygous loss-of-function variants in SYNGAP1 cause SYNGAP1-related intellectual disability (SRID), a severe neurodevelopmental disorder characterized by epilepsy, developmental delay, and autism. SYNGAP1 mutations often result in haploinsufficiency, providing a strong rationale for gene-targeted therapies. However, no treatment currently addresses the underlying genetic cause of SRID. Here, we developed a CRISPR-mediated transcriptional activation (CRISPRa) approach to upregulate the functional Syngap1 allele in a SRID mouse model. CRISPRa activated Syngap1, normalized SynGAP protein expression and downstream signaling, and rescued working memory deficits. We validated the translational potential of this strategy in human induced pluripotent stem cell (hiPSC)-derived excitatory cortical neurons. CRISPRa rescued SYNGAP1 in two distinct loss-of-function variant lines. Together, these findings demonstrate the feasibility of mutation-independent transcriptional activation as a therapeutic approach for SRID and its broader applicability to haploinsufficiency disorders.

neuroscience↗

bioRxiv2024

Single-cell transcriptomics reveal synergistic and antagonistic effects of T21 and GATA1s on hematopoiesis

Trisomy 21 (T21), or Down syndrome (DS), is associated with baseline macrocytic erythrocytosis, thrombocytopenia, and neutrophilia, as well as transient abnormal myelopoiesis (TAM) and myeloid leukemia of DS (ML-DS). TAM and ML-DS blasts both arise from an aberrant megakaryocyte-erythroid progenitor and exclusively express GATA1s, the truncated isoform of GATA1, while germline GATA1s mutations in a non-T21 context lead to congenital cytopenia(s) without a leukemic predisposition. This suggests that T21 and GATA1s both perturb hematopoiesis in multipotent progenitors, but studying their individual effects is challenging due to limited access to relevant human progenitor populations. To dissect individual developmental impacts, we used single-cell RNA-sequencing to interrogate hematopoietic progenitor cells (HPCs) from isogenic human induced pluripotent stem cells differing only by chromosome 21 and/or GATA1 status. The transcriptomes of these HPCs revealed significant heterogeneity and lineage skew dictated by T21 and/or GATA1s. T21 and GATA1s each disrupted temporal regulation of lineage-specific transcriptional programs and specifically perturbed cell cycle genes. Trajectory inference revealed that GATA1s nearly eliminated erythropoiesis, slowed MK maturation, and promoted myelopoiesis in the euploid context, while in T21 cells, GATA1s competed with the enhanced erythropoiesis and impaired megakaryopoiesis driven by T21 to promote production of immature erythrocytes, MKs, and myeloid cells. The use of isogenic cells revealed distinct transcriptional programs that can be attributed specifically to T21 and GATA1s, and how they independently and synergistically result in HPC proliferation at the expense of maturation, consistent with a pro-leukemic phenotype.

molecular biology↗

bioRxiv2023

A novel iPSC model reveals selective vulnerability of neurons in Multiple Sulfatase Deficiency

Multiple sulfatase deficiency (MSD) is an ultra-rare, inherited lysosomal storage disease caused by mutations in the gene sulfatase modifying factor 1 (SUMF1). MSD is characterized by the functional deficiency of all sulfatase enzymes, leading to the storage of sulfated substrates including glycosaminoglycans (GAGs), sulfolipids, and steroid sulfates. Patients with MSD experience severe neurological impairment, hearing loss, organomegaly, corneal clouding, cardiac valve disease, dysostosis multiplex, contractures, and ichthyosis. Here, we generated a novel human model of MSD by reprogramming patient peripheral blood mononuclear cells to establish an MSD iPSC line (SUMF1 p.A279V). We also generated an isogenic control iPSC line by correcting the pathogenic variant with CRISPR/Cas9 gene editing. We successfully differentiated these iPSC lines into neural progenitor cells (NPCs) and NGN2-induced neurons (NGN2-iN) to model the neuropathology of MSD. Mature neuronal cells exhibited decreased SUMF1 gene expression, increased lysosomal stress, impaired neurite outgrowth and maturation, reduced sulfatase activities, and GAG accumulation. Interestingly, MSD iPSCs and NPCs did not exhibit as severe of phenotypes, suggesting that as neurons differentiate and mature, they become more vulnerable to loss of SUMF1. In summary, we demonstrate that this human iPSC-derived neuronal model recapitulates the cellular and biochemical features of MSD. These cell models can be used as tools to further elucidate the mechanisms of MSD pathology and for the development of therapeutics.

cell biology↗

bioRxiv2023

Cross-site reproducibility of human cortical organoids reveals consistent cell type composition and architecture

BackgroundReproducibility of human cortical organoid (hCO) phenotypes remains a concern for modeling neurodevelopmental disorders. While guided hCO protocols reproducibly generate cortical cell types in multiple cell lines at one site, variability across sites using a harmonized protocol has not yet been evaluated. We present an hCO cross-site reproducibility study examining multiple phenotypes. MethodsThree independent research groups generated hCOs from one induced pluripotent stem cell (iPSC) line using a harmonized miniaturized spinning bioreactor protocol. scRNA-seq, 3D fluorescent imaging, phase contrast imaging, qPCR, and flow cytometry were used to characterize the 3 month differentiations across sites. ResultsIn all sites, hCOs were mostly cortical progenitor and neuronal cell types in reproducible proportions with moderate to high fidelity to the in vivo brain that were consistently organized in cortical wall-like buds. Cross-site differences were detected in hCO size and morphology. Differential gene expression showed differences in metabolism and cellular stress across sites. Although iPSC culture conditions were consistent and iPSCs remained undifferentiated, primed stem cell marker expression prior to differentiation correlated with cell type proportions in hCOs. ConclusionsWe identified hCO phenotypes that are reproducible across sites using a harmonized differentiation protocol. Previously described limitations of hCO models were also reproduced including off-target differentiations, necrotic cores, and cellular stress. Improving our understanding of how stem cell states influence early hCO cell types may increase reliability of hCO differentiations. Cross-site reproducibility of hCO cell type proportions and organization lays the foundation for future collaborative prospective meta-analytic studies modeling neurodevelopmental disorders in hCOs.

neuroscience↗

bioRxiv2023

Generation of a human Tropomyosin 1 knockout iPSC line

The CHOPWT17_TPM1KOc28 iPSC line was generated to interrogate the functions of Tropomyosin 1 (TPM1) in primary human cell development. This line was reprogrammed from a previously published wild type control iPSC line.

cell biology↗

bioRxiv2022

Mapping PTBP splicing in human brain identifies targets for therapeutic splice switching including SYNGAP1

Alternative splicing of neuronal genes is controlled in part by the coordinated action of the polypyrimidine tract binding proteins (PTBP1 and PTBP2). While PTBP1 is ubiquitously expressed, PTBP2 is predominantly neuronal, controlling the expression of such targets as DLG4, which encodes PSD95, a protein important in synaptic function whose deficiency causes neurodevelopmental disorders. Here, we fully define the PTBP2 footprint in the human transcriptome using both human brain tissue and neurons derived from human induced pluripotent stem cells (iPSC-neurons). We identify direct PTBP2 binding sites and define PTBP2-dependent alternative splicing events, finding novel targets such as STXBP1 and SYNGAP1, which are synaptic genes also associated with neurodevelopmental disorders. The resultant PTBP2 binding and splicing maps were used to test if PTBP2 binding could be manipulated to increase gene expression in PTBP-targeted genes that cause disease when haploinsufficient. We find that PTBP2 binding to SYNGAP1 mRNA promotes alternative splicing and non-sense mediated decay. Antisense oligonucleotides that disrupt PTBP binding sites on SYNGAP1 redirect splicing and increase gene and protein expression. Collectively, our data provide a comprehensive view of PTBP2-dependent alternative splicing in human neurons and human cerebral cortex, guiding the development of novel therapeutic tools that may benefit a range of neurodevelopmental disorders.

molecular biology↗

bioRxiv2021

PPARdelta signaling activation improves metabolic and contractile maturation of human pluripotent stem cell-derived cardiomyocytes.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) provide an unprecedented opportunity to study human heart development and disease. A major caveat however is that they remain functionally and structurally immature in culture, limiting their potential for disease modeling and regenerative approaches. Here, we address the question of how different metabolic pathways can be modulated in order to induce efficient hPSC-CM maturation. We show that PPAR signaling acts in an isoform-specific manner to balance glycolysis and fatty acid oxidation (FAO). PPARD activation or inhibition results in efficient respective up- or down-regulation of the gene regulatory networks underlying FAO in hPSC-CMs. PPARD induction further increases mitochondrial and peroxisome content, enhances mitochondrial cristae formation and augments FAO flux. Lastly PPARD activation results in enhanced myofibril organization and improved contractility. Transient lactate exposure, commonly used in hPSC-CM purification protocols, induces an independent program of cardiac maturation, but when combined with PPARD activation equally results in a metabolic switch to FAO. In summary, we identify multiple axes of metabolic modifications of hPSC-CMs and a role for PPARD signaling in inducing the metabolic switch to FAO in hPSC-CMs. Our findings provide new and easily implemented opportunities to generate mature hPSC-CMs for disease modeling and regenerative therapy.

cell biology↗

bioRxiv2019

Machine learning-based identification and cellular validation of Tropomyosin 1 as a genetic inhibitor of hematopoiesis.

Identifying causal variants and genes from human genetics studies of hematopoietic traits are important to enumerate basic regulatory mechanisms underlying these traits, and could ultimately augment translational efforts to generate platelets and/or red blood cells in vitro. To identify putative causal genes from these data, we performed computational modelling using available genome-wide association data sets for platelet traits. Our model identified a joint collection of genomic features enriched at established platelet trait associations and plausible candidate variants. Additional studies associating variation at these loci with change in gene expression highlighted the Tropomyosin 1 (TPM1) among our top-ranked candidate genes. CRISPR/Cas9-mediated TPM1 knockout in human induced pluripotent stem cells (iPSCs) enhanced hematopoietic progenitor development, increasing total megakaryocyte as well as erythroid cell yields. Our findings may help explain human genetics associations and identify a novel genetic strategy to enhance in vitro hematopoiesis.

genomics↗