Biology subjects

Psatha, N.

Publications and source records attributed to Psatha, N..

4 recordsLinked to original sources

bioRxiv2026

In vivo HSPC gene therapy of hemoglobinopathies without drug selection of corrected cells

In vivo hematopoietic stem/progenitor cell (HSPC) gene therapy remains limited by low gene-editing efficiency and a lack of clinically applicable selection strategies to enrich therapeutically corrected progeny. We used in vivo base and prime editing to introduce a nonpathogenic EPOR variant into HSPCs, conferring erythropoietin hypersensitivity and promoting preferential expansion of gene-corrected erythroid cells. EPOR editing was combined with three therapeutic approaches for the correction of hemoglobinopathies: {gamma}-globin gene addition, {gamma}-globin reactivation, or correction of the sickle cell disease mutation. Tropism-modified helper-dependent adenoviral vectors (HDAd6/3+) targeting HSPCs were used to simultaneously deliver the EPOR-editing machinery and the corresponding therapeutic components. In vitro studies in an erythroid progenitor cell line and primary CD34+ cells demonstrated that the EPORW439* variant conferred a strong proliferative advantage to therapeutically modified erythroid progenitors. Mice humanized with CD34+ cells from a {beta}/{beta}-thalassemia patient were subjected to EPORW439*-mediated EPO hypersensitivity alongside a therapeutic {gamma}-globin transgene which resulted in >70% HbF-positive erythroid cells and substantial reversion of the disease-associated phenotype, including reduced oxidative stress, near-complete elimination of splenic iron deposition, and reduced splenomegaly. Importantly, these effects were achieved after simple intravenous administration of the vectors following HSPC mobilization and cytokine prophylaxis, without subsequent pharmacologic selection. Together, these findings establish a strategy to amplify the therapeutic benefit of otherwise limited in vivo HSPC gene editing for hemoglobinopathies.

bioengineering↗

bioRxiv2023

Large-scale discovery of potent, compact and lineage specific enhancers for gene therapy vectors

Regulation of gene expression during cell development and differentiation is chiefly orchestrated by distal noncoding regulatory elements that precisely modulate cell selective gene activity. Gene therapy vectors rely on the cellular and context specificity of regulatory DNA elements to express therapeutic transgenes in the correct location and time. Here, we develop a straight-forward, one-shot approach to screen putative regulatory sequences identified in large-scale epigenomics profiling experiments for precise and programmable control of transgenes encoded within gene therapy viral vectors. We designed a library of 15,000 short sequences ([~]200bp) derived from a set of developmentally active DHS elements during human ex vivo erythropoiesis and cloned them into a GFP reporter lentiviral vector. In an erythroid progenitor cell line, these elements display a gradient of transcriptional enhancer activity, with some demonstrating equivalent activity to the canonical {beta}-globin LCR despite a 9-fold smaller size. We show that these elements are both highly cell type restricted and developmental stage specific both in vitro and in vivo. Finally, we replace the LCR element with one of the novel short enhancers in a {beta}-thalassemia lentiviral therapeutic vector and efficiently correct the thalassemic phenotype in patient-derived HSPCs. More broadly, our approach provides further insights into enhancer biology with wider implications into the development of highly cell type specific and efficacious viral vectors for human gene therapy.

genomics↗

bioRxiv2022

Enhancer grammar of liver cell types and hepatocyte zonation states

Cell type identity is encoded by gene regulatory networks (GRN), in which transcription factors (TFs) bind to enhancers to regulate target gene expression. In the mammalian liver, lineage TFs have been characterized for the main cell types, including hepatocytes. Hepatocytes cover a relatively broad cellular state space, as they differ significantly in their metabolic state, and function, depending on their position with respect to the central or portal vein in a liver lobule. It is unclear whether this spatially defined cellular state space, called zonation, is also governed by a well-defined gene regulatory code. To address this challenge, we have mapped enhancer-GRNs across liver cell types at high resolution, using a combination of single cell multiomics, spatial omics, GRN inference, and deep learning. We found that cell state changes in transcription and chromatin accessibility in hepatocytes, liver sinusoidal endothelial cells and hepatic stellate cells depend on zonation. Enhancer-GRN mapping suggests that zonation states in hepatocytes are driven by the repressors Tcf7l1 and Tbx3, that modulate the core hepatocyte GRN, controlled by Hnf4a, Cebpa, Hnf1a, Onecut1 and Foxa1, among others. To investigate how these TFs cooperate with cell type TFs, we performed an in vivo massively parallel reporter assay on 12,000 hepatocyte enhancers and used these data to train a hierarchical deep learning model (called DeepLiver) that exploits both enhancer accessibility and activity. DeepLiver confirms Cebpa, Onecut, Foxa1, Hnf1a and Hnf4a as drivers of enhancer specificity in hepatocytes; Tcf7l1/2 and Tbx3 as regulators of the zonation state; and Hnf4a, Hnf1a, AP-1 and Ets as activators. Finally, taking advantage of in silico mutagenesis predictions from DeepLiver and enhancer assays, we confirmed that the destruction of Tcf7l1/2 or Tbx3 motifs in zonated enhancers abrogates their zonation bias. Our study provides a multi-modal understanding of the regulatory code underlying hepatocyte identity and their zonation state, that can be exploited to engineer enhancers with specific activity levels and zonation patterns.

genomics↗

bioRxiv2020

Chromatin dynamics during hematopoiesis reveal discrete regulatory modules instructing differentiation

Lineage commitment and differentiation is driven by the concerted action of master transcriptional regulators at their target chromatin sites. Multiple efforts have characterized the key transcription factors (TFs) that determine the various hematopoietic lineages. However, the temporal interactions between individual TFs and their chromatin targets during differentiation and how these interactions dictate lineage commitment remains poorly understood. We performed dense, daily, temporal profiling of chromatin accessibility (DNase I-seq) and gene expression changes (total RNA-seq) along ex vivo human erythropoiesis to comprehensively define developmentally regulated DNase I hypersensitive sites (DHSs) and transcripts. We link both distal DHSs to their target gene promoters and individual TFs to their target DHSs, revealing that the regulatory landscape is organized in distinct sequential regulatory modules that regulate lineage restriction and maturation. Finally, direct comparison of transcriptional dynamics (bulk and single-cell) and lineage potential between erythropoiesis and megakaryopoiesis uncovers differential fate commitment dynamics between the two lineages as they exit pluripotency. Collectively, these data provide novel insights into the global regulatory landscape during hematopoiesis.

developmental biology↗