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Abstract Hematopoietic stem cell (HSC) mutations can result in clonal hematopoiesis (CH) with heterogeneous clinical outcomes. Here, we investigate how the cell state preceding Tet2 mutation impacts the pre-malignant phenotype. Using an inducible system for clonal analysis of myeloid progenitors, we find that the epigenetic features of clones at similar differentiation status are highly heterogeneous and functionally respond differently to Tet2 mutation. Cell differentiation stage also influences Tet2 mutation response indicating that the cell of origin’s epigenome modulates clone-specific behaviors in CH. Molecular features associated with higher risk outcomes include Sox4 that sensitizes cells to Tet2 inactivation, inducing dedifferentiation, altered metabolism and increasing the in vivo clonal output of mutant cells, as confirmed in primary GMP and HSC models. Our findings validate the hypothesis that epigenetic features can predispose specific clones for dominance, explaining why identical genetic mutations can result in different phenotypes.

A major goal of biological imaging is localization of biomolecules inside a cell. Fluorescence microscopy can localize biomolecules inside whole cells and tissues, but its ability to count biomolecules and accuracy of the spatial coordinates is limited by the wavelength of visible light. Cryo-electron microscopy (cryo-EM) provides highly accurate position and orientation information of biomolecules but is often confined to small fields of view inside a cell, limiting biological context. In this study, we use a new data-acquisition scheme called Defocus-Corrected Large-Area cryo-EM (DeCo-LACE) to collect high-resolution images of entire sections (100- to 250-nm-thick lamellae) of neutrophil-like mouse cells, representing 1–2% of the total cellular volume. We use 2D template matching (2DTM) to determine localization and orientation of the large ribosomal subunit in these sections. These data provide maps of ribosomes across entire sections of mammalian cells. This high-throughput cryo-EM data collection approach together with 2DTM will advance visual proteomics and provide biological insight that cannot be obtained by other methods.

Abstract Precise correction of the CD40LG gene in T cells and hematopoietic stem/progenitor cells (HSPC) holds promise for treating X‐linked hyper‐IgM Syndrome (HIGM1), but its actual therapeutic potential remains elusive. Here, we developed a one‐size‐fits‐all editing strategy for effective T‐cell correction, selection, and depletion and investigated the therapeutic potential of T‐cell and HSPC therapies in the HIGM1 mouse model. Edited patients’ derived CD4 T cells restored physiologically regulated CD40L expression and contact‐dependent B‐cell helper function. Adoptive transfer of wild‐type T cells into conditioned HIGM1 mice rescued antigen‐specific IgG responses and protected mice from a disease‐relevant pathogen. We then obtained ~ 25% CD40LG editing in long‐term repopulating human HSPC. Transplanting such proportion of wild‐type HSPC in HIGM1 mice rescued immune functions similarly to T‐cell therapy. Overall, our findings suggest that autologous edited T cells can provide immediate and substantial benefits to HIGM1 patients and position T‐cell ahead of HSPC gene therapy because of easier translation, lower safety concerns and potentially comparable clinical benefits.

Abstract Gene targeting constitutes a new step in the development of gene therapy for inherited diseases. Although previous studies have shown the feasibility of editing fibroblasts from Fanconi anemia (FA) patients, here we aimed at conducting therapeutic gene editing in clinically relevant cells, such as hematopoietic stem cells (HSCs). In our first experiments, we showed that zinc finger nuclease (ZFN)‐mediated insertion of a non‐therapeutic EGFP‐reporter donor in the AAVS1 “safe harbor” locus of FA‐A lymphoblastic cell lines (LCLs), indicating that FANCA is not essential for the editing of human cells. When the same approach was conducted with therapeutic FANCA donors, an efficient phenotypic correction of FA‐A LCLs was obtained. Using primary cord blood CD34+ cells from healthy donors, gene targeting was confirmed not only in in vitro cultured cells, but also in hematopoietic precursors responsible for the repopulation of primary and secondary immunodeficient mice. Moreover, when similar experiments were conducted with mobilized peripheral blood CD34+ cells from FA‐A patients, we could demonstrate for the first time that gene targeting in primary hematopoietic precursors from FA patients is feasible and compatible with the phenotypic correction of these clinically relevant cells.