Lentiviral vector (LV)-mediated ex vivo gene therapy for haematopoietic stem and progenitor cells (HSPCs) has delivered on the promise of a 'one-and-done' treatment for several genetic diseases1. However, ex vivo manipulation and patient conditioning before transplantation are major hurdles that could be overcome by an in vivo approach. Here we demonstrate that in vivo gene delivery to HSPCs after systemic LV administration is enabled by the substantial trafficking of these cells from the liver to the bone marrow in newborn mice. We improved gene-transfer efficiency using a phagocytosis-shielded LV, successfully reaching bona fide HSPCs capable of long-term multilineage output and engraftment after serial transplantation, as confirmed by clonal tracking. HSPC mobilization further increased gene transfer, extending the window of intervention, although permissiveness to LV transduction declined with age. We successfully tested this in vivo strategy in mouse models of adenosine deaminase deficiency, autosomal recessive osteopetrosis and Fanconi anaemia. Interestingly, in vivo gene transfer provided a selective advantage to corrected HSPCs in Fanconi anaemia, leading to near-complete haematopoietic reconstitution and prevention of bone marrow failure. Given that circulating HSPCs in humans are also most abundant shortly after birth, in vivo HSPC gene transfer holds strong translational potential across multiple diseases.
© 2025. The Author(s).

Mechanisms governing the maintenance of blood-producing hematopoietic stem and multipotent progenitor cells (HSPCs) are incompletely understood, particularly those regulating fate, ensuring long-term maintenance, and preventing aging-associated stem cell dysfunction. We uncovered a role for transitory free cytoplasmic iron as a rheostat for adult stem cell fate control. We found that HSPCs harbor comparatively small amounts of free iron and show the activation of a conserved molecular response to limited iron-particularly during mitosis. To study the functional and molecular consequences of iron restriction, we developed models allowing for transient iron bioavailability limitation and combined single-molecule RNA quantification, metabolomics, and single-cell transcriptomic analyses with functional studies. Our data reveal that the activation of the limited iron response triggers coordinated metabolic and epigenetic events, establishing stemness-conferring gene regulation. Notably, we find that aging-associated cytoplasmic iron loading reversibly attenuates iron-dependent cell fate control, explicating intervention strategies for dysfunctional aged stem cells.
Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.

Live bacteria-mediated antitumor therapies mark a pivotal point in cancer immunotherapy. However, the difficulty in reconciling the safety and efficacy of bacterial therapies has limited their application. Improving bacterial tumor-targeted delivery while maintaining biosafety is a critical hurdle for the clinical translation of live microbial therapy for cancer. Here, we developed "dead" yet "functional" Salmonella-loaded macrophages using liquid nitrogen cold shock of an attenuated Salmonella typhimurium VNP20009-contained macrophage cell line. The obtained "dead" macrophages achieve an average loading of approximately 257 live bacteria per 100 cells. The engineered cells maintain an intact cellular structure but lose their original pathogenicity, while intracellular bacteria retain their original biological activity and are delay freed, followed by proliferation. This "Trojan horse"-like bacterial camouflage strategy avoids bacterial immunogenicity-induced neutrophil recruitment and activation in peripheral blood, reduces the clearance of bacteria by neutrophils and enhances bacterial tumor enrichment efficiently after systemic administration. Furthermore, this strategy also strongly activated the tumor microenvironment, including increasing antitumor effector cells (including M1-like macrophages and CD8+ Teffs) and decreasing protumor effector cells (including M2-like macrophages and CD4+ Tregs), and ultimately improved antitumor efficacy in a subcutaneous H22 tumor-bearing mouse model. The cryo-shocked macrophage-mediated bacterial delivery strategy holds promise for expanding the therapeutic applications of living bacteria for cancer.© 2023. The Author(s).

Despite potential impact on the graft vs. leukemia (GVL) effect, immunotherapy targeting CTLA-4 and/or PD-1 has not been successfully combined with bone marrow transplant (BMT) because it exacerbates graft vs. host disease (GVHD). Here, using models of GVHD and leukemia, we demonstrate that targeting hypoxia-inducible factor 1α (HIF1α) via pharmacological or genetic approaches reduces GVHD by inducing PDL1 expression on host tissue while selectively inhibiting PDL1 in leukemia cells to enhance the GVL effect. More importantly, combination of HIF1α inhibition with anti-CTLA-4 antibodies allows simultaneous inhibition of both PDL1 and CTLA-4 checkpoints to achieve better outcomes in models of mouse and human BMT-leukemia settings. These findings provide an approach to enhance the curative effect of BMT for leukemia and broaden the impact of cancer immunotherapy.
Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.

Mutations in the gene encoding DNA methyltransferase 3A (DNMT3A) are the most common cause of clonal hematopoiesis and are among the most common initiating events of acute myeloid leukemia (AML). Studies in germline and somatic Dnmt3a knockout mice have identified focal, canonical hypomethylation phenotypes in hematopoietic cells; however, the kinetics of methylation loss following acquired DNMT3A inactivation in hematopoietic cells is essentially unknown. Therefore, we evaluated a somatic, inducible model of hematopoietic Dnmt3a loss, and show that inactivation of Dnmt3a in murine hematopoietic cells results in a relatively slow loss of methylation at canonical sites throughout the genome; in contrast, remethylation of Dnmt3a deficient genomes in hematopoietic cells occurs much more quickly. This data suggests that slow methylation loss may contribute, at least in part, to the long latent period that characterizes clonal expansion and leukemia development in individuals with acquired DNMT3A mutations in hematopoietic stem cells.
© 2022 The Authors.