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).

In vivo gene therapy to the liver using lentiviral vectors (LV) may represent a one-and-done therapeutic approach for monogenic diseases. Increasing LV gene therapy potency is crucial for reducing the effective doses, thus alleviating dose-dependent toxicities and facilitating manufacturing. LV-mediated liver transduction may be enhanced by positively selecting LV-transduced hepatocytes after treatment (a posteriori) or by augmenting the initial fraction of LV-targeted hepatocytes (a priori). We show here that the a posteriori enhancement increased transgene output without expansion of hepatocytes bearing LV genomic integrations near cancer genes, in mouse models of hemophilia, an inherited coagulation disorder. Furthermore, we enhanced hepatocyte transduction a priori in mice by transiently inhibiting antiviral pathways and/or through a fasting regimen. The most promising transduction-enhancer combination synergized with phagocytosis-shielded LV, resulting in a remarkable 40-fold increase in transgene output. Overall, our work highlights the potential of minimally invasive, cost-effective treatments capable of improving the potency of in vivo LV gene therapy to hepatocytes, in order to expand its applicability and ease clinical translation.
© 2025. The Author(s).

With increasing age, the risk for age-related type-2-diabetes also increases due to impaired glucose tolerance and insulin secretion. This disease process may be influenced by various factors, including immune cell triggered inflammation and fibrosis. Although immune cells are a necessary component of islets, little is known about immune cell accumulation, immune cell subtype shifts and subsequent influence on glucose metabolism in healthy aging. However, this is critical for understanding the mechanisms that influence β-cell health. Therefore, we studied young and old male C57BL/6J mice, focusing on immune cell composition, patterns of accumulation, and the presence of fibrosis within the pancreatic islets. Our findings demonstrate that insulitis occurs in healthy aged mice without immediate development of a diabetic phenotype. Aged islets exhibited an increase in leukocytes and a shift in immune cell composition. While insulitis typically involves excessive immune cell accumulation, we observed a moderate increase in macrophages and T-cells during aging, which may support β-cell proliferation via cytokine secretion. In fact, aged mice in our study showed an increase in β-cell mass as well as a partially higher insulin secretory capacity, which compensated for the loss of β-cell functionality in insulitic islets and led to improved glucose tolerance. Furthermore, fibrosis which is normally triggered by immune cells, increased with age but appears to reach a steady state, emphasizing the importance of counter-regulatory mechanisms and immune system regulation. Our results suggest, that immune cell subtypes change with age and that non-pathological accumulation of immune-cells may regulate glucose metabolism through secretion of cytokines.
Copyright © 2025 The Authors. Published by Elsevier B.V. All rights reserved.

Premetastatic cancer cells often spread from the primary lesion through the lymphatic vasculature and, clinically, the presence or absence of lymph node metastases impacts treatment decisions. However, little is known about cancer progression via the lymphatic system or of the effect that the lymphatic environment has on cancer progression. This is due, in part, to the technical challenge of studying lymphatic vessels and collecting lymph fluid. Here we provide a step-by-step procedure to collect both lymph and tumor-draining lymph in mouse models of cancer metastasis. This protocol has been adapted from established methods of lymph collection and was developed specifically for the collection of lymph from tumors. The approach involves the use of mice bearing melanoma or breast cancer orthotopic tumors. After euthanasia, the cisterna chyli and the tumor are exposed and viewed using a stereo microscope. Then, a glass cannula connected to a 1 mL syringe is inserted directly into the cisterna chyli or the tumor-draining lymphatics for collection of pure lymph. These lymph samples can be used to analyze the lymph-derived cancer cells using highly sensitive multiomics approaches to investigate the impact of the lymph environment during cancer metastasis. The procedure requires 2 h per mouse to complete and is suitable for users with minimal expertise in small animal handling and use of microsurgical tools under a stereo microscope.
© 2025. Springer Nature Limited.

Fibro-adipogenic progenitor cells (FAPs) are a heterogeneous population of multipotent mesenchymal cells that give rise to fibroblasts and adipocytes. In response to muscle injury, FAPs are activated and cooperate with inflammatory and muscle stem cells to promote muscle regeneration. In pathological conditions, such as muscular dystrophies, this coordinated response is partially lost and an accumulation of FAPs is observed that is responsible for maladaptive fibrosis, ectopic fat deposition, and impaired muscle regeneration. The role of intracellular thyroid hormone (TH) signaling in this cellular context is largely unknown. Here we show that intracellular 3,5,3'-triiodothyronine (T3) concentration in FAPs is increased in vitro during adipogenic differentiation via the increase of the T3-producing type 2 deiodinase (D2). The adipogenic potential is reduced in FAPs cultured in the presence of 3,3,5'-triiodothyronine (rT3), a specific D2 inhibitor, while exogenous administration of THs is able to induce the expression of relevant adipogenic genes. Accordingly, on genetic D2 depletion in vivo, adipogenesis was significantly reduced in D2KO compared to control mice. These data were confirmed using a FAP-inducible specific D2-KO mouse model, suggesting that a cell-specific D2-depletion in FAPs is sufficient to decrease fatty muscle infiltration and to improve muscle regeneration. Taken together, these data show that TH signaling is dynamically modulated in FAPs wherein D2-produced T3 is required to promote maturation of FAPs into adipocytes.
© The Author(s) 2025. Published by Oxford University Press on behalf of the Endocrine Society.