Clinical response to systemic AAV gene therapy in a large animal model of late-stage lysosomal storage disease

Gene Therapy · 2026-05-04

The benefit of early diagnosis and treatment has been demonstrated in animal models of several lysosomal storage diseases. In a clinical setting, however, diagnoses are often not made until after patients become symptomatic. The lysosomal storage disease alpha-mannosidosis is caused by a genetic deficiency of lysosomal alpha-mannosidase, leading to the widespread presence of storage lesions throughout the brain and other tissues. In a feline model of alpha-mannosidosis, we previously demonstrated complete correction of the brain following delivery of AAVhu.32-fMANB via the carotid artery in the early symptomatic stage. Here, we investigate the efficacy of AAV gene therapy on globally distributed storage lesions in animals with advanced disease. Some improvements in clinical parameters were observed, however these improvements were less than in animals with less advanced disease. Although the treated animals were improved compared to untreated animals, increasing the vector dose did not further improve clinical outcomes. These results further demonstrate the importance of early detection and treatment of a lysosomal storage disease to successful outcomes. Despite this, partial correction extended the lifespan of diseased cats and may be medically beneficial to patients by slowing or stabilizing the progressive degenerative course of disease.

Tailoring inter-core distance of clustered SPIONs using silica spacers for enhanced magnetic particle imaging (MPI)

Nanoscale · 2026-01-01

An emulsion-based self-assembly strategy for the formation of Superparamagnetic Iron Oxide Nanoparticles (SPION) clusters with precisely tunable silica spacers is reported. Systematic control of the silica shell thickness enables modulation of magnetic inter-core spacing within the clusters. This results in a pronounced enhancement in Magnetic Particle Imaging (MPI) signal generation, attributed to a combination of enhanced long-range magnetic dipole-dipole coupling interaction, the suppression of short-range exchange-like coupling interaction and the passivation of SPION surface spin disorder. The interplay between effects gives rise to an increase in harmonic content of clustered core@shell particles compared with their non-clustered counterparts, resulting in brighter MPI images.

Lysosomal abundance in young and aged mouse hearts assessed by In Vivo Imaging Systems (IVIS) Lysotracker imaging and autophagy-related gene expression

bioRxiv (Cold Spring Harbor Laboratory) · 2026-02-17

Abstract Lysosomal function is essential for cardiac proteostasis and cellular health, yet its regulation during ageing remains poorly defined. We hypothesised that ageing alters both the abundance of acidic organelles and the machinery supporting their acidification. Using fluorescence-based In Vivo Imaging Systems (IVIS) with Lysotracker™ Red in young (2–4 months) and aged (18 months) mouse hearts, we quantified whole-heart acidic-vesicle signals and assessed expression of lysosomal and autophagy-related genes ( Lamp2, Atp6v1a, Sqstm1, Cd63, Atg12, Nfe2l2, M6pr ) by RT-qPCR. Whole-heart labelled Lysotracker fluorescence did not differ significantly between age groups, indicating preservation of the total acidic-vesicle pool. No changes in Atp6v1a and Lamp2 expression suggest acidification capacity and structural stability are maintained, whereas the minor, upregulation of Sqstm1 might indicate increased autophagic demand and altered vesicle trafficking, which warrants further investigation. No statistical significant changes in M6pr, Atg12, or Nfe2l2 were detected, suggesting transcriptional stability in enzyme trafficking, core autophagy, and oxidative stress pathways. Regionally, atria showed higher Lysotracker signal than ventricles, consistent with known enrichment of acidic vesicular stores in atrial physiology. These findings highlight the utility of IVIS imaging of Lysotracker-labelled hearts, providing rapid whole-organ assessment of acidic vesicle distribution, albeit with limited depth resolution. Complementary techniques such as RT-qPCR analysis is essential to interpret IVIS findings, enabling insight into underlying molecular changes in lysosomal and autophagy pathways during cardiac ageing.

ITRF / LhARA conceptual design report

Science and Technology Facilities Council · 2026-01-01

Imaging vascular characteristics and glycolytic metabolism of glioblastoma in a chick embryo model using 1H MRI and [18F]FDG-PET

Molecular Imaging and Biology · 2026-02-23

Abstract Purpose To assess hypoxia-associated host-tumour vascular adaptations and glycolytic metabolism in the chick chorioallantoic membrane (CAM) glioblastoma model. Procedures U251 GBM cells were conditioned under normoxia (21% O₂) or hypoxia (1% O₂) for 72 h before implantation onto the CAM on embryonic day 7 (E7). Imaging was performed on E13 using MRI (control-CAM n = 8, normoxic-tumour n = 7, hypoxic-tumour n = 6) and brightfield microscopy (control-CAM n = 7, normoxic-tumour n = 8, hypoxic-tumour n = 7). Tumours were harvested on E14 for histology and gene expression analyses. In a separate cohort of 25 GBM-CAM tumours grown under normoxic conditioning, the correlation of glucose metabolism was assessed using [ 18 F]FDG-PET on E12 followed by lactate MRS on E13 (n = 8). Results Normoxia- and hypoxia-conditioned tumour-bearing CAMs exhibited vascular remodelling and significant upregulation of VEGFA and ADM compared to cultured cells. αSMA staining confirmed vessel infiltration in normoxia-conditioned tumours. CAIX staining revealed a hypoxic core in these tumours while hypoxia-conditioned tumours displayed heterogeneous staining. In both conditions, GLUT1 staining colocalised with CAIX staining, indicating hypoxia-associated glycolysis. GLUT1, PDK1 and LDHA expression was elevated in CAM tumours relative to tumour cells in vitro. In the metabolic imaging cohort, most tumours exhibited [ 18 F]FDG uptake and lactate signal. However, no statistically significant relationship was observed between the two methods. Conclusions The CAM model provides a versatile platform for investigating GBM vascularisation and metabolism. Hypoxic conditioning amplifies transcriptional and vascular changes to the CAM. Although both [ 18 F]FDG uptake and lactate were measurable, no significant correlation between the two was observed, potentially reflecting variability in tumour engraftment, vascular delivery of [ 18 F]FDG, and microenvironmental influences on lactate accumulation.

Evaluating Labelling Efficiency of Commercial SPIONs in Mesenchymal Stem/Stromal Cells for Magnetic Particle Imaging Applications

bioRxiv (Cold Spring Harbor Laboratory) · 2025-08-02

Abstract Magnetic Particle Imaging (MPI) is a state-of-the-art, highly sensitive modality for non-invasive cell tracking. This study evaluated labelling efficiency, biocompatibility, intracellular localization, and MPI detection sensitivity of four commercial superparamagnetic iron oxide nanoparticles (SPIONs)—ProMag, VivoTrax, SynoMag-D, and Ferumoxytol—in mouse mesenchymal stem/stromal cells. SPION labelling efficiency and cytotoxicity was assessed at varying concentrations and incubation times using Prussian blue staining and ATP-based viability assays, respectively. MPI characterization and transmission electron microscopy (TEM) evaluations were performed for cells labelled for two-hour with ProMag or VivoTrax. For >90% labelling efficiency, ProMag required 20□µg Fe/mL across all time points. VivoTrax, however, required ≥240□µg Fe/mL, reducing cell viability by >20% necessitating a reduction to 120 µg/mL for further analyses. Transfection agents improved SynoMag-D and Ferumoxytol labelling but compromised viability. MPI analysis revealed linear dependence of signal intensity on labelled cell numbers for ProMag and VivoTrax (r 2 =0.99). ProMag yielded higher signal intensity due to greater iron uptake, although VivoTrax exhibited higher signal per unit iron. TEM confirmed intracellular SPION localization, with ProMag present as individual particles and VivoTrax as aggregates within endocytic vesicles. Low-temperature assays confirmed energy-dependent endocytosis as the primary uptake mechanism. Despite ProMag’s stronger MPI signals and lower detection threshold (12,500 cells), VivoTrax’s superior magnetization per iron suggests its potential following further optimization of cell uptake. Overall, ProMag and VivoTrax emerged as optimal candidates for MPI-based stem cell tracking. These findings underscore the importance of optimizing both nanoparticle selection and labelling protocols to maximize MPI performance and inform future in vivo applications.

Clustering-Based Multiparametric MRI for Differentiation Between True Tumor Progression and Pseudoprogression in Glioblastoma

Proceedings on CD-ROM - International Society for Magnetic Resonance in Medicine. Scientific Meeting and Exhibition/Proceedings of the International Society for Magnetic Resonance in Medicine, Scientific Meeting and Exhibition · 2025-09-16

Motivation: Clustering analysis in brain tumor aims to improve differentiation between true progression (TP) and pseudoprogression (PsP) in glioblastoma using multiparametric MRI-based pharmacokinetic and diffusion parameters, addressing a critical diagnostic challenge to enhance treatment assessment and planning. Goal(s): The objective is to distinguish between true progression (TP) from pseudoprogression (PsP) in glioblastoma using multiparametric MRI-based clustering analysis. Approach: K-means ++ Clustering based Multiparametric MRI data analysis for segregating the tumor into low and high intensity regions. Results: Mean Ktrans and Mean Kep are statistically significant DCE-MRI parameters for differentiating true progression from pseudoprogression of glioblastoma. Impact: These results demonstrate the importance of vascular permeability in tumor assessment and establish Ktrans and Kep along with tumor volume as essential parameters for differentiating true progression from treatment effects.

Methodology for generating chorioallantoic membrane patient-derived xenograft (CAM-PDX) models of pleural mesothelioma and performing preclinical imaging for the translation of cancer studies and drug screening.

F1000Research · 2025-08-14 · 1 citations

<ns3:p> Background Pleural mesothelioma is a cancer of the lung lining associated with asbestos exposure. Platinum/pemetrexed chemotherapy has been used for many years but provides little benefit and, despite recent immunotherapy advances, prognosis remains poor underpinning the need for development of novel therapeutics or drug repurposing. Fertilized hens’ eggs provide a rapid and cost-effective alternative to murine models of pleural mesothelioma which are commonly used in preclinical studies, with chorioallantoic membrane (CAM) xenografts being a partial replacement for mouse flank xenografts. Here we describe methods to generate mesothelioma patient-derived xenografts on the CAM (CAM-PDX), and to subsequently assess these PDX nodules by preclinical imaging and histology. Methods Fragments of surplus mesothelioma tissue obtained from patient biopsies were implanted onto the CAM on embryonic day 7 (E7), fresh or following cryopreservation, with the established PDX dissected on E14 and fixed for histological/immunohistochemical analysis. The optimal freezing method was determined by comparing tissue integrity and cellular content of cryopreserved tissue fragments with paired fresh samples via histological/immunohistochemical analyses. [ <ns3:sup>18</ns3:sup> F]-fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) was used to assess viability of PDXs <ns3:italic>in ovo.</ns3:italic> Results Methodologies for processing, cryopreservation, re-animation, and engraftment of mesothelioma tissue fragments were established. Cryopreservation of biopsy samples and parallel processing of contiguous sections allows for assessment of mesothelioma cellularity. CAM-PDXs, generated from fresh or slow-frozen tissue, were well vascularized whilst maintaining the architecture and cellular composition of the patient tissue. Furthermore, uptake of [ <ns3:sup>18</ns3:sup> F]-FDG following intravenous injection could be visualized and quantified. Conclusions The CAM is a rapid platform for engrafting patient-derived tissue, maintaining elements of the tumor microenvironment and recapitulating heterogeneity observed in mesothelioma. Combining the CAM-PDX model and FDG-PET/CT provides a quantitative <ns3:italic>in vivo</ns3:italic> platform for pre-screening of novel treatment strategies and drug combinations, with the potential for development of patient tumor avatars for predicting clinical response. </ns3:p>

Hypoxia Dependent Inhibition of Glioblastoma Cell Proliferation, Invasion, and Metabolism by the Choline-Kinase Inhibitor JAS239

Metabolites · 2025-01-26 · 1 citations

Background: Elevated choline kinase alpha (ChoK) levels are observed in most solid tumors, including glioblastomas (GBM), and ChoK inhibitors have demonstrated limited efficacy in GBM models. Given that hypoxia is associated with resistance to GBM therapy, we hypothesized that tumor hypoxia could be responsible for the limited response. Therefore, we evaluated the effects of hypoxia on the function of JAS239, a potent ChoK inhibitor in four GBM cell lines. Methods: Rodent (F98 and 9L) and human (U-87 MG and U-251 MG) GBM cell lines were subjected to 72 h of hypoxic conditioning and treated with JAS239 for 24 h. NMR metabolomic measurements and analyses were performed to evaluate the signaling pathways involved. In addition, cell proliferation, cell cycle progression, and cell invasion parameters were measured in 2D cell monolayers as well as in 3D cell spheroids, with or without JAS239 treatment, in normoxic or hypoxic cells to assess the effect of hypoxia on JAS239 function. Results: Hypoxia and JAS239 treatment led to significant changes in the cellular metabolic pathways, specifically the phospholipid and glycolytic pathways, associated with a reduction in cell proliferation via induced cell cycle arrest. Interestingly, JAS239 also impaired GBM invasion. However, effects from JAS239 were variable depending on the cell line, reflecting the inherent heterogeneity of GBMs. Conclusions: Our findings indicate that JAS239 and hypoxia can deregulate cellular metabolism, inhibit cell proliferation, and alter cell invasion. These results may be useful for designing new therapeutic strategies based on ChoK inhibition, which can act on multiple pro-tumorigenic features.

1H MR spectroscopy to evaluate the effects of lonidamine and temozolomide treatment in a mouse model of glioblastoma

Proceedings on CD-ROM - International Society for Magnetic Resonance in Medicine. Scientific Meeting and Exhibition/Proceedings of the International Society for Magnetic Resonance in Medicine, Scientific Meeting and Exhibition · 2025-09-16

Motivation: To develop an alternate effective therapy for treating glioblastomas. Goal(s): To assess metabolic changes as therapeutic indices of treatment response to Lonidamine (LND)and temozolomide (TMZ) against glioblastoma (GBM). Approach: C57BL6 mice implanted with GL261 GBM cells were treated with saline or LND+TMZ. Metabolic changes were monitored using longitudinal MRS. Results: Significant reductions in lactate, tCho/NAA, Lip+Lac/tCr and mI/tCr ratios, were observed post-treatment, suggesting early indicators of treatment response. Impact: This combination therapy might be a suitable alternative for better treatment of glioblastomas.