IRCCS San Raffaele Scientific Institute Showcase

"Chimeric enzymes enhance treatment potential for globoid cell leukodystrophy through hematopoietic stem cell gene therapy" - PMID: 40988335 - DOI: 10.1016/j.ymthe.2025.09.030 Globoid cell leukodystrophy (GLD) is a fatal lysosomal storage disorder caused by a deficiency in the β-galactosylceramidase (GALC) enzyme, leading to severe demyelination and neurodegeneration, and often death before the age of 2 years. Hematopoietic stem/progenitor cell transplantation (HSPC-T) has limited efficacy due to inadequate GALC delivery to the central (CNS) and peripheral nervous systems (PNS) and associated risks. In vivo gene therapy (GT) using adeno-associated viral vectors shows promise, but safety concerns persist. This research presents a strategy using lentiviral (LV) vector-mediated ex vivo HSPC-GT with a chimeric GALC enzyme that incorporates peptides from α-L-iduronidase (IDUA) and apolipoprotein E II (APO) to enhance expression and blood-brain barrier penetration. The chimeric IDUAsp.GALC.APO enzyme exhibited superior production and secretion compared to native GALC and previous chimeric variants in LV-transduced HSPCs, resulting in improved cross-correction and normalization of GALC activity in GLD neural cells. Proof-of-concept studies demonstrated effective enzyme production, secretion, and cross-correction capability of macrophages from GLD patients. In vivo results showed stable gene marking, sustained enzyme production, and efficient delivery of the chimeric GALC in affected organs, including the CNS and PNS. These findings highlight the potential of HSPC-GT using chimeric GALC enzymes as an innovative therapeutic approach for treating GLD. Data related to the paper are available in the folder with the same name. "Enhancement of expression, bioavailability and cross-correction of chimeric GALC enzyme to refine gene therapy approaches for globoid cell leukodystrophy" - GR 2019-12369357 - Final Report In this project, we propose a lentiviral vector-mediated gene therapy with a chimeric GALC enzyme engineered for improved expression and blood-brain barrier penetration as therapeutic approach for GLD. Our findings demonstrate that the chimeric enzyme, when expressed in LV-transduced human HSPCs and GLD iPSC derived NPC progeny, exhibits enhanced production and secretion compared to native GALC and prior chimeric variants. This leads to significant cross-correction of GALC activity in GLD neural cells. In vivo studies in immunodeficient mice and a severe GLD mouse model confirmed stable gene marking and sustained enzyme production, emphasizing our strategy's potential for effective therapeutic delivery and cross-correction of affected tissues. The data related to the final report of GR 2019-12369357 are available in the folder with the same name.

Data generated under the umbrella of the research grant “RF-2019-12368847” have been included in the pubblication "Specific types of male infertility are correlated with T cell exhaustion or senescence signatures" (https://doi.org/10.1038/s41467-025-56193-2). Below are the links to the open-access data repository where the data have been published. The first link to GEO Repository contains all the data generated by single cell RNA sequencing The second link to the San Raffaele Open Research Data Repository contains all the other data generated and used to build manuscript figures.

β-thalassemia is a genetic disorder caused by mutations in the β-globin gene, and characterized by anemia, ineffective erythropoiesis and iron overload. Patients affected by the most severe transfusion-dependent form of the disease (TDT) require lifelong blood transfusions and iron chelation therapy, a symptomatic treatment associated with several complications. Other therapeutic opportunities are available, but none is fully effective and/or applicable to all patients, calling for the identification of novel strategies. Transferrin receptor 2 (TFR2) balances red blood cells production according to iron availability, being an activator of the iron-regulatory hormone hepcidin in the liver and a modulator of erythropoietin signaling in erythroid cells. Selective Tfr2 deletion in the BM improves anemia and iron-overload in non-TDT mice, both as a monotherapy and, even more strikingly, in combination with iron restricting approaches. However, whether Tfr2 targeting might represent a therapeutic option for TDT has never been investigated so far. Here, we prove that BM Tfr2 deletion improves anemia, erythrocytes morphology and ineffective erythropoiesis in the Hbbth1/th2 murine model of TDT. This effect is associated with a decrease in the expression of α-globin, which partially corrects the unbalance with β-globin chains and limits the precipitation of misfolded hemoglobin, and with a decrease in the activation of unfolded protein response. Remarkably, BM Tfr2 deletion is also sufficient to avoid long-term blood transfusions required for survival of Hbbth1/th2 animals, preventing mortality due to chronic anemia and reducing transfusion-associated complications, such as progressive iron-loading. Altogether, these findings prove that TFR2 targeting might represent a promising therapeutic option also for TDT.

This dataset contains raw, replicate-level measurements generated in a multicenter study assessing the commutability of External Quality Assessment (EQA) control materials across routine clinical chemistry platforms. No post-processing, outlier removal, bias calculations, uncertainty propagation, or commutability classifications are included here; all values are as reported by the participating instruments in the single analytical run per site. Study design (for data context) Setting: Ten routine clinical laboratories participating in the Lombardy Regional EQA program. Analytes (measurands): ALP, ALT, AST, CK, LDH, CHE, creatinine, calcium. Specimens: Three lyophilized control materials (CM-1/CM-2/CM-3) and fifteen human serum pools. Each site performed one analytical run. CMs were measured in six replicates; pools in three replicates. Routine calibration and IQC preceded each run

Extracellular vesicles (EVs) are crucial for intercellular communication and are found in various biological fluids. The identification and immunophenotyping of such small particles continue to pose significant challenges. Here, we have developed a workflow for the optimisation of a next-generation panel for in-depth immunophenotyping of circulating plasma EVs using spectral flow cytometry. Our data collection followed a multistep optimisation phase for both instrument setup and 21-colour panel design, thus maximising fluorescent signal recovery. This spectral approach enabled the identification of novel EV subpopulations. Indeed, besides common EVs released by erythrocytes, platelets, leukocytes and endothelial cells, we observed rare and poorly known EV subsets carrying antigens related to cell activation or exhaustion. Notably, the unsupervised data analysis of major EV subsets revealed subpopulations expressing up to five surface antigens simultaneously. However, the majority of EVs expressed only a single surface antigen, suggesting they may not fully represent the phenotype of their parent cells. This is likely due to the small surface area or the biogenesis of EVs rather than antibody steric hindrance. Finally, we tested our workflow by analysing the plasma EV landscape in a cohort of systemic lupus erythematosus (SLE) patients. Interestingly, we observed a significant increase in CD54+ EVs, supporting the notion of elevated circulating ICAM under SLE conditions. To our knowledge, these are the first data highlighting the importance of a spectral flow cytometry approach in deciphering the heterogeneity of plasma EVs paving the way for the routine use of a high-dimensional immunophenotyping in EV research.

Supporting information and datasets to the paper titled "Testis Molecular Pathways in CAIS Unveil Testosterone/Estradiol on Germ Cell Tumor Risk in Non-Obstructive Azoospermia". DOI: 10.1210/clinem/dgaf404

Cell migration is orchestrated by molecular networks supporting motility. The scaffolds ERC1/ELKS and Liprin-α1 sustain cell migration and invasion by assembling dynamic plasma membrane-associated platforms. ERC1/ELKS forms cytoplasmic condensates with liquid–like behavior. In this study we tested whether the ability of ERC1 to form condensates is relevant to its function in cell motility. We identified the shortest N-terminal region of ERC1 sufficient to drive phase separation in vitro and in cells. Fluorescence recovery after photobleaching confirmed the dynamic behavior of ERC1(1-244) condensates. Surprisingly, deletion of ERC1(1-244) including an intrinsically disordered region did not abolish the ability of ERC1DΔN to form condensates. Although the interactions of ERC1ΔN with partners were unaffected, the biophysical properties of ERC1ΔN condensates were altered, with consequences on cell motility. These findings highlight the importance of ERC1/ELKS to assemble functional networks, and show that altering the properties of ERC1–driven condensates interferes with tumor cell motility.

Spinal cord atrophy is a key contributor to disability in multiple sclerosis (MS), with early and progressive loss of cervical cord volume correlating with worsening clinical outcomes. This study explored whether aerobic capacity moderates the association between spinal cord atrophy and clinical disability in people with MS. A cross-sectional analysis was conducted on 51 MS patients with mild to moderate disability (Expanded Disability Status Scale [EDSS] ≤ 6.0) and 33 age- and sex-matched healthy controls (HCs). All participants underwent 3T brain MRI to assess normalized mean upper cervical cord area (nMUCCA), a surrogate for spinal cord atrophy. Aerobic capacity was assessed via peak oxygen consumption (VO₂peak) measured during cardiopulmonary exercise testing (CPET), with low aerobic capacity defined as a z-score < –1.64. MS patients demonstrated significantly reduced nMUCCA and VO₂peak compared to HCs. Within the MS cohort, 61% were classified as having low aerobic capacity. Although no major differences in brain volumes or clinical characteristics (aside from body mass index) were observed between low and high aerobic capacity subgroups, a significant negative correlation between nMUCCA and EDSS was found only in those with low aerobic capacity. Moderation analysis confirmed a significant interaction between spinal cord atrophy and aerobic capacity in predicting disability (β = –0.099, p = 0.012). Specifically, reduced nMUCCA was associated with higher disability only in MS patients with low aerobic capacity, but not in those with high aerobic capacity. These findings suggest that greater aerobic capacity may attenuate the detrimental impact of spinal cord atrophy on clinical disability in MS, acting as a physical reserve factor. This highlights the potential of aerobic fitness enhancement as a non-pharmacological strategy to buffer neurodegenerative effects and support functional outcomes in MS, especially in the early stages of the disease.

Fatigue is a prevalent and disabling symptom in people with multiple sclerosis (pwMS), affecting both cognitive and physical domains. This study explored the relationship between aerobic capacity, thalamic nuclei volumes, and fatigue, and assessed whether thalamic integrity mediates the effect of aerobic fitness on fatigue perception. The study retrospectively enrolled 83 pwMS (36 with relapsing–remitting MS and 47 with secondary progressive MS) and 63 age- and sex-matched healthy controls (HC). All participants underwent 3T brain MRI to quantify thalamic and global brain volumes. Fatigue was evaluated using the Modified Fatigue Impact Scale (MFIS), and aerobic capacity was assessed via peak oxygen uptake (VO₂peak) from cardiopulmonary exercise testing (CPET), available for all pwMS and 22 HC. Compared to HC, pwMS exhibited significantly lower VO₂peak and thalamic volumes and higher global, physical, and cognitive fatigue scores. In pwMS, higher VO₂peak was associated with lower fatigue (MFIS and pMFIS) and with greater volume in the laterodorsal thalamic nuclei cluster (Dor). Furthermore, lower Dor volume was linked to greater fatigue in all MFIS domains. Mediation analyses revealed that the Dor cluster partially mediated the beneficial effects of VO₂peak on global fatigue (21% indirect effect) and cognitive fatigue (32% indirect effect), but not physical fatigue. These findings suggest that aerobic capacity influences fatigue in pwMS, partly via structural preservation of specific thalamic subregions, especially the laterodorsal nuclei. While physical fatigue may be more directly influenced by systemic or extra-thalamic factors, cognitive fatigue appears partially dependent on thalamic integrity. This underscores the potential neuroprotective role of cardiorespiratory fitness and highlights the Dor thalamic cluster as a critical neural substrate in MS-related fatigue. Targeting aerobic capacity through rehabilitation may not only alleviate fatigue but also support thalamic structural integrity in pwMS.

Spinal cord (SC) involvement, particularly cervical SC (cSC) atrophy and lesions, is increasingly recognized as a key determinant of disability in multiple sclerosis (MS), yet its role in specific sensorimotor impairments remains underexplored. This cross-sectional multiparametric MRI study aimed to assess the relative contribution of brain and cSC lesion burden and atrophy to global disability and specific sensorimotor functions in MS. A total of 151 MS patients and 69 age- and sex-matched healthy controls underwent 3T brain and cSC MRI and clinical evaluations including Expanded Disability Status Scale (EDSS), pyramidal and sensory functional system (P-FS, S-FS) scores, motor performance tests (9-hole peg test [9-HPT], finger tapping test [FTT], timed 25-foot walk test [T25FWT]), and vibration detection threshold (VDT). Random forest models identified MRI features most predictive of disability milestones (EDSS ≥3.0, ≥4.0, and ≥6.0), functional system impairments, and motor/sensory test performance. Across all outcomes, cSC gray matter cross-sectional area (GM CSA) emerged as the most informative variable, outperforming brain volumetric and lesion metrics. GM CSA was the strongest predictor of progressive MS phenotype and higher EDSS scores (AUC up to 0.900). It also best explained impairments in lower limb motor function (T25FWT), upper limb repetitive motor performance (FTT), and vibratory sensation (VDT). In contrast, 9-HPT impairment, a more complex dexterity task, was associated with a broader combination of cSC and brain abnormalities, including lesion burden and cortical and deep gray matter atrophy. VDT impairment was linked to volume loss in both cSC GM and white matter (WM), supporting the role of dorsal columns and posterior horns in somatosensory processing. Brain metrics, particularly thalamic volume, also contributed to VDT variability. The findings highlight the predominant role of cSC GM atrophy as a structural correlate of sensorimotor impairment and disability in MS, especially at higher levels of functional decline. These results support integrating spinal cord MRI, particularly quantification of GM damage, into routine clinical assessments to enhance prognostic accuracy and guide personalized interventions in MS management.