IRCCS San Raffaele Scientific Institute Showcase

Raw data associated to the manuscript by Fiumara et al. Base and prime editors (BEs and PEs) may provide more precise genetic engineering than nuclease-based approaches because they bypass the dependence on DNA double-strand breaks. However, little is known about their cellular responses and genotoxicity. Here, we compared state-of-the-art BEs and PEs and Cas9 in human hematopoietic stem and progenitor cells with respect to editing efficiency, cytotoxicity, transcriptomic changes and on-target and genome-wide genotoxicity. BEs and PEs induced detrimental transcriptional responses that reduced editing efficiency and hematopoietic repopulation in xenotransplants and also generated DNA double-strand breaks and genotoxic byproducts, including deletions and translocations, at a lower frequency than Cas9. These effects were strongest for cytidine BEs due to suboptimal inhibition of base excision repair and were mitigated by tailoring delivery timing and editor expression through optimized mRNA design. However, BEs altered the mutational landscape of hematopoietic stem and progenitor cells across the genome by increasing the load and relative proportions of nucleotide variants. These findings raise concerns about the genotoxicity of BEs and PEs and warrant further investigation in view of their clinical application.

Mobilized peripheral blood is increasingly used instead of bone marrow as a source of autologous hematopoietic stem/progenitor cells for ex vivo gene therapy. Here, we present an unplanned exploratory analysis evaluating the hematopoietic reconstitution kinetics, engraftment and clonality in 13 pediatric Wiskott-Aldrich syndrome patients treated with autologous lentiviral-vector transduced hematopoietic stem/progenitor cells derived from mobilized peripheral blood (n=7), bone marrow (n=5) or the combination of the two sources (n=1). 8 out of 13 gene therapy patients were enrolled in an open-label, non-randomized, phase 1/2 clinical study(NCT01515462) and the remaining 5 patients were treated under expanded access programs. Although mobilized peripheral blood- and bone marrow- hematopoietic stem/progenitor cells display similar capability of being gene-corrected, maintaining the engineered grafts up to 3 years after gene therapy, mobilized peripheral blood-gene therapy group shows faster neutrophil and platelet recovery, higher number of engrafted clones and increased gene correction in the myeloid lineage which correlate with higher amount of primitive and myeloid progenitors contained in hematopoietic stem/progenitor cells derived from mobilized peripheral blood. In vitro differentiation and transplantation studies in mice confirm that primitive hematopoietic stem/progenitor cells from both sources have comparable engraftment and multilineage differentiation potential. Altogether, our analyses reveal that the differential behavior after gene therapy of hematopoietic stem/progenitor cells derived from either bone marrow or mobilized peripheral blood is mainly due to the distinct cell composition rather than functional differences of the infused cell products, providing new frames of references for clinical interpretation of hematopoietic stem/progenitor cell transplantation outcome. Data supporting the current study are part of a registered clinical trial (NCT01515462). These data are available under restricted access for the sensitive nature of the clinical data, access can be obtained by request to the corresponding author (Prof. Alessandro Aiuti, San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCCS San Raffaele Scientific Institute, Milan, 20132 Italy.). We intend to reply to any requests within two weeks and we will share the deposited data only for research purposes. The non-clinical data generated in this study are also provided as the Source Data file associated to the published work.

Rett syndrome (RTT) is a X-linked neurodevelopmental disorder which represents the leading cause of severe incurable intellectual disability in females worldwide. The vast majority of RTT cases are caused by mutations in the X-linked MECP2 gene, and preclinical studies on RTT largely benefit from the use of mouse models of Mecp2, which present a broad spectrum of symptoms phenocopying those manifested by RTT patients. Neurons represent the core targets of the pathology; however, neuroanatomical abnormalities that regionally characterize the Mecp2 deficient mammalian brain remain ill-defined. Neuroimaging techniques, such as MRI and MRS, represent a key approach for assessing in vivo anatomic and metabolic changes in brain. Being non-invasive, these analyses also permit to investigate how the disease progresses over time through longitudinal studies. To foster the biological comprehension of RTT and identify useful biomarkers, we have performed a thorough in vivo longitudinal study of MRI and MRS in Mecp2 deficient mouse brains. Analyses were performed on both genders of two different mouse models of RTT, using an automatic atlas-based segmentation tool that permitted to obtain a detailed and unbiased description of the whole RTT mouse brain. We found that the most robust alteration of the RTT brain consists in an overall reduction of the brain volume. Accordingly, Mecp2 deficiency generally delays brain growth, eventually leading, in heterozygous older animals, to stagnation and/or contraction. Most but not all brain regions participate to the observed deficiency in brain size; similarly, the volumetric defect progresses diversely in different brain areas also depending on the specific Mecp2 genetic lesion and gender. Interestingly, in some regions volumetric defects anticipate overt symptoms, possibly revealing where the pathology originates and providing a useful biomarker for assessing drug efficacy in pre-clinical studies. DOI: 10.1016/j.nbd.2023.106083

DOI: 10.1371/journal.pone.0287670 Cell migration requires a complex array of molecular events to promote protrusion at the front of motile cells. The scaffold protein LL5beta interacts with the scaffold ERC1, and recruits it at plasma membrane–associated platforms that form at the front of migrating tumor cells. LL5beta and ERC1 proteins support protrusion during migration as shown by the finding that depletion of either endogenous protein impairs tumor cell motility and invasion. In this study we have tested the hypothesis that interfering with the interaction between LL5beta and ERC1 may be used to interfere with the function of the endogenous proteins to inhibit tumor cell migration. For this, we identified ERC1(270-370) and LL5beta(381-510) as minimal fragments required for the direct interaction between the two proteins. The biochemical characterization demonstrated that the specific regions of the two proteins, including predicted intrinsically disordered regions, are implicated in a reversible, high affinity direct heterotypic interaction. NMR spectroscopy further confirmed the disordered nature of the two fragments and also support the occurrence of interaction between them. We tested if the LL5beta protein fragment interferes with the formation of the complex between the two full-length proteins. Coimmunoprecipitation experiments showed that LL5beta(381-510) hampers the formation of the complex in cells. Moreover, expression of either fragment is able to specifically delocalize endogenous ERC1 from the edge of migrating MDA-MB-231 tumor cells. Coimmunoprecipitation experiments show that the ERC1-binding fragment of LL5beta interacts with endogenous ERC1 and interferes with the binding of endogenous ERC1 to full length LL5beta. Expression of LL5beta(381-510) affects tumor cell motility with a reduction in the density of invadopodia and inhibits transwell invasion. These results provide a proof of principle that interfering with heterotypic intermolecular interactions between components of plasma membrane–associated platforms forming at the front of tumor cells may represent a new approach to inhibit cell invasion.

In the manuscript "Tolerogenic IL-10-engineered dendritic cell-based therapy to restore antigen-specific tolerance in T cell mediated diseases" published in Journal of Autoimmunity on May 22 2023 (doi: 10.1016/j.jaut.2023.103051). We describe the in vitro generation of dendritic cells genetically modified to render them pro-tolerogenic and able to modulate unwanted immune responses to auto-antigens both in vitro and in vivo. Raw data for each main figure published in the manuscript are available in the relative.csv file. Read the Read_me.rtf file for more details and for the accession numbers of NGS data deposited at GEO.

we performed a functional analysis of the BrS familial rare variant NM_198056.2:c.3673G>A (NP_932173.1:p.Glu1225Lys), which has been never functionally characterized before in a cardi-ac-relevant context, as the human cardiomyocyte. Using a specific lentiviral vector encoding a GFP-tagged SCN5A gene carrying the specific c.3673G>A variant and CMs differentiated from control PSCs (PSC-CMs), we demonstrated an impairment of the mutated Nav1.5, thus suggesting the pathogenicity of the rare BrS detected variant.

In the manuscript "Aryl hydrocarbon receptor activity downstream of IL-10 signaling is required to promote regulatory functions in human dendritic cells" published in Cell Reports on March 03, 2023 https://doi.org/10.1016/j.celrep.2023.112193 we describe a novel role for AHR in controlling the establishment of tolerogenic gene expression patterns and functional features down-stream IL-10 signaling in human dendritic cells. Raw data for each main figure published in the manuscript are available in the relative folder containing a .csv file for each figure panel. Read the Description.rtf file for more details and for the accession numbers of NGS data deposited at GEO.

Scaled input dataset for training the Long Short-Term Memory (LSTM) recurrent neural networks for prediction of gestational age at birth developed and included in the RF-2016-02364081 project titled "A generalized prediction framework of preterm birth: The combination of maternal risk factors, fetal and newborn functional and structural brain connectivity for predicting neurodevelopmental outcome".

Raw data to reprlicate the analyses conducted in "68Ga-PSMA and 68Ga-DOTA-RM2 PET/MRI in Recurrent Prostate Cancer: Diagnostic Performance and Association with Clinical and Histopathological Data"

Raw data, not included in the pubblished supplemental materials, that generated the figures of the manuscript by Raia et al. "Ceruloplasmin-deficient mice show dysregulation of lipid metabolism in liver and adipose tissue reduced by a protein replacement" International Journal of Molecular Sciences, 2023, 24, 1150 (https://doi.org/10.3390/ijms24021150).