Biology subjects

Broccoli, V.

Publications and source records attributed to Broccoli, V..

13 recordsLinked to original sources

bioRxiv2026

Engineered α-Synuclein-specific nanobody CAR iTregs restrain neuroinflammation and proteinopathy in Parkinson's disease mice

Parkinson's disease (PD) is characterized by progressive DAergic neurodegeneration and the accumulation of aggregated -Synuclein (Syn), which drives chronic neuroinflammation through sustained activation of innate and adaptive immune responses. Regulatory T cells (Tregs) exert potent immunosuppressive functions and have shown neuroprotective effects in preclinical PD models; however, clinical translation of polyclonal Treg therapies has been limited by poor tissue specificity and insufficient therapeutic efficacy. To overcome these limitations, we engineered induced human Tregs (iTregs) expressing chimeric antigen receptors (CARs) directed against pathological Syn aggregates. Among the CAR designs tested, only a nanobody-based construct incorporating NbSyn87 displayed selective antigen-dependent activation in response to Syn preformed fibrils (PFFs). Intriguingly, despite the ability of the parental NbSyn87 nanobody to bind both monomeric and aggregated Syn, incorporation into the CAR architecture conferred functional selectivity for aggregated conformers. This feature enabled discrimination between pathological extracellular aggregates and physiological monomeric Syn, providing an important safety advantage. To evaluate therapeutic activity in vivo, we established an immunodeficient mouse model of synucleinopathy permissive to human cell engraftment. iTregs preferentially accumulated within Syn-rich brain regions and, in the presence of astrocyte-derived human IL-2 with antigen-independent mechanism. Conversely, only CAR iTregs directed against Syn significantly reduced microglial and astrocytic activation, decreased pro-inflammatory cytokine expression, and attenuated Syn pathology. Collectively, these findings demonstrate that Syn-specific CAR iTregs can selectively exert potent local immunomodulatory effects, establishing a promising antigen-specific cellular immunotherapy platform for PD and other synucleinopathies.

neuroscience↗

bioRxiv2026

Suppression of YAP/TAZ molecular targets by epigenetic editing interferes with GBM growth and invasiveness

Glioblastoma (GBM) is a highly aggressive brain tumor characterized by extensive heterogeneity, diffuse invasion, and recurrence despite multimodal therapy. Aberrant transcriptional programs driven by oncogenic signaling pathways sustain GBM growth, stemness, and therapy resistance, yet targeting individual molecular nodes has yielded limited clinical benefit. Here, we introduce a transcriptional rewiring strategy based on an engineered epigenetic silencer factor (ESF) targeting the YAP/TAZ-TEAD axis. We developed a TEAD1 Epigenetic Silencer (TES) by fusing the DNA-binding domain of TEAD1 to repressive epigenetic modules. TES selectively binds TEAD genomic targets and imposes stable transcriptional repression of YAP/TAZ-dependent gene programs through chromatin remodeling and DNA methylation. Genome-wide analyses revealed that TES preserves TEAD1 DNA-binding specificity while converting an oncogenic transcriptional platform into a repressive state. Functionally, TES impaired proliferation, induced cell death, and reduced migratory and invasive properties in glioma cell lines and patient-derived cancer stem-like cells. In vivo, TES significantly reduced tumor growth in orthotopic GBM xenograft models and enhanced the therapeutic efficacy of temozolomide. Importantly, TES was well tolerated by normal neural cells in vitro and in the adult mouse brain in vivo. These findings establish TES as a proof-of-concept epigenetic therapy to durably suppress oncogenic transcriptional networks in GBM.

cancer biology↗

bioRxiv2025

Fully Human Stem Cell-Derived BBB Model for Clinical Disease Modeling and Neurotropic AAV selection

The blood-brain barrier (BBB) is a highly functionalized vascular interface which regulates the exchange of substances between the neural parenchyma and its periphery. BBB leakage, leading to its uncontrolled permeability, is increasingly recognized to facilitate the onset of neuropathologies and aggravate their clinical progression. In vitro models of the BBB have rapidly evolved into elaborated structures that mimic its spatial architecture and multicellular nature. However, their cellular components are currently highly heterogeneous in origin and maturation state. Here, we have developed novel procedures to establish reproducible and scalable sources of endothelial, mural and astroglial cells generating a fully human pluripotent stem cell (hPSC)-derived BBB model, termed thBBBA. hPSC-derived BBB cell types are readily assembled into thBBBAs that develop mature functional properties with high barrier impermeability. Mature thBBBAs can also be generated by frozen hPSC-derived cell samples, providing a simple and scalable off-the-shelf system for general use. thBBBAs were instrumental in identifying the critical pathological role of an IL-6 autocrine source in disrupting thBBBA integrity, increasing its permeability to NMDAR antibodies from autoimmune encephalitis patients, and revealing the therapeutic effects of tocilizumab in this setting. Additionally, we have shown that thBBBAs are an invaluable system for ranking the clinical readiness of novel engineered AAV neurotropic capsids, previously selected in animal models or in vitro systems.

neuroscience↗

bioRxiv2025

Targeted and efficient AAV therapy for neuroblastoma via direct capsid-antibody coupling

Adeno-associated viral (AAV) vectors are widely used in gene therapy for their versatility and safety, but their broad tropism limits cell-specific applications such as targeting primary or metastatic tumor cells. To address this, we developed AAV-STITCH, a strategy using SpyTag technology to covalently attach polypeptides to the AAV capsid. This allows precise, dose-dependent coupling of an anti-GD2 ScFv to a galactose-binding-deficient AAV9-W503A capsid, redirecting tropism specifically to GD2-expressing neuroblastoma (NB) cells. In pseudometastatic xenograft mouse models, AAV-STITCHGD2 selectively transduced NB tumor cells without transduction of healthy tissues. Furthermore, delivery of a suicide gene via AAV-STITCHGD2 suppressed tumor growth and extended survival in mice with subcutaneous and pseudometastatic NB xenografts. These findings establish the feasibility of engineering AAVs with cell-type-specific transduction properties, providing a powerful and adaptable platform for the selective elimination of NB tumor cells. This technology marks a meaningful advance toward next-generation, targeted cancer therapies with strong clinical potential.

bioengineering↗

bioRxiv2023

A WFS1 variant disrupting acceptor splice site uncovers the impact of alternative splicing on ER-stress independent β cell apoptosis in a patient with Wolfram syndrome.

Aims/hypothesisWolfram Syndrome 1 (WS1) is an inherited condition mainly manifesting in childhood-onset diabetes mellitus and progressive optic nerve atrophy. The causative gene, WFS1, encodes for Wolframin, a master regulator of several cellular responses, whose mutations associate with clinical variability. Indeed, nonsense/frameshift variants correlate with more severe symptoms than missense/in-frame ones. As achieving a genotype-phenotype correlation is crucial to deal with disease outcome, works investigating the impact of transcriptional and translational landscapes stemming from such mutations are needed. Therefore, we sought to elucidate the molecular determinants behind the pathophysiological alterations in a WS1 patient carrying compound heterozygous mutations in WFS1 gene: c.316-1G>A, affecting the acceptor splice site (ASS) upstream exon 4, and c.757A>T, introducing a premature termination codon (PTC) in exon 7. MethodsBioinformatic analysis was carried out to infer the alternative splicing events occurring after disruption of ASS, followed by RNAseq and PCR to validate the transcriptional landscape. Patient-derived induced Pluripotent Stem Cells (iPSCs) were used as an in vitro model of WS1 and to investigate the WFS1 alternative splicing isoforms into pancreatic {beta} cells. CRISPR/Cas9 technology was employed to correct ASS mutation and generate a syngeneic control for the ER-stress induction and immunotoxicity assays. ResultsWe showed that patient-derived iPSCs retained the ability to differentiate into pancreatic {beta} cells. We demonstrated that the allele carrying the ASS mutation c.316-1G>A originates two PTC-containing alternative splicing transcripts (c.316del and c.316-460del), and two ORF-conserving mRNAs (c.271-513del and c.316-456del) leading to N-terminally truncated polypeptides. By retaining the C-terminal domain, these isoforms sustained the endoplasmic reticulum (ER)-stress response in {beta} cells. Otherwise, PTC-carrying transcripts were regulated by the nonsense mediated decay (NMD) in basal conditions. Exposure to cell stress inducers and pro-inflammatory cytokines affected the NMD-related gene SMG7 expression levels (>2 fold decrease; p<0.001) without eliciting a robust unfolded protein response in WFS1 {beta} cells, thus resulting in a dramatic accumulation of the PTC-containing isoforms c.316del (>100-fold increase over basal; p<0.001) and c.316-460del (>20-fold increase over basal; p<0.001) and predisposing affected {beta} cells to undergo apoptosis. Cas9-mediated recovery of ASS retrieved the canonical transcriptional landscape, rescuing the normal phenotype in patient-derived {beta} cells. Conclusions/interpretationThis study represents a new model to study Wolframin, highlighting how each single mutation of WFS1 gene can determine dramatically different functional outcomes. Our data point to increased vulnerability of WFS1 {beta} cells to stress and inflammation, and we postulate that this is triggered by escaping NMD and accumulation of mutated transcripts and truncated proteins. These findings pave the way for further studies on the molecular basis of genotype-phenotype relationship in WS1, to uncover the key determinants that might be targeted to ameliorate the clinical outcome of patients affected by this rare disease.

genetics↗

bioRxiv2023

New AAV9 engineered variants with enhanced neurotropism and reduced liver off-targeting in mice and marmosets

Adeno-Associated Virus 9 (AAV9) is a delivery platform highly exploited to develop gene-based treatments for neurological disorders given its low pathogenicity and brain tissue tropism. However, the efficacy of this vector is dampened by its relatively low efficiency to cross the adult blood-brain barrier (BBB) and inherent targeting to the liver upon intravenous delivery. We generated a new peptide display library starting from a galactose binding-deficient AAV9 capsid and selected two new AAV9 engineered capsids, named AAV-Se1 and AAV-Se2, with an enhanced targeting in mouse and marmoset brains after intravenous delivery. Interestingly, the loss of the galactose binding strongly reduced the undesired targeting to peripheral organs, and above all liver, while not compromising the transduction of the brain vasculature. However, we had to reconstitute the galactose binding in order to efficiently infect non-endothelial brain cells. Thus, the combinatorial actions of the galactose-binding domain and the installed exogenous displayed peptide are crucial to enhance BBB crossing together with brain cell transduction. We also identified Ly6C1 as primary receptor for AAV-Se2 which is a Ly6A homologue highly expressed in the brain endothelial cells. This study describes a new strategy to select neurotropic AAV9 variants and identifies two novel capsids with high brain endothelial infectivity and extremely low liver targeting based on manipulating the AAV9 galactose binding domain.

bioengineering↗

bioRxiv2022

Balanced SET levels favor the correct enhancer repertoire during cell fate acquisition

Within the chromatin, distal elements interact with promoters to regulate specific transcriptional programs. Histone acetylation, interfering with the net charges of the nucleosomes, is a key player in this regulation. Here, we report that the onco-protein SET is a critical determinant for the levels of histone acetylation within enhancers. We disclose that conditions in which SET is accumulated, including the severe Schinzel-Giedion Syndrome (SGS), are characterized by a failure in the usage of the distal regulatory regions typically employed during fate commitment. This is accompanied by the usage of alternative enhancers leading to a massive rewiring of the distal control of the gene transcription. This represents a (mal)adaptive mechanism that, on one side, allows to achieve a certain degree of differentiation, while on the other affects the fine and corrected maturation of the cells. Thus, we propose the differential in cis-regulation as a contributing factor to the pathological basis of the SET-related disorders in humans, including SGS, neurodevelopmental disorders, myeloproliferative diseases, and cancer.

developmental biology↗

bioRxiv2022

MCT1-dependent energetic failure and neuroinflammation underlie optic nerve degeneration in Wolfram syndrome mice

Wolfram syndrome 1 (WS1) is a rare genetic disorder caused by mutations in the WFS1 gene leading to a wide spectrum of clinical dysfunctions, among which blindness, diabetes and neurological deficits are the most prominent. WFS1 encodes for the endoplasmic reticulum (ER) resident transmembrane protein Wolframin with multiple functions in ER processes. However, the WFS1-dependent etiopathology in retinal cells is unknown. Herein, we showed that Wfs1 mutant mice developed early retinal electrophysiological impairments followed by marked visual loss. Interestingly, axons and myelin disruption in the optic nerve preceded the degeneration of the retinal ganglion cell bodies in the retina. Transcriptomics at pre-degenerative stage revealed the STAT3-dependent activation of proinflammatory glial markers with reduction of the homeostatic and pro-survival factors Glutamine synthetase and BDNF. Furthermore, label-free comparative proteomics identified a significant reduction of the monocarboxylate transport isoform 1 (MCT1) and its partner Basigin that are highly enriched on retinal astrocytes and myelin-forming oligodendrocytes in optic nerve together with Wolframin. Loss of MCT1 caused a failure in lactate transfer from glial to neuronal cell bodies and axons leading to a chronic hypometabolic state. Thus, this bioenergetic impairment is occurring concurrently both in the axonal regions and cell bodies of the retinal ganglion cells, selectively endangering their survival while impacting less on other retinal cells. This metabolic dysfunction occurs months before the frank RGC degeneration suggesting an extended time window for intervening with new therapeutic strategies focused on boosting retinal and optic nerve bioenergetics in WS1.

neuroscience↗

bioRxiv2021

Controlled administration of aerosolized SARS-CoV-2 to K18-hACE2 transgenic mice uncouples respiratory infection and anosmia from fatal neuroinvasion

The development of a tractable small animal model faithfully reproducing human COVID-19 pathogenesis would arguably meet a pressing need in biomedical research. Thus far, most investigators have used transgenic mice expressing the human ACE2 in epithelial cells (K18-hACE2 transgenic mice) that are intranasally instilled with a liquid SARS-CoV-2 suspension under deep anesthesia. Unfortunately, this experimental approach results in disproportionate high CNS infection leading to fatal encephalitis, which is rarely observed in humans and severely limits this models usefulness. Here, we describe the use of an inhalation tower system that allows exposure of unanesthetized mice to aerosolized virus under controlled conditions. Aerosol exposure of K18-hACE2 transgenic mice to SARS-CoV-2 resulted in robust viral replication in the respiratory tract, anosmia, and airway obstruction, but did not lead to fatal viral neuroinvasion. When compared to intranasal inoculation, aerosol infection resulted in a more pronounced lung pathology including increased immune infiltration, fibrin deposition and a transcriptional signature comparable to that observed in SARS-CoV-2- infected patients. This model may prove useful for studies of viral transmission, disease pathogenesis (including long-term consequences of SARS-CoV-2 infection) and therapeutic interventions.

immunology↗

bioRxiv2020

A human stem cell-derived neurosensory-epithelial circuitry on-a-chip to model herpes simplex virus reactivation

Both emerging viruses and well-known viral pathogens endowed with neurotropism can either impair directly the neuronal functions or induce physio-pathological changes by diffusing from the periphery through neurosensory-epithelial connections. However, the current lack of an in vitro system modeling the connectivity between human neurons and peripheral tissues excludes the analysis of viral latency and reactivation and the assessment of natural/artificial induced anti-viral immunity. In this study, we developed the first stable topographic neurosensory-epithelial connection on-a-chip using human stem cell derived dorsal root ganglia (DRG) sensory neurons. Bulk and single cell transcriptomics showed that different combinations of key receptors for Herpes Simplex Virus 1 (HSV-1) are expressed by each sensory neuronal cell type. This neuronal-epithelial circuitry enabled a detailed analysis of the HSV infectivity faithfully modeling its dynamics and cell type specificity. The reconstitution of an organized connectivity between human sensory neurons and keratinocytes into microfluidic chips provides for the first time a powerful in vitro platform to model viral latency and reactivation of human viral pathogens.

microbiology↗

bioRxiv2019

Whole brain delivery of an instability-prone Mecp2 transgene improves behavioral and molecular pathological defects in mouse models of Rett syndrome

Rett syndrome (RTT) is an incurable neurodevelopmental disorder caused by mutations in the gene encoding for methyl-CpG binding-protein 2 (MeCP2). Gene therapy for this disease presents inherent hurdles since MECP2 is expressed throughout the brain and its duplication leads to severe neurological conditions as well. However, the recent introduction of AAV-PHP.eB, an engineered capsid with an unprecedented efficiency in crossing the blood-brain barrier upon intravenous injection, has provided an invaluable vehicle for gene transfer in the mouse nervous system. Herein, we use AAV-PHP.eB to deliver an instability-prone Mecp2 (iMecp2) transgene cassette which, increasing RNA destabilization and inefficient protein translation of the viral Mecp2 transgene, limits supraphysiological Mecp2 protein levels in transduced neural tissues. Intravenous injections of the PHP.eB-iMecp2 virus in symptomatic male and female Mecp2 mutant mice significantly ameliorated the disease progression with improved locomotor activity, coordination, lifespan and normalization of altered gene expression and mTOR signaling. Remarkably, PHP.eB-iMecp2 administration did not result in severe toxicity effects either in female Mecp2 mutant or in wild-type animals. In contrast, we observed a strong immune response to the transgene in treated male Mecp2 mutant mice that was overcome by immunosuppression. Overall, PHP.eB-mediated delivery of the iMecp2 cassette provided widespread and efficient gene transfer maintaining physiological Mecp2 protein levels in the brain. This combination defines a novel viral system with significant therapeutic efficacy and increased safety which can contribute to overcome the hurdles that are delaying clinical applications of gene therapy for RTT.\n\nOne Sentence SummaryGlobal brain transduction of the instability-prone Mecp2 transgene by systemic AAV-PHP.eB administration is both safe and effective in protecting male and female Mecp2 mutant mice from the RTT disease phenotype.

neuroscience↗

bioRxiv2019

SULTA4A1 modulates synaptic development and function by promoting the formation of PSD-95/NMDAR complex

Sulfotransferase 4A1 (SULT4A1) is a cytosolic sulfotransferase, that is highly conserved across species and extensively expressed in the brain. However, the biological function of SULT4A1 is unclear. SULT4A1 has been implicated in several neuropsychiatric disorders, such as Phelan-McDermid Syndrome and schizophrenia. Here, we investigate the role of SULT4A1 within neuron development and function. Our data demonstrate that SULT4A1 modulates neuronal branching complexity and dendritic spines formation. Moreover, we show that SULT4A1, by negatively regulating the catalytic activity of Pin1 towards PSD-95, facilitates NMDAR synaptic expression and function. Finally, we demonstrate that the pharmacological inhibition of Pin1 reverses the pathological phenotypes of SULT4A1 knockdown neurons by specifically restoring dendritic spine density and rescuing NMDAR-mediated synaptic transmission. Together, these findings identify SULT4A1 as a novel player in neuron development and function by modulating dendritic morphology and synaptic activity.

neuroscience↗

bioRxiv2018

CRISPRa-mediated Kcna1 upregulation decreases neuronal excitability and suppresses seizures in a rodent model of temporal lobe epilepsy

Epilepsy is a major health burden, calling for new mechanistic and therapeutic insights. CRISPR-mediated gene editing shows promise to cure genetic pathologies, although hitherto it has mostly been applied ex-vivo. Its translational potential for treating non-genetic pathologies is still unexplored. Furthermore, neurological diseases represent an important challenge for the application of CRISPR, because of the need in many cases to manipulate gene function of neurons in situ. A variant of CRISPR, CRISPRa, offers the possibility to modulate the expression of endogenous genes by directly targeting their promoters. We asked if this strategy can effectively treat acquired focal epilepsy, focusing on ion channels because their manipulation is known be effective in changing network hyperactivity and hypersynchronisation. We applied a doxycycline-inducible CRISPRa technology to increase the expression of the potassium channel gene Kcna1 (encoding Kv1.1) in mouse hippocampal excitatory neurons. CRISPRa-mediated Kv1.1 upregulation led to a substantial decrease in neuronal excitability. Continuous video-EEG telemetry showed that AAV9-mediated delivery of CRISPRa, upon doxycycline administration, decreased spontaneous generalized tonic-clonic seizures in a model of temporal lobe epilepsy, and rescued cognitive impairment and transcriptomic alterations associated with chronic epilepsy. The focal treatment minimizes concerns about off-target effects in other organs and brain areas. This study provides the proof of principle for a translational CRISPR-based approach to treat neurological diseases characterized by abnormal circuit excitability.

neuroscience↗