Biology subjects

Zinghirino, F.

Publications and source records attributed to Zinghirino, F..

6 recordsLinked to original sources

bioRxiv2026

In-Depth Characterization of Stem Cell Potency and Genotoxicity for Clinical-Scale Ex Vivo CRISPR/Cas9 Gene Editing

Translating CRISPR/Cas9-based homology-directed repair (HDR) strategies into clinical application remains a major challenge due to limited standardization, concerns over safety, and efficacy issues. Here, we present a comprehensive and clinically compliant preclinical framework for the ex vivo correction of Wiskott-Aldrich Syndrome (WAS) using a CRISPR/Cas9-AAV6 platform targeting hematopoietic stem and progenitor cells (HSPCs). In this study, we established a clinical-compatible platform enabling large-scale manufacturing while preserving HSPC viability, stemness, and multilineage functionality. To overcome low HSPC long-term engraftment, we fine-tuned AAV dosing and transiently modulated p53 signalling, achieving significantly improved in vivo correction and repopulation. Importantly, we implemented a multi-tiered genotoxicity assessment strategy, integrating in silico, genome-wide, and orthogonal assays, revealing a largely favorable safety profile with minimal off-target risks and no signs of clonal dominance or transformation. Longitudinal in vivo safety monitoring revealed donor specific rare off-target events and structural variants. This highlights the crucial importance of patient monitoring after transplantation, further emphasized by the identification of a de novo chromosomal rearrangements that could be detected exclusively following cell engraftment in mice. This work offers a robust and adaptable roadmap for future HDR-based gene editing platforms, establishing critical benchmarks for efficacy, safety, and regulatory readiness in the development of advanced therapeutic medicinal products.

molecular biology↗

bioRxiv2023

CRISPR/Cas9-based disease modelling and functional correction of Interleukin 7 Receptor alpha Severe Combined Immunodeficiency in T-lymphocytes and hematopoietic stem cells

Interleukin 7 Receptor Severe Combined Immunodeficiency (IL7R-SCID) is a life-threatening disorder caused by homozygous mutations in the IL7RA gene. Defective IL7R expression in humans hampers T cell precursors proliferation and differentiation during lymphopoiesis resulting in absence of T cells in newborns, who succumb to severe infections and death early after birth. Previous attempts to tackle IL7R-SCID by viral gene therapy have shown that unregulated IL7R expression predisposes to leukaemia, suggesting the application of targeted gene editing to insert a correct copy of the IL7RA gene in its genomic locus and mediate its physiological expression as a more feasible therapeutic approach. To this aim, we have first developed a CRISPR/Cas9-based IL7R-SCID disease modelling system that recapitulates the disease phenotype in primary human T cells and hematopoietic stem and progenitor cells (HSPCs). Then, we have designed a knock-in strategy that targets IL7RA exon 1 and introduces via homology directed repair a corrective, promoterless IL7RA cDNA followed by a reporter cassette through AAV6 transduction. Targeted integration of the corrective cassette in primary T cells restored IL7R expression and rescued functional downstream IL7R signalling. When applied to HSPCs further induced to differentiate into T cells in an Artificial Thymic Organoid system, our gene editing strategy overcame the T cell developmental block observed in IL7R-SCID patients, while promoting full maturation of T cells with physiological and developmentally regulated IL7R expression. Finally, genotoxicity assessment of the CRISPR/Cas9 platform in HSPCs using biased and unbiased technologies confirmed the safety of the strategy, paving the way for a new, efficient, and safe therapeutic option for IL7R-SCID patients.

molecular biology↗

bioRxiv2023

Human VDAC pseudogenes: an emerging role for VDAC1P8 pseudogene in acute myeloid leukemia

BackgroundVoltage-dependent anion selective channels (VDACs) are the most abundant mitochondrial outer membrane proteins, encoded in mammals by three genes, VDAC1, 2 and 3, mostly ubiquitously expressed. As mitochondrial gatekeepers, VDACs control organelle and cell metabolism and are involved in many diseases. Despite the presence of numerous VDAC pseudogenes in the human genome, their significance and possible role in VDAC protein expression has not yet been considered. ResultsWe investigated the relevance of processed pseudogenes of human VDAC genes, both in physiological and in pathological contexts. Using high-throughput tools and querying many genomic and transcriptomic databases, we show that some VDAC pseudogenes are transcribed in specific tissues and pathological contexts. The obtained experimental data confirm an association of the VDAC1P8 pseudogene with acute myeloid leukemia (AML). ConclusionsOur in-silico comparative analysis between the VDAC1 gene and its VDAC1P8 pseudogene, together with experimental data produced in AML cellular models, indicate a specific over-expression of the VDAC1P8 pseudogene in AML, correlated with a downregulation of the parental VDAC1 gene.

genomics↗

bioRxiv2020

The secret of VDAC isoforms is in their gene regulation? Characterization of human VDAC genes expression profile, promoter activity, and transcriptional regulators.

BackgroundVDACs (Voltage-Dependent Anion-selective Channels) are pore-forming proteins of the outer mitochondrial membrane, whose permeability is primarily due to their presence. In higher eukaryotes three isoforms raised during the evolution: they have the same exon-intron organization and the proteins show the same channel-forming activity. We provide a comprehensive analysis of the three human VDAC genes (VDAC1-3), their expression profiles, promoter activity, and potential transcriptional regulators. ResultsVDAC isoforms are broadly but also specifically expressed in various human tissues at different levels with a predominance of VDAC1 and VDAC2 over VDAC3. However, RNA-seq CAGE approach revealed a higher level of transcription activation of VDAC3 gene. We experimentally confirmed this information by reporter assay of VDACs promoter activity. Transcription Factor Binding Sites (TFBSs) distribution in the promoters was investigated. The main regulators common to the three VDAC genes were identified as E2FF, NRF1, KLFS, EBOX transcription factors family members. All of them are involved in cell cycle and growth, proliferation, differentiation, apoptosis, and metabolism. More transcription factors specific for each isoform gene were identified, supporting the results in the literature, indicating a general role of VDAC1, as actor of apoptosis for VDAC2, and the involvement in sex determination and development of VDAC3. ConclusionsFor the first time, we propose a comparative analysis of human VDAC promoters to investigate their specific biological functions. Bioinformatics and experimental results confirm the essential role of VDAC protein family in mitochondrial functionality. Moreover, insights about a specialized function and different regulation mechanisms arise for the three isoforms genes.

molecular biology↗

bioRxiv2020

NRF-1 and HIF-1α modulate activity of human VDAC1 gene promoter during starvation and hypoxia in HeLa cells

VDAC (Voltage Dependent Anion Channel) is a family of pore forming protein located in the outer mitochondrial membrane. Its channel property ensures metabolites exchange between mitochondria and the rest of the cell resulting in metabolism and bioenergetics regulation, and in cell death and life switch. VDAC1 is the best characterized and most abundant isoform, and is involved in many pathologies, as cancer or neurodegenerative diseases. However, little information is available about its gene expression regulation in normal and/or pathological conditions. In this work, we explored VDAC1 gene expression regulation in normal conditions and in the contest of metabolic and energetic mitochondrial dysfunction and cell stress. The most active area of the putative promoter region was characterized in terms of transcription factors responsive elements both by bioinformatic studies and promoter activity experiments. In particular, we found a predominant presence of NRF-1 together with other transcription factors binding sites, involved in cell growth, proliferation, development and we studied their prevalence in gene activity. Furthermore, upon depletion of nutrients or controlled hypoxia, as reported in various pathologies, we found that VDAC1 transcripts levels were significantly increased in a time related manner. VDAC1 promoter activity was also validated by gene reporter assays. According to PCR real-time data, it was confirmed that VDAC1 promoter activity is further stimulated when are exposed to stress. A bioinformatic survey suggested NRF-1 e HIF-1 as the most active TFBS. Their validation was obtained by mutagenesis and overexpression experiments. In conclusion, we demonstrated experimentally the involvement of both NRF-1 and HIF-1 in the regulation of VDAC1 promoter activation at basal level and in cell stress conditions.

molecular biology↗

bioRxiv2020

Emergence of stealth polymorphs that escape α-synuclein amyloid monitoring, take over and acutely spread in neurons

The conformational strain diversity characterizing -synuclein (-syn) amyloid fibrils is possibly at the origin of the different clinical presentations of synucleinopathies. Experimentally, various -syn fibril polymorphs have been obtained from distinct fibrillization conditions by altering the medium constituents and were selected by amyloid monitoring using the probe Thioflavin T (ThT). We report here that besides classical ThT positive products, fibrillization in saline simultaneously gives rise to competing fibril polymorphs that are invisible to ThT (stealth polymorphs), and that can take over. Due to competition, spontaneous generation of such stealth polymorphs bears on the apparent fibrillization kinetics and on the final plateau values. Their emergence has thus been ignored so far or mistaken for fibrillization inhibitions/failures. Compared to their ThT-positive counterparts, and as judged from their chemical shift resonances fingerprint, these new stealth polymorphs present a yet undescribed atomic organization and show an exacerbated propensity (approx. 20-fold) towards self-replication in cortical neurons. They also trigger a long distance synucleinopathic spread along nigro-striatal projections in vivo. In order to rapidly screen fibrillization products for the presence of such stealth polymorphs, we designed a simple multiplexed assay that can be easily and rapidly operated. This assay allows us to demonstrate the sustainability of the conformational replication of these novel and particularly invasive strains. It should also be of help to avoid erroneous upstream interpretations of fibrillization rates based on sole ThT, and to expedite further structural and functional characterization of stealth amyloid assemblies. One Sentence Summarystealth -synuclein fibrils take over

neuroscience↗