Efficient correction of ABCA4 variants by CRISPR-Cas9 in hiPSCs derived from Stargardt disease patients.

Inherited retinal dystrophies comprise a broad group of genetic eye diseases without effective treatment. Among them, Stargardt disease is the second most prevalent pathology. This pathology triggers progressive retinal degeneration and vision loss in children and adults. In recent years, the evolution of several genome editing technologies, such as the CRISPR-Cas9 system, has revolutionized disease modeling and personalized medicine. Human induced pluripotent stem cells also provide a valuable tool for in vitro disease studies and therapeutic applications. Here, we show precise correction of two ABCA4 pathogenic variants in human induced pluripotent stem cells from two unrelated patients affected with Stargardt disease. Gene editing was achieved with no detectable off-target genomic alterations, demonstrating efficient ABCA4 gene correction without deleterious effects. These results will contribute to the development of emerging gene and cell therapies for inherited retinal dystrophies.

The microphthalmia-associated transcription factor is involved in gastrointestinal stromal tumor growth.

Gastrointestinal stromal tumors (GISTs) are the most common neoplasms of mesenchymal origin, and most of them emerge due to the oncogenic activation of KIT or PDGFRA receptors. Despite their relevance in GIST oncogenesis, critical intermediates mediating the KIT/PDGFRA transforming program remain mostly unknown. Previously, we found that the adaptor molecule SH3BP2 was involved in GIST cell survival, likely due to the co-regulation of the expression of KIT and Microphthalmia-associated transcription factor (MITF). Remarkably, MITF reconstitution restored KIT expression levels in SH3BP2 silenced cells and restored cell viability. This study aimed to analyze MITF as a novel driver of KIT transforming program in GIST. Firstly, MITF isoforms were characterized in GIST cell lines and GIST patients' samples. MITF silencing decreases cell viability and increases apoptosis in GIST cell lines irrespective of the type of KIT primary or secondary mutation. Additionally, MITF silencing leads to cell cycle arrest and impaired tumor growth in vivo. Interestingly, MITF silencing also affects ETV1 expression, a linage survival factor in GIST that promotes tumorigenesis and is directly regulated by KIT signaling. Altogether, these results point to MITF as a key target of KIT/PDGFRA oncogenic signaling for GIST survival and tumor growth.

SH3BP2 Silencing Increases miRNAs Targeting ETV1 and Microphthalmia-Associated Transcription Factor, Decreasing the Proliferation of Gastrointestinal Stromal Tumors.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. Gain of function in receptor tyrosine kinases type III, KIT, or PDGFRA drives the majority of GIST. Previously, our group reported that silencing of the adaptor molecule SH3 Binding Protein 2 (SH3BP2) downregulated KIT and PDGFRA and microphthalmia-associated transcription factor (MITF) levels and reduced tumor growth. This study shows that SH3BP2 silencing also decreases levels of ETV1, a required factor for GIST growth. To dissect the SH3BP2 pathway in GIST cells, we performed a miRNA array in SH3BP2-silenced GIST cell lines. Among the most up-regulated miRNAs, we found miR-1246 and miR-5100 to be predicted to target MITF and ETV1. Overexpression of these miRNAs led to a decrease in MITF and ETV1 levels. In this context, cell viability and cell cycle progression were affected, and a reduction in BCL2 and CDK2 was observed. Interestingly, overexpression of MITF enhanced cell proliferation and significantly rescued the viability of miRNA-transduced cells. Altogether, the KIT-SH3BP2-MITF/ETV1 pathway deserves to be considered in GIST cell survival and proliferation.

Impaired Bestrophin Channel Activity in an iPSC-RPE Model of Best Vitelliform Macular Dystrophy (BVMD) from an Early Onset Patient Carrying the P77S Dominant Mutation.

Best Vitelliform Macular dystrophy (BVMD) is the most prevalent of the distinctive retinal dystrophies caused by mutations in the BEST1 gene. This gene, which encodes for a homopentameric calcium-activated ion channel, is crucial for the homeostasis and function of the retinal pigment epithelia (RPE), the cell type responsible for recycling the visual pigments generated by photoreceptor cells. In BVMD patients, mutations in this gene induce functional problems in the RPE cell layer with an accumulation of lipofucsin that evolves into cell death and loss of sight. In this work, we employ iPSC-RPE cells derived from a patient with the p.Pro77Ser dominant mutation to determine the correlation between this variant and the ocular phenotype. To this purpose, gene and protein expression and localization are evaluated in iPSC-RPE cells along with functional assays like phagocytosis and anion channel activity. Our cell model shows no differences in gene expression, protein expression/localization, or phagocytosis capacity, but presents an increased chloride entrance, indicating that the p.Pro77Ser variant might be a gain-of-function mutation. We hypothesize that this variant disturbs the neck region of the BEST1 channel, affecting channel function but maintaining cell homeostasis in the short term. This data shed new light on the different phenotypes of dominant mutations in BEST1, and emphasize the importance of understanding its molecular mechanisms. Furthermore, the data widen the knowledge of this pathology and open the door for a better diagnosis and prognosis of the disease.

MYO1F Regulates IgE and MRGPRX2-Dependent Mast Cell Exocytosis.

The activation and degranulation of mast cells is critical in the pathogenesis of allergic inflammation and modulation of inflammation. Recently, we demonstrated that the unconventional long-tailed myosin, MYO1F, localizes with cortical F-actin and mediates adhesion and migration of mast cells. In this study, we show that knockdown of MYO1F by short hairpin RNA reduces human mast cell degranulation induced by both IgE crosslinking and by stimulation of the Mas-related G protein-coupled receptor X2 (MRGPRX2), which has been associated with allergic and pseudoallergic drug reactions, respectively. Defective degranulation was accompanied by a reduced reassembly of the cortical actin ring after activation but reversed by inhibition of actin polymerization. Our data show that MYO1F is required for full Cdc42 GTPase activation, a critical step in exocytosis. Furthermore, MYO1F knockdown resulted in less granule localization in the cell membrane and fewer fissioned mitochondria along with deficient mitochondria translocation to exocytic sites. Consistent with that, AKT and DRP1 phosphorylation are diminished in MYO1F knockdown cells. Altogether, our data point to MYO1F as an important regulator of mast cell degranulation by contributing to the dynamics of the cortical actin ring and the distribution of both the secretory granules and mitochondria.

Mutation in KARS: A novel mechanism for severe anaphylaxis.

BACKGROUND: Anaphylaxis is a severe allergic reaction that can be lethal if not treated adequately. The underlying molecular mechanisms responsible for the severity are mostly unknown. OBJECTIVE: This study is based on a clinical case of a patient with extremely severe anaphylaxis to paper wasp venom. This patient has a mutation in the KARS gene, which encodes lysyl-tRNA synthetase (LysRS), a moonlight protein with a canonical function in protein synthesis and a noncanonical function in antigen dependent-FcεRI activation in mast cells. In this study, the objective was to characterize the mutation at the molecular level. METHODS: Analysis of the KARS mutation was carried out using biochemical and functional approaches, cell transfection, Western blot, confocal microscopy, cell degranulation, prostaglandin D2 secretion, and proteases gene transcription. Structural analysis using molecular dynamics simulations and well-tempered metadynamics was also performed. RESULTS: The mutation found, P542R (proline was replaced by arginine at aminoacid 542), affects the location of the protein as we show in biochemical and structural analyses. The mutation resembles active LysRS and causes a constitutive activation of the microphthalmia transcription factor, which is involved in critical mast cell functions such as synthesis of mediators and granule biogenesis. Moreover, the structural analysis provides insights into how LysRS works in mast cell activation. CONCLUSIONS: A link between the aberrant LysRS-P542R function and mast cell-exacerbated activation with increase in proinflammatory mediator release after antigen-IgE-dependent response could be established.

Myo1f, an Unconventional Long-Tailed Myosin, Is a New Partner for the Adaptor 3BP2 Involved in Mast Cell Migration.

Mast cell chemotaxis is essential for cell recruitment to target tissues, where these cells play an important role in adaptive and innate immunity. Stem cell factor (SCF) is a major chemoattractant for mast cells. SCF binds to the KIT receptor, thereby triggering tyrosine phosphorylation in the cytoplasmic domain and resulting in docking sites for SH2 domain-containing molecules, such as Lyn and Fyn, and the subsequent activation of the small GTPases Rac that are responsible for cytoskeletal reorganization and mast cell migration. In previous works we have reported the role of 3BP2, an adaptor molecule, in mast cells. 3BP2 silencing reduces FcεRI-dependent degranulation, by targeting Lyn and Syk phosphorylation, as well as SCF-dependent cell survival. This study examines its role in SCF-dependent migration and reveals that 3BP2 silencing in human mast cell line (LAD2) impairs cell migration due to SCF and IgE. In that context we found that 3BP2 silencing decreases Rac-2 and Cdc42 GTPase activity. Furthermore, we identified Myo1f, an unconventional type-I myosin, as a new partner for 3BP2. This protein, whose functions have been described as critical for neutrophil migration, remained elusive in mast cells. Myo1f is expressed in mast cells and colocalizes with cortical actin ring. Interestingly, Myo1f-3BP2 interaction is modulated by KIT signaling. Moreover, SCF dependent adhesion and migration through fibronectin is decreased after Myo1f silencing. Furthermore, Myo1f silencing leads to downregulation of ß1 and ß7 integrins on the mast cell membrane. Overall, Myo1f is a new 3BP2 ligand that connects the adaptor to actin cytoskeleton and both molecules are involved in SCF dependent mast cell migration.

MRGPRX2-mediated mast cell response to drugs used in perioperative procedures and anaesthesia.

The study of anaphylactoid reactions during perioperative procedures and anaesthesia represents a diagnostic challenge for allergists, as many drugs are administered simultaneously, and approximately half of them trigger allergic reactions without a verifiable IgE-mediated mechanism. Recently, mast cell receptor MRGPRX2 has been identified as a cause of pseudo-allergic drug reactions. In this study, we analyse the ability of certain drugs used during perioperative procedures and anaesthesia to induce MRGPRX2-dependent degranulation in human mast cells and sera from patients who experienced an anaphylactoid reaction during the perioperative procedure. Using a ß-hexosaminidase release assay, several drugs were seen to cause mast cell degranulation in vitro in comparison with unstimulated cells, but only morphine, vancomycin and cisatracurium specifically triggered this receptor, as assessed by the release of ß-hexosaminidase in the control versus the MRGPRX2-silenced cells. The same outcome was seen when measuring degranulation based on the percentage of CD63 expression at identical doses. Unlike that of the healthy controls, the sera of patients who had experienced an anaphylactoid reaction induced mast-cell degranulation. The degranulation ability of these sera decreased when MRGPRX2 was silenced. In conclusion, MRGPRX2 is a candidate for consideration in non-IgE-mediated allergic reactions to some perioperative drugs, reinforcing its role in mast cell responses and their pathophysiology.

Silencing of adaptor protein SH3BP2 reduces KIT/PDGFRA receptors expression and impairs gastrointestinal stromal tumors growth.

Gastrointestinal stromal tumors (GISTs) represent about 80% of the mesenchymal neoplasms of the gastrointestinal tract. Most GISTs contain oncogenic KIT (85%) or PDGFRA (5%) receptors. The kinase inhibitor imatinib mesylate is the preferential treatment for these tumors; however, the development of drug resistance has highlighted the need for novel therapeutic strategies. Recently, we reported that the adaptor molecule SH3 Binding Protein 2 (SH3BP2) regulates KIT expression and signaling in human mast cells. Our current study shows that SH3BP2 is expressed in primary tumors and cell lines from GIST patients and that SH3BP2 silencing leads to a downregulation of oncogenic KIT and PDGFRA expression and an increase in apoptosis in imatinib-sensitive and imatinib-resistant GIST cells. The microphthalmia-associated transcription factor (MITF), involved in KIT expression in mast cells and melanocytes, is expressed in GISTs. Interestingly, MITF is reduced after SH3BP2 silencing. Importantly, reconstitution of both SH3BP2 and MITF restores cell viability. Furthermore, SH3BP2 silencing significantly reduces cell migration and tumor growth of imatinib-sensitive and imatinib-resistant cells in vivo. Altogether, SH3BP2 regulates KIT and PDGFRA expression and cell viability, indicating a role as a potential target in imatinib-sensitive and imatinib-resistant GISTs.

IgE-Related Chronic Diseases and Anti-IgE-Based Treatments.

IgE is an immunoglobulin that plays a central role in acute allergic reactions and chronic inflammatory allergic diseases. The development of a drug able to neutralize this antibody represents a breakthrough in the treatment of inflammatory pathologies with a probable allergic basis. This review focuses on IgE-related chronic diseases, such as allergic asthma and chronic urticaria (CU), and on the role of the anti-IgE monoclonal antibody, omalizumab, in their treatment. We also assess the off-label use of omalizumab for other pathologies associated with IgE and report the latest findings concerning this drug and other new related drugs. To date, omalizumab has only been approved for severe allergic asthma and unresponsive chronic urticaria treatments. In allergic asthma, omalizumab has demonstrated its efficacy in reducing the dose of inhaled corticosteroids required by patients, decreasing the number of asthma exacerbations, and limiting the effect on airway remodeling. In CU, omalizumab treatment rapidly improves symptoms and in some cases achieves complete disease remission. In systemic mastocytosis, omalizumab also improves symptoms and its prophylactic use to prevent anaphylactic reactions has also been discussed. In other pathologies such as atopic dermatitis, food allergy, allergic rhinitis, nasal polyposis, and keratoconjunctivitis, omalizumab significantly improves clinical manifestations. Omalizumab acts in two ways: by sequestering free IgE and by accelerating the dissociation of the IgE-Fcε receptor I complex.