Unravelling the Regions of Mutant F508del-CFTR More Susceptible to the Action of Four Cystic Fibrosis Correctors.

Cystic fibrosis (CF) is a genetic disease associated with the defective function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein that causes obstructive disease and chronic bacterial infections in airway epithelia. The most prevalent CF-causing mutation, the deletion of phenylalanine at position 508 (F508del), leads to CFTR misfolding, trafficking defects and premature degradation. A number of correctors that are able to partially rescue F508del-CFTR processing defects have been identified. Clinical trials have demonstrated that, unfortunately, mono-therapy with the best correctors identified to date does not ameliorate lung function or sweat chloride concentration in homozygous F508del patients. Understanding the mechanisms exerted by currently available correctors to increase mutant F508del-CFTR expression is essential for the development of new CF-therapeutics. We investigated the activity of correctors on the mutant F508del and wild type (WT) CFTR to identify the protein domains whose expression is mostly affected by the action of correctors, and we investigated their mechanisms of action. We found that the four correctors under study, lumacaftor (VX809), the quinazoline derivative VX325, the bithiazole compound corr4a, and the new molecule tezacaftor (VX661), do not influence either the total expression or the maturation of the WT-CFTR transiently expressed in human embryonic kidney 293 (HEK293) cells. Contrarily, they significantly enhance the expression and the maturation of the full length F508del molecule. Three out of four correctors, VX809, VX661 and VX325, seem to specifically improve the expression and the maturation of the mutant CFTR N-half (M1N1, residues 1-633). By contrast, the CFTR C-half (M2N2, residues 837-1480) appears to be the region mainly affected by corr4a. VX809 was shown to stabilize both the WT- and F508del-CFTR N-half isoforms, while VX661 and VX325 demonstrated the ability to enhance the stability only of the mutant F508del polypeptide.

CD32 captures committed haemogenic endothelial cells during human embryonic development.

During embryonic development, blood cells emerge from specialized endothelial cells, named haemogenic endothelial cells (HECs). As HECs are rare and only transiently found in early developing embryos, it remains difficult to distinguish them from endothelial cells. Here we performed transcriptomic analysis of 28- to 32-day human embryos and observed that the expression of Fc receptor CD32 (FCGR2B) is highly enriched in the endothelial cell population that contains HECs. Functional analyses using human embryonic and human pluripotent stem cell-derived endothelial cells revealed that robust multilineage haematopoietic potential is harboured within CD32+ endothelial cells and showed that 90% of CD32+ endothelial cells are bona fide HECs. Remarkably, these analyses indicated that HECs progress through different states, culminating in FCGR2B expression, at which point cells are irreversibly committed to a haematopoietic fate. These findings provide a precise method for isolating HECs from human embryos and human pluripotent stem cell cultures, thus allowing the efficient generation of haematopoietic cells in vitro.

Exonic knockout and knockin gene editing in hematopoietic stem and progenitor cells rescues RAG1 immunodeficiency.

Recombination activating genes (RAGs) are tightly regulated during lymphoid differentiation, and their mutations cause a spectrum of severe immunological disorders. Hematopoietic stem and progenitor cell (HSPC) transplantation is the treatment of choice but is limited by donor availability and toxicity. To overcome these issues, we developed gene editing strategies targeting a corrective sequence into the human RAG1 gene by homology-directed repair (HDR) and validated them by tailored two-dimensional, three-dimensional, and in vivo xenotransplant platforms to assess rescue of expression and function. Whereas integration into intron 1 of RAG1 achieved suboptimal correction, in-frame insertion into exon 2 drove physiologic human RAG1 expression and activity, allowing disruption of the dominant-negative effects of unrepaired hypomorphic alleles. Enhanced HDR-mediated gene editing enabled the correction of human RAG1 in HSPCs from patients with hypomorphic RAG1 mutations to overcome T and B cell differentiation blocks. Gene correction efficiency exceeded the minimal proportion of functional HSPCs required to rescue immunodeficiency in Rag1-/- mice, supporting the clinical translation of HSPC gene editing for the treatment of RAG1 deficiency.

HyperIgE in hypomorphic recombination-activating gene defects.

Increased immunogloblulin-E (IgE) levels associated with eosinophilia represent a common finding observed in Omenn syndrome, a severe immunodeficiency caused by decreased V(D)J recombination, leading to restricted T- and B-cell receptor repertoire. V(D)J recombination is initiated by the lymphoid-restricted recombination-activating gene (RAG) recombinases. The lack of RAG proteins causes a block in lymphocyte differentiation, resulting in T-B- severe combined immunodeficiency. Conversely, hypomorphic mutations allow the generation of few T and B cells, leading to a spectrum of immunological phenotypes, in which immunodeficiency associates to inflammation, immune dysregulation, and autoimmunity. Elevated IgE levels are frequently observed in hypomorphic RAG patients. Here, we describe the role of RAG genes in lymphocyte differentiation and maintenance of immune tolerance.

In Silico and In Vitro Evaluation of the Mechanism of Action of Three VX809-Based Hybrid Derivatives as Correctors of the F508del CFTR Protein.

Cystic fibrosis (CF), the most common autosomal recessive fatal genetic disease in the Caucasian population, is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel that regulates salt and water transport across a variety of secretory epithelia. Deletion of phenylalanine at position 508, F508del, the most common CF-causing mutation, destabilises the CFTR protein, causing folding and trafficking defects that lead to a dramatic reduction in its functional expression. Small molecules called correctors have been developed to rescue processing-defective F508del CFTR. We have combined in silico and in vitro approaches to investigate the mechanism of action and potential as CFTR correctors of three hybrid derivatives (2a, 7a, and 7m) obtained by merging the amino-arylthiazole core with the benzodioxole carboxamide moiety characterising the corrector lumacaftor. Molecular modelling analyses suggested that the three hybrids interact with a putative region located at the MSD1/NBD1 interface. Biochemical analyses confirmed these results, showing that the three molecules affect the expression and stability of the F508del NBD1. Finally, the YFP assay was used to evaluate the influence of the three hybrid derivatives on F508del CFTR function, assessing that their effect is additive to that of the correctors VX661 and VX445. Our study shows that the development and testing of optimised compounds targeting different structural and functional defects of mutant CFTR is the best strategy to provide more effective correctors that could be used alone or in combination as a valuable therapeutic option to treat an even larger cohort of people affected by CF.

NBD2 Is Required for the Rescue of Mutant F508del CFTR by a Thiazole-Based Molecule: A Class II Corrector for the Multi-Drug Therapy of Cystic Fibrosis.

Cystic fibrosis (CF) is caused by loss-of-function mutations in the CF transmembrane conductance regulator (CFTR) protein, an anion channel that regulates epithelial surface fluid secretion. The deletion of phenylalanine at position 508 (F508del) is the most common CFTR mutation. F508del CFTR is characterized by folding and trafficking defects, resulting in decreased functional expression of the protein on the plasma membrane. Several classes of small molecules, named correctors, have been developed to rescue defective F508del CFTR. Although individual correctors failed to improve the clinical status of CF patients carrying the F508del mutation, better results were obtained using correctors combinations. These results were obtained according to the premise that the administration of correctors having different sites of action should enhance F508del CFTR rescue. We investigated the putative site of action of an aminoarylthiazole 4-(3-chlorophenyl)-N-(3-(methylthio)phenyl)thiazol-2-amine, named FCG, with proven CFTR corrector activity, and its synergistic effect with the corrector VX809. We found that neither the total expression nor the maturation of WT CFTR transiently expressed in human embryonic kidney 293 cells was influenced by FCG, administrated alone or in combination with VX809. On the contrary, FCG was able to enhance F508del CFTR total expression, and its combination with VX809 provided a further effect, being able to increase not only the total expression but also the maturation of the mutant protein. Analyses on different CFTR domains and groups of domains, heterologously expressed in HEK293 cells, show that NBD2 is necessary for FCG corrector activity. Molecular modelling analyses suggest that FCG interacts with a putative region located into the NBD2, ascribing this molecule to class II correctors. Our study indicates that the continuous development and testing of combinations of correctors targeting different structural and functional defects of mutant CFTR is the best strategy to ensure a valuable therapeutic perspective to a larger cohort of CF patients.

A novel L1CAM isoform with angiogenic activity generated by NOVA2-mediated alternative splicing.

The biological players involved in angiogenesis are only partially defined. Here, we report that endothelial cells (ECs) express a novel isoform of the cell-surface adhesion molecule L1CAM, termed L1-ΔTM. The splicing factor NOVA2, which binds directly to L1CAM pre-mRNA, is necessary and sufficient for the skipping of L1CAM transmembrane domain in ECs, leading to the release of soluble L1-ΔTM. The latter exerts high angiogenic function through both autocrine and paracrine activities. Mechanistically, L1-ΔTM-induced angiogenesis requires fibroblast growth factor receptor-1 signaling, implying a crosstalk between the two molecules. NOVA2 and L1-ΔTM are overexpressed in the vasculature of ovarian cancer, where L1-ΔTM levels correlate with tumor vascularization, supporting the involvement of NOVA2-mediated L1-ΔTM production in tumor angiogenesis. Finally, high NOVA2 expression is associated with poor outcome in ovarian cancer patients. Our results point to L1-ΔTM as a novel, EC-derived angiogenic factor which may represent a target for innovative antiangiogenic therapies.