Modified U1snRNAs as innovative therapeutic strategy for inherited coagulation factor deficiencies

The theoretical framework in which this study is framed concerns the internationalization of production dynamics of firms abroad. The international literature in this field argues that there is not a conventional definition nor a universally tested theory that can explain all forms of foreign-owned production (see, among others, Dunning, 1988b and 1993a, Ietto-Gillies, 2005 and 2007). In this complex context, a large amount of Italian literature has been published around the theoretical framework of internationalization of production of Italian companies abroad (Tattara, Corò and Volpe, 2006, Mariotti and Mutinelli, 2005, et.al.). Nevertheless, a limited number of quantitative studies are available about the topic of Italian firms “migration” in Romania - as a particular expression of productive internationalization of our companies in this Country (Majocchi, 2002, Unimpresa Romania, 2005 and Antenna Veneto Romania, 2005). Thus, in order to investigate it by an industrial policy perspective a multilevel modelling approach has been applied. In particular, a two level model has been used to determine the effects of Romanian regions (romanian judets) in which Italian manufacturing firms internationalized the production in 2009 on their industrial performance. Based on a business register-based survey a business register of the Italian business community in Romania has been created. The data on Italian manufacturing firms located in Romania in 2009 have been selected from this register. The dataset used for the analysis includes 796 cases of firms internationalized in Romania. Therefore, a two level model has been carried out in order to determine the effects of intra-industry characteristics of these firms (i.e. Firm size and Firm Industrial specialization) on their industrial performance in 2009 both at individual and area level. The model results have shown that: as the firms dimension increase they have to be extremely sensible in the choice of the romanian region in which they want to (de)localize the production. Furthermore, the choice of the romanian region of productive delocalization has to be chosen by firms according to their sectors of activity

Hypogeal fauna of the military subterranean fortification Forte di Vernante Opera 11 "Tetto Ruinas" (Piedmont, Italy)

The military subterranean fortification Forte di Vernante Opera 11 "Tetto Ruinas" is not mentioned in the extensive specific literature about the underground fortifications of the NW Alps. Only the project in the Military Engineers Corps archives is known, however, the schematic survey recently detected by our research association Biologia Sotterranea Piemonte – Gruppo di Ricerca highlights differences from the original map. On the contrary, biological research and scientific literature about the hypogeal fauna carried out in this artificial cavity are numerous. Two new species of spiders with remarkable adaptations to the underground environment were discovered by Angelo Morisi (1943-2016): the Linyphiidae Troglohyphantes konradi Brignoli, 1975 and the Nesticidae Typhlonesticus morisii (Brignoli, 1975). Subsequently, other spiders adapted to this particular environment were observed too, such as Kryptonesticus eremita (Simon, 1880) and Leptoneta crypticola Simon, 1907. Other spiders with less evident adaptations were observed in this artificial cavity such as Tegenaria silvestris L. Koch, 1872, Pimoa rupicola (Simon, 1884), Meta menardi (Latreille, 1804), Metellina merianae (Scopoli, 1763). Terrestrial Crustacean Isopods adapted to underground environment are present in this fort: Buddelundiella zimmeri Verhoeff, 1930 and Trichoniscus voltai Arcangeli, 1948. Among the Miriapods were observed the Chilopoda Eupolybothrus longicornis (Risso, 1826) and Lithobius microps Meinert, 1868, and the Diplopoda Plectogona vignai draco (Strasser, 1975), very specialized subspecies organism, and the larger and less adapted Callipus foetidissimus (Savi, 1819). The troglophile grasshopper Dolichopoda azami Saulcy, 1893 is very common in the cavity. Of considerable interest are the anophthalmous Trechini Carabidae Duvalius carantii (Sella, 1874), and the very rare insect Staphylinidae Blepharhymenus mirandus Fauvel, 1899. Sometimes at the entrances it is possible to observe the cave salamander Speleomantes strinatii (Aellen, 1958). The association Biologia Sotterranea Piemonte – Gruppo di Ricerca deals mainly the study of subterranean species and environments and their conservations. Our last twenty years research in this subterranean military cavity have allowed to observe and monitor the presence of all the aforementioned organisms and to document the presence of numerous other ones. Our studies in this and other Piedmontese artificial cavities highlights the presence of numerous species extremely adapted to the hypogeal environment, reiterating the importance of the subterranean fauna monitoring in the artificial cavities too, and of the presence of these organisms due to some biotic and abiotic environmental factors, regardless of the natural or artificial origin of the cavity

DNA base editing corrects common Hemophilia A mutations and restores factor VIII expression in vitro and ex-vivo models

Background: Replacement and non-replacement therapies effectively control bleedings in Hemophilia A (HA) but imply lifelong interventions. The authorized gene addition therapy could provide a cure but still poses questions on durability. F8 gene correction would definitively restore factor VIII (FVIII) production, as shown in animal models through nucleases mediating homologous recombination (HR). However, low efficiency and potential off-target double-strand break (DSB) still limit HR translatability. Objectives: To correct common model single point mutations leading to severe HA through the recently developed DSB/HR-independent base (BE) and prime (PE) editing approaches. Methods: Screening for efficacy of BE/PE systems in HEK293T transiently expressing FVIII variants and validation at DNA (sequencing) and protein (ELISA; aPTT) level in stable clones. Evaluation of rescue in engineered blood outgrowth endothelial cells (BOEC) by lentiviral-mediated delivery of BE. Results and conclusions: Transient assays identified the best-performing BE/PE systems for each variant, with the highest rescue of FVIII expression (up to 25% of rFVIIIwt) for the p.R2166* and p.R2228Q mutations. In stable clones we demonstrated that the mutation reversion on DNA (∼24%) was consistent with the rescue of FVIII secretion and activity 20-30%). The lentiviral-mediated delivery of the selected BE systems was attempted in engineered BOEC harboring the p.R2166* and p.R2228Q variants, which led to an appreciable and dose-dependent rescue of secreted functional FVIII. Overall data provide the first proof-of-concept for effective BE/PE-mediated correction of HA-causing mutations, which encourage studies in mouse models to develop a personalized cure for large cohorts of patients though a single intervention

An exon-specific U1 small nuclear RNA (snRNA) strategy to correct splicing defects

A significant proportion of disease-causing mutations affect precursor-mRNA splicing, inducing skipping of the exon from the mature transcript. Using F9 exon 5, CFTR exon 12 and SMN2 exon 7 models, we characterized natural mutations associated to exon skipping in Haemophilia B, cystic fibrosis and spinal muscular atrophy (SMA), respectively, and the therapeutic splicing rescue by using U1 small nuclear RNA (snRNA). In minigene expression systems, loading of U1 snRNA by complementarity to the normal or mutated donor splice sites (5′ss) corrected the exon skipping caused by mutations at the polypyrimidine tract of the acceptor splice site, at the consensus 5′ss or at exonic regulatory elements. To improve specificity and reduce potential off-target effects, we developed U1 snRNA variants targeting non-conserved intronic sequences downstream of the 5′ss. For each gene system, we identified an exon-specific U1 snRNA (ExSpeU1) able to rescue splicing impaired by the different types of mutations. Through splicing-competent cDNA constructs, we demonstrated that the ExSpeU1-mediated splicing correction of several F9 mutations results in complete restoration of secreted functional factor IX levels. Furthermore, two ExSpeU1s for SMA improved SMN exon 7 splicing in the chromosomal context of normal cells. We propose ExSpeU1s as a novel therapeutic strategy to correct, in several human disorders, different types of splicing mutations associated with defective exon definition

C6orf10 low-frequency and rare variants in italian multiple sclerosis patients

In light of the complex nature of multiple sclerosis (MS) and the recently estimated contribution of low-frequency variants into disease, decoding its genetic risk components requires novel variant prioritization strategies. We selected, by reviewing MS Genome Wide Association Studies (GWAS), 107 candidate loci marked by intragenic single nucleotide polymorphisms (SNPs) with a remarkable association (p-value <= 5 x 10(-6)). A whole exome sequencing (WES)-based pilot study of SNPs with minor allele frequency (MAF) <= 0.04, conducted in three Italian families, revealed 15 exonic low-frequency SNPs with affected parent-child transmission. These variants were detected in 65/120 Italian unrelated MS patients, also in combination (22 patients). Compared with databases (controls gnomAD, dbSNP150, ExAC, Tuscany-1000 Genome), the allelic frequencies of C6orf10 rs 16870005 and IL2RA rs12722600 were significantly higher (i.e., controls gnomAD, p = 9.89 x 10(-7) and p < 1 x 10(-20)). TET2 rs61744960 and TRAF3 rs138943371 frequencies were also significantly higher, except in Tuscany-1000 Genome. Interestingly, the association of C6orf10 rs16870005 (Ala431Thr) with MS did not depend on its linkage disequilibrium with the HLA-DRB1 locus. Sequencing in the MS cohort of the C6orf10 3' region revealed 14 rare mutations (10 not previously reported). Four variants were null, and significantly more frequent than in the databases. Further, the C6orf10 rare variants were observed in combinations, both intra-locus and with other low-frequency SNPs. The C6orf10 Ser389Xfr was found homozygous in a patient with early onset of the MS. Taking into account the potentially functional impact of the identified exonic variants, their expression in combination at the protein level could provide functional insights in the heterogeneous pathogenetic mechanisms contributing to MS.In light of the complex nature of multiple sclerosis (MS) and the recently estimated contribution of low-frequency variants into disease, decoding its genetic risk components requires novel variant prioritization strategies. We selected, by reviewing MS Genome Wide Association Studies (GWAS), 107 candidate loci marked by intragenic single nucleotide polymorphisms (SNPs) with a remarkable association (p-value ≤ 5 × 10−6). A whole exome sequencing (WES)-based pilot study of SNPs with minor allele frequency (MAF) ≤ 0.04, conducted in three Italian families, revealed 15 exonic low-frequency SNPs with affected parent-child transmission. These variants were detected in 65/120 Italian unrelated MS patients, also in combination (22 patients). Compared with databases (controls gnomAD, dbSNP150, ExAC, Tuscany-1000 Genome), the allelic frequencies of C6orf10 rs16870005 and IL2RA rs12722600 were significantly higher (i.e., controls gnomAD, p = 9.89 × 10−7 and p < 1 × 10−20). TET2 rs61744960 and TRAF3 rs138943371 frequencies were also significantly higher, except in Tuscany-1000 Genome. Interestingly, the association of C6orf10 rs16870005 (Ala431Thr) with MS did not depend on its linkage disequilibrium with the HLA-DRB1 locus. Sequencing in the MS cohort of the C6orf10 3′ region revealed 14 rare mutations (10 not previously reported). Four variants were null, and significantly more frequent than in the databases. Further, the C6orf10 rare variants were observed in combinations, both intra-locus and with other low-frequency SNPs. The C6orf10 Ser389Xfr was found homozygous in a patient with early onset of the MS. Taking into account the potentially functional impact of the identified exonic variants, their expression in combination at the protein level could provide functional insights in the heterogeneous pathogenetic mechanisms contributing to MS

Elucidation of aberrant SBDS splicing mechanisms to design RNA-therapeutics for Shwachman-Diamond Syndrome

Il progetto si pone l'obiettivo di investigare i meccanismi di splicing alterato in pazienti di con la Sindorme di Shwachman-Diamond per il successivo sviluppo di nuovi approcci correttiv

RNA-Based therapeutics for OTC deficiency

Il progetto si propone di valutare la capacità correttiva di una nuova molecola ingegnerizzata nel modello murino della deficienza congenita di Ornitina Carbamilas

F8 splicing-swithcing molecules for tailored hemophilia A therapies

Il seguente progetto propone di sviluppare nuovi approcci correttivi per mutazioni di splicing e basato su approcci ad RNA. Tali approcci verranno valutati sia in modelli cellulari che animali di Emofilia A

La terapia genica nelle Malattie emorragiche e trombotiche

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Molecular Mechanisms and Determinants of Innovative Correction Approaches in Coagulation Factor Deficiencies

Molecular strategies tailored to promote/correct the expression and/or processing of defective coagulation factors would represent innovative therapeutic approaches beyond standard substitutive therapy. Here, we focus on the molecular mechanisms and determinants underlying innovative approaches acting at DNA, mRNA and protein levels in inherited coagulation factor deficiencies, and in particular on: (i) gene editing approaches, which have permitted intervention at the DNA level through the specific recognition, cleavage, repair/correction or activation of target sequences, even in mutated gene contexts; (ii) the rescue of altered pre-mRNA processing through the engineering of key spliceosome components able to promote correct exon recognition and, in turn, the synthesis and secretion of functional factors, as well as the effects on the splicing of missense changes affecting exonic splicing elements; this section includes antisense oligonucleotide- or siRNA-mediated approaches to down-regulate target genes; (iii) the rescue of protein synthesis/function through the induction of ribosome readthrough targeting nonsense variants or the correction of folding defects caused by amino acid substitutions. Overall, these approaches have shown the ability to rescue the expression and/or function of potentially therapeutic levels of coagulation factors in different disease models, thus supporting further studies in the future aimed at evaluating the clinical translatability of these new strategies