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Objective Nonsense mutations are responsible for 15% of Cystic Fibrosis (CF) patients due to the introduction of a premature stop codon (PTC) in the mRNA and the production of a truncated CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) protein1. A promising therapeutic approach for stop mutations is the suppression therapy by Translational Readthrough Inducing Drugs (TRIDs) to restore the expression of the protein2,3. Recently three new TRIDS (NV848, NV914, NV930) have been proposed and validated by several assays. Our work was focused on TRIDs NV848, NV914, NV930. Important aspects of TRIDs to be evaluated are their specificity towards PTC, to demonstrate that TRIDs do not interfere with the normal translation process4 in vitro, and their in vivo toxicity in mice models, to assess their tolerability at high doses. Methods For the toxicity study, mice were treated with an acute dose as reported in standard protocols5, monitored and then sacrificed to collect organs. To study the effects of the TRIDs on natural termination codons (NTC) we have examined the molecular weight and functional activity of a panel of proteins. Results Acute toxicity studies evidenced that all three molecules used have been well tolerated in all subjects treated. Histological analyses have not shown any tissue damage. So, we can assess TRIDs are safe in vivo also at the high doses tested. About NTC preservation, experiments have not shown any impairment or dysfunction in all samples. Indeed, no aberrant protein form has been detected. Conclusions In conclusion, our experiments identified the acute dosage for each TRIDs and their tolerability also at elevated doses, encouraging to test their efficacy in in vivo CF models. Accordingly, the in vitro analyses have suggested that these TRIDs do not lead to jeopardise NTC integrity, assessing their promising use for PTC readthrough. Consequently, our results could be indicative of TRIDs safety in in vitro models, laying the basis for further investigations. Bibliography 1. Giordani B, Amato A, Majo F, Ferrari G, Quattrucci S, Minicucci L, Padoan R, Floridia G, Salvatore D, Carnovale V, Puppo Fornaro G, Taruscio D, Salvatore M; Gruppo di lavoro RIFC. Italian Cystic Fibrosis Registry (ICFR). Report 2015-2016]. Epidemiol Prev. 2019 Jul-Aug;43(4S1):1-36. 2. Pibiri I., A., Tutone, M., Lentini, L., Melfi, R., & DI LEONARDO, A. et al. Oxadiazole Derivatives For The Treatment Of Genetic Diseases Due To Nonsense Mutations, PCT Int. Appl. (2019), WO 2019/101709 A1 20190531. 3. Pibiri I, Melfi R, Tutone M, Di Leonardo A, Pace A, Lentini L. Targeting Nonsense: Optimization of 1,2,4-Oxadiazole TRIDs to Rescue CFTR Expression and Functionality in Cystic Fibrosis Cell Model Systems. Int J Mol Sci. 2020 Sep 3;21(17):6420. doi: 10.3390/ijms21176420. PMID: 32899265; PMCID: PMC7504161 4. Grover R, Candeias MM, Fåhraeus R, Das S. p53 and little brother p53/47: linking IRES activities with protein functions. Oncogene. 2009 Jul 30;28(30):2766-72. doi: 10.1038/onc.2009.138. Epub 2009 Jun 1. PMID: 19483723. 5. OECD (1992). OECD Guidelines for the Testing of Chemicals No. 420: Acute Oral Toxicity Fixed Dose Method, 7pp. Paris, France: OECD.

The only Italian population of false smooth snakes is found on Lampedusa, a small island located in the Sicilian Channel and part of the African continental shelf. The taxonomic identity of this population is currently uncertain, although it is most often attributed to Macroprotodon cucullatus textilis on a morphological basis. We present here the first genetic data on this population. The analysis carried out on the mitochondrial cytochrome b gene shows that the Lampedusan false smooth snake belongs to a clade shared with a single sample from central Tunisia. The genetic distance between this lineage and its sister group (M. abubakeri) is comparable to or higher than that found among many reptile species. To define the identity of this distinctive lineage, as well as the Macroprotodon taxonomic structure, further sampling efforts within the undersampled distribution area of this genus and more extensive analyses will be necessary.

Drosophila GAGA factor (GAF) is a nuclear protein, conserved along evolution, with multiple roles in gene regulation, chromatin remodelling, Polycomb-dependent silencing, and insulator functions (1). GAF recognizes and specifically binds GAGAG consensus DNA motif by its C2H2-type zinc-finger domain, and interacts with other regulatory factors by its BTB/POZ domain. We have identified plGaga, a cDNA coding for a putative GAF of the sea urchin P. lividus, which showed significant sequence similarity with Drosophila and vertebrate GAFs. Real time RT-PCR revealed that the plGaga RNA is always present during embryo development decreasing rapidly in abundance at the larval pluteus stage. We raised a specific antibody against the sea urchin GAF and used it in whole-mount immunofluorescence assays, showing that the protein was distributed predominantly to the nuclei of the whole embryo. By Chromatin Immunoprecipitation experiments, we showed that the sea urchin GAF not only binds the sns5 insulator, which is essential for the regulation of the sea urchin early-histone genes (2,3), but also to other GAGA containing sequences in the early-histone gene cluster. 1 Matharu NK et al. 2010 JMB 400 (3): 434–447 2 Melfi R et Al. 2001 JMB (1); 304:753-63 3 Cavalieri V et al. 2013 Nucleic Acids Res 37: 7407-7415

Cystic Fibrosis patients with nonsense-mutation in h-CFTR gene generally make virtually no CFTR protein and thus often have a more severe form of CF. Ataluren (PTC124) was suggested to induce read-through of premature but not normal termination codons. Despite the promising results there is not a general consensus on the mechanism of its action (protein stabilization or codon read-through) and its efficacy, the identification of new PTC124 analogues and the study of the mechanism of action may led to a new strategy for the development of a pharmacologic approach to the cure of CF.

Obiettivi specifici: Le mutazioni nonsenso (mutazioni STOP), un difetto genetico frequente negli individui affetti da Fibrosi Cistica (CF), causano la sintesi di proteine CFTR tronche e non funzionanti che sono associate ad un fenotipo più severo della CF (McKone EF. et al., Chest 2006). L’obiettivo del nostro studio è stato quello di disegnare derivati dell’Ataluren (PTC124), una ‘small molecule’ a cui è stata attribuita attività readthrough, e valutarne l’attività su tre differenti sistemi modello sperimentali contenenti codoni di STOP prematuri (UGA, UAG, UAA). Materiali e metodi: Sono state sintetizzate 24 molecole derivate dal PTC124 e analizzate mediante tecniche spettroscopiche per valutare la loro struttura molecolare e il loro grado di purezza. Per valutare la capacità readthrough, è stato usato (1) un vettore reporter (FLuc190) in cui è presente la mutazione UGA nella porzione codificante FLuc e (2) il plasmide pBOS-H2BGFP in cui, mediante mutagenesi sito specifica, sono stati alternativamente introdotti nella porzione codificante la proteina GFP i tre diversi tipi di codoni di stop (UGA, UAG, UAA). Il DNA plasmidico è stato purificato e isolato dai batteri mediante ‘Colony PCR’, sottoposto a PCR-selettiva e sequenziato. Cellule HeLa sono state quindi trasfettate con i vettori contenenti la mutazione STOP, inoltre sono state anche utilizzate cellule IB3.1 (FC) per valutare la ri-espressione della proteina CFTR. Entrambe i tipi cellulari sono stati trattati con i differenti derivati del PTC124 la cui attività è stata verificata mediante osservazione diretta al microscopio a fluorescenza, saggi enzimatici e immunofluorescenza indiretta. Risultati: Lo screening funzionale che fa uso del vettore in cui è clonato il gene della Luciferasi contenente la mutazione di stop UGA (opal), sui 24 derivati del PTC124 ha permesso l’identificazione di tre molecole (derivati: #1, #3 e #5) che mostrano una attività readthrough superiore al PTC124. Tale risposta risulta evidente anche nel ‘recoding’ dell’espressione della proteina H2BGFP in cellule HeLa esprimenti il vettore reporter pBOS-H2BGFP-opal, la cui presenza è stata messa in evidenza in vivo/in vitro e mediante analisi di immunofluorescenza indiretta. Per quanto riguarda gli altri due codoni di stop, UAG, UAA (amber e ochre), i nostri risultati dimostrano che una delle molecole (#3) che aveva dato un buon risultato sulla mutazione UGA (opal) sembra avere anche un buon effetto nel recupero della proteina H2BGFP nelle cellule HeLa H2BGFP-amber (UAG). Al contrario nelle cellule HeLa H2BGFP-ochre (UAA), nessuna delle molecole testate ha dato un risultato positivo. Esperimenti condotti sulle cellule IB3.1 (F508/W1282X) hanno messo in evidenza a 24 ore dal trattamento con il derivato #5 l’incremento della quantità della proteina CFTR attribuibile probabilmente a readthrough della mutazione nonsense. Questo risultato è ancora più evidente dopo 10 giorni di trattamento continuato in cui il derivato #5 è stato aggiunto ogni 24 ore. Conclusioni: La ricerca ha identificato tre molecole, tra le 24 da noi disegnate e sintetizzate, in grado di indurre il readthrough delle mutazioni di stop premature, in particolare della mutazione UGA (opal). Inoltre, al contrario degli amminoglicosidi, i trattamenti con i derivati del PTC124 non presentano tossicità a livello cellulare, indice del fatto che tali molecole potrebbero trovare una migliore applicazione in sostituzione alla terapia con gli antibiotici amminoglicosidi.

Circa il 10% dei pazienti affetti da fibrosi cistica (FC) presenta nel gene CFTR mutazioni non senso (o 'stop': UGA, UAG or UAA, mutazioni di classe I) che bloccano prematuramente la sintesi della proteina. Attualmente non esiste una cura per questo tipo di mutazioni ma si sta cercando di individuare delle molecole che siano in grado di indurre la traduzione di codoni di stop prematuri (readthrough) che, rispetto a molecole già note come il G418, abbiano effetti collaterali ridotti ed una maggiore specificità per uno specifico codone. Una piccola molecola che sembra possedere una tale attività è il PTC124 (Welch, 2007). Ad oggi però non c’è ancora un consenso generale sul meccanismo di azione di questa molecola (stabilizzazione proteica, superamento del codone stop o altri meccanismi) (Welch, 2007; Auld, 2009). Al fine di chiarirlo abbiamo introdotto nel gene codificante la proteina GFP del plasmide pBOS-H2BGFP un codone di stop TGA mediante mutagenesi sito diretta. Cellule HeLa trasfettate con questo vettore e cellule IB3.1 (FC) sono state utilizzate per valutare la capacità di indurre readthrough del PTC124.

Nucleotide changes within an exon may alter the trinucleotide normally encoding a particular amino acid, such that a new “stop” signal is transcribed into the mRNA open reading frame. This causes the ribosome to prematurely terminate its reading of the mRNA, leading to the lack of production of a normal full-length protein. Such premature termination codon (PTC) mutations occur in an estimated 10% to 15% of many genetically based disorders (1). Pathological nonsense mutations resulting in TAG (40.4%), TGA (38.5%), and TAA (21.1%) occur in different proportions to naturally occurring stop codons (2). Several genetic disorders are characterized by opal (TGA; Cystic fibrosis, Duchenne/Becker muscular dystrophy), amber (TAG; -thalassemia, emphysema, cystic fibrosis) and ochre mutations (TAA; APC gastric cancer, Haemophilia B, Hypothyroidism) (3). Messenger RNA containing a nonsense mutation is often degraded rapidly through the process of nonsense-mutation-mediated decay (NMD) resulting in the lack of the protein (4). A recent approach to directly overcome the deleterious effects caused by nonsense mutations is represented by readthrough strategies which take advantage of the known properties of aminoglycosides that can suppress stop codons (5). Several aminoglycosides (gentamicin, amikacin, hygromycin, etc.) can suppress the accurate identification of translation termination codons in cultured eukaryotic cells. Unfortunately, aminoglycoside action lacks specificity resulting in readthrough of many correctly positioned stop codons. Consequently, long- term use of aminoglycosides may originate toxic aggregates or dominant negative readthrough products (6). By an high throughput screening it was identified the PTC124 (Ataluren), a small molecule that has been suggested to allow PTCs readthrough (7). However, despite the results obtained on opal mutation it was shown that it has a lower activity against ochre and amber nonsense mutations (7). In the attempt to identify molecules with an activity against ochre and amber nonsense mutations, we designed and synthesized new PTC124 derivatives to be tested in human cultured cells to see if they have higher and wider activity towards PTCs than PTC124. To this aim we generated a reporter vector with non-sense mutation by introducing in the pBOS-H2BGFP plasmid a TAG codon (amber) and TAA codon (ochre) by site-directed mutagenesis. PCR and sequencing analyses confirmed the presence of the stop codons in the plasmids that were transfected in HeLa cells to explore the ability of the derivatives to promote the translational read-through. Immunofluorescence analyses showed that one of the analyzed derivatives was able to restore GFP fluorescence in HeLa H2BGFP-amber cells, as indicated by GFP-localization in the nuclei of treated cells (figure 1). This positive response also confirmed the correct functioning of the model system which will allow us to perform the screening of a greater number of molecules with read-through action.

Background Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). Approximately 10% (worldwide) of patients have in-frame nonsense mutations (UAA, UAG or UGA class I mutations) in the CF trans-membrane regulator (CFTR) gene that result in premature stop codons (PTCs) in the messenger RNA (mRNA) generating truncated CFTR protein responsible for a severe CF phenotype. Pharmacological approaches have been proposed to directly overcome PTCs. Ataluren (PTC124) a small molecule that mimics the activity of aminoglycosides has been suggested to allow PTCs readthrough and to partially restore the protein function. However, despite the results obtained "in vitro" and "in vivo" as well the clinical trials done with PTC124, some caveats exist. In particular Ataluren showed a lower activity against UAA and UAG than UGA nonsense mutations and also there is no general consensus about its mechanism of action. Hypothesis and Objectives Our proposal is aimed to design and synthesize new small molecules, structurally related to PTC124 and to establish a new cell based approach to evaluate their ability to promote translational read-through of PTCs present in a reporter plasmid generated by us and transfected in human cells. Preliminary results Twelve derivatives were designed and synthesized and tested in HeLa cells transfected with a plasmid harbouring an in-frame PTC (TGA). To this aim we generate a reporter vector with non-sense mutation by introducing in the pBOS-H2BGFP plasmid a TGA codon (opal) by site-directed mutagenesis. PCR and sequencing analyses confirmed the presence of the TGA codon in the plasmid that was transfected in HeLa cells to explore the ability of the derivatives to promote the translational read-through. Treatments with PTC124 of HeLa transfected cells confirmed the correct functioning of the model system. Moreover, HeLa cells transfected with a reporter vector encoding the mutated FLuc (opal mutation) when treated with the derivatives showed a positive response. Spin-off for research and clinical purpose Since there is not a general consensus on the mechanism of action of the PTC124 (protein stabilization or PTCs read-through) development of molecules capable of promoting the read-through of all PTCs without adverse effects, as Ataluren is suggested to do for the TGA codon, would be helpful as therapeutic strategy for other nonsense mutations found in CF.

Background Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). Approximately 10% (worldwide) of patients have in-frame nonsense mutations (UAA, UAG or UGA class I mutations) in the CF trans-membrane regulator (CFTR) gene that result in premature stop codons (PTCs) in the messenger RNA (mRNA) generating truncated CFTR protein responsible for a severe CF phenotype. Pharmacological approaches have been proposed to directly overcome PTCs. Ataluren (PTC124) a small molecule that mimics the activity of aminoglycosides has been suggested to allow PTCs readthrough and to partially restore the protein function. However, despite the results obtained "in vitro" and "in vivo" as well the clinical trials done with PTC124, some caveats exist. In particular Ataluren showed a lower activity against UAA and UAG than UGA nonsense mutations and also there is no general consensus about its mechanism of action. Hypothesis and Objectives Our proposal is aimed to design and synthesize new small molecules, structurally related to PTC124 and to establish a new cell based approach to evaluate their ability to promote translational read-through of PTCs present in a reporter plasmid generated by us and transfected in human cells. Preliminary results Twelve derivatives were designed and synthesized and tested in HeLa cells transfected with a plasmid harbouring an in-frame PTC (TGA). To this aim we generate a reporter vector with non-sense mutation by introducing in the pBOS-H2BGFP plasmid a TGA codon (opal) by site-directed mutagenesis. PCR and sequencing analyses confirmed the presence of the TGA codon in the plasmid that was transfected in HeLa cells to explore the ability of the derivatives to promote the translational read-through. Treatments with PTC124 of HeLa transfected cells confirmed the correct functioning of the model system. Moreover, HeLa cells transfected with a reporter vector encoding the mutated FLuc (opal mutation) when treated with the derivatives showed a positive response. Spin-off for research and clinical purpose Since there is not a general consensus on the mechanism of action of the PTC124 (protein stabilization or PTCs read-through) development of molecules capable of promoting the read-through of all PTCs without adverse effects, as Ataluren is suggested to do for the TGA codon, would be helpful as therapeutic strategy for other nonsense mutations found in CF.

Enhancers are DNA elements which increase the transcription of associated gene in a position and distance independent manner relative to the transcription initiation site. Molecular mechanisms must operate to direct enhancers to specific promoters in complex genetic loci. The sea urchin a-histone genes are organized in several hundred tandem repeated units, each containing one copy of the five histone genes in the order 5’-H4-H2B-H3-H2A-H1-3’. Transcription is limited to the early cleavage and reaches its maximum at morula stage. After hatching these genes are repressed and maintained in the silenced state for whole life cycle of the animal. In Paracentrotus lividus, the MBF-1 activator binds to a 30 bp sequence of the H2A modulator, the only cis-acting element of the histone unit for which an enhancer activity has unambiguously demonstrated. Here we assessed the specificity of the MBF-1 factor and the molecular mechanisms that eventually limit its interaction with the adjacent promoters. We find by Chromatin ImmunePrecipitation and in vivo functional studies that the MBF-1 activator binds both the H3 and H2A regulatory sequences, suggesting that this activator is needed for the maximal expression of both genes. In addition, we find that the enhancer blocking activity of the sns5 insulator buffers the downstream H1 promoter from the H2A enhancer bound MBF-1 factor. These results suggest that the block of the H2A enhancer by the sns5 insulator might be the mechanism that regulate the ratio of 2 to 1 of core to linker histones during early development of the sea urchin embryo.

Core promoters and chromatin insulators (ins) may direct a transcriptional enhancer (enh) to prefer a specific promoter in complex genetic loci. Enh and ins flank the sea urchin Paracentrotus lividus α-histone H2A transcription unit in a tandem repeated cluster containing the five histone genes. In vivo competition assays of enh and ins functions reveal that the H2A enh-bound MBF-1 activator participates also in the expression of the H3 gene and that the sns5 ins buffers the downstream H1 promoter from the H2A enh. These results suggest that both the H2A enh and the sns5 ins may account for the diverse accumulation of the linker vs core nucleosomal histones during early development of the sea urchin embryo. Finally, preliminary results indicate that non-coding RNAs originate from the insulator region.