I difetti di splicing possono essere considerati causa diretta di diverse patologie e l’evento patologico più frequente di splicing alternativo e' exon skipping (salto dell'esone). Le mutazioni di splicing che inducono exon skipping possono influenzare diversi elementi in cis, comprese le sequenze di consenso del sito di splicing (3' sito di splicing – ss, e 5'ss), il tratto di polipirimidine a ridosso della regione al 3’ss o gli elementi regolatori esonici / intronici. U1snRNA modificati rappresentano uno strumento interessante per recuperare le mutazioni patologiche legate ad exon skipping per scopi terapeutici. Denominati Exon Specific U1 (ExSpeU1s), si basano su modifiche genetiche del loro sito di legame al 5’ss del pre-mRNA, legandosi per complementarità alle sequenze introniche a valle del sito donatore. La loro applicazione ha dimostrato promuovere il recupero e l’inclusione degli esoni skippati, ma ad oggi non sono noti i determinanti strutturali che sono coinvolti nella regolazione di questo fenomeno. In questo progetto di dottorato, utilizzando Factor IX e FANCA come modelli, ho esplorato gli elementi presenti negli esoni difettosi che influenzano negativamente la risposta agli ExSpeU1. Inoltre, ho valutato in dettaglio il potenziale terapeutico di ExSpeU1s nella Fibrosi Cistica (CF) investigando un pannello rappresentativo di mutazioni di splicing. Per studiare i determinanti coinvolti nel recupero di exon skipping mediato da ExSpeU1, mi sono concentrato sull'esone 5 del fattore IX (FIX) e sull'esone 8 FANCA (FAN) che differiscono nell’efficienza di recupero ad opera di ExSpeU1. Attraverso l'analisi di una serie di minigeni chimerici FIX-FANCA ed esperimenti di mutagenesi sito-diretta ho identificato un elemento esonico critico, denominato ExSpeU1 Inhibitory Element (ExSpeU1-IE) presente nel contesto FANCA che inibisce la risposta ExSpeU1. Esperimenti di co-trasfezione con fattori di splicing hanno suggerito che questo elemento ha un meccanismo di regolazione dello splicing dipendente dal contesto: a seconda dell'esone, questa strategia potrebbe I) o contrastare la funzione di potenziamento dei fattori di splicing o II) promuovere l'attività dei fattori di silenziamento. Mi sono inoltre concentrato sull’applicazione della strategia degli ExSpeU1 nella patologia Fibrosi Cistica (FC). Negli ultimi anni, la ricerca sulla FC ha avuto molto successo nello sviluppo di terapie personalizzate basate sulla correzione delle mutazioni di base sottostanti a livello proteico. Ciò ha portato alla scoperta di farmaci che agiscono su specifici mutanti della proteina CFTR sia come potenziatori che come correttori. Tuttavia, gli individui FC con mutazioni che alterano l'elaborazione del pre-mRNA di CFTR non possono beneficiare di queste terapie personalizzate. Per dimostrare la potenziale attività terapeutica di ExSpeU1 su CFTR e per stabilire una piattaforma utile da applicare a questa malattia, mi sono concentrato su dieci mutazioni di splicing relativamente frequenti che causano skipping dei corrispondenti esoni 5, 10, 13, 16 e 18. Mutazioni di splicing 711 + 3A> C / G e 711 + 5G> A si trovano nel 5'ss dell'esone 5; 1863C> T (p.Y577Y) e 1898 + 3A> G in un elemento regolatore esonico e nel 5'ss dell'esone 13, rispettivamente; 2789 + 5G> A e 3120G> A si trovano nel 5‘ss dell'esone 16 e 18 rispettivamente, mentre TG13T3, TG13T5, TG12T5 sono varianti del tratto polipirimidinico dell'esone 10. Usando il saggio di splicing in minigeni, dai miei esperimenti si evince che ExSpeU1s può correggere efficacemente i difetti di splicing in tutte queste mutazioni, ripristinando i livelli di proteina CFTR. Questi risultati rappresentano il primo passo verso lo sviluppo di un approccio genico personalizzato basato sulla strategia ExSpeU1 per il recupero delle mutazioni di splicing in Fibrosi Cistica, non solo in mutazioni appartenenti ad un singolo esone, ma anche su piu’ esoni dello stesso gene.

Splicing defects can be the direct cause or contribute to the severity of several diseases and exon skipping represents the most frequent pathological event. Splicing mutations that induce exon skipping can affect different cis-acting elements including the splice site consensus sequences (3’ss and 5’ss), the polypyrimidine tract or exonic/ intronic regulatory elements. Modified U1snRNA represents an interesting and promising tool to rescue exon skipping mutations for therapeutic purposes. Modified U1s, named Exon Specific U1s (ExSpeU1s), are based on the genetic engineering of their 5’tail that bind by complementarity to intronic sequences downstream the donor site. This loading on defective exons has been shown to promote their definition but the determinants that regulate this rescue are not known. In this study, using Factor IX and FANCA as models, I explored the elements present in defective exons that negatively affect their response to ExSpeU1s. In addition, I evaluated in detail the therapeutic potential of ExSpeU1s in Cystic Fibrosis (CF) studying a representative panel of splicing mutations. To study the determinants involved in ExSpeU1-mediated rescue of defective exons I focussed on Factor IX (FIX) exon 5 and FANCA (FAN) exon 8 that differ in their ExSpeU1 rescue efficiency. Through the analysis of a series of chimeric FIX-FANCA minigenes and site-directed mutagenesis experiments I have identified a critical cis- acting exonic elements, named ExSpeU1 Inhibitory Element (ExSpeU1-IE) present in the FANCA context that inhibits the ExSpeU1 response. In addition, co- transfection experiments with splicing factors suggested that this element has a context-dependent splicing regulatory mechanism: depending on the exon it might either counteract the function of enhancing splicing factors or promote the activity of silencing factors. To provide a useful rescuing approach in a specific disease, I focussed on Cystic Fibrosis (CF). In the last few years, CF research has been very successful in developing personalised therapies based on the correction of the underlying basic mutations at the protein level. This led to the discovery of drugs that act on specific CFTR protein mutants either as potentiators or correctors. However, CF individuals with mutations that disrupt CFTR RNA processing cannot benefit of these personalized therapies. To prove the potential therapeutic activity of ExSpeU1 on CFTR and to establish a useful platform that can be applied to this disease, I focussed on ten relatively frequent splicing mutations that cause skipping of corresponding exons 5, 10, 13, 16 and 18. Splicing mutations 711+3A>C/G and 711+5G>A are located in the 5’ss consensus of exon 5; 1863C>T (p.Y577Y) and 1898+3A>G in an exonic regulatory element and in the 5’ss consensus of exon 13, respectively; 2789+5G>A and 3120G>A are located at the 5’ss consensus of exon 16 and 18 respectively, whereas TG13T3, TG13T5, TG12T5 are variants at the polypyrimidine tract of exon 10. Using minigene splicing assay, I show that ExSpeU1s can efficiently correct the splicing defects in all these mutations, restoring the CFTR protein levels. This result represents the first step towards the development of a personalized approach based on the ExSpeU1 strategy for rescuing CFTR splicing mutations. With ten mutations and five exons analysed, it is the first time that an exon rescue strategy is shown to work efficiently in a single gene on several exon skipping mutations and different exons.

U1snRNA-mediated rescue of splicing defects: exon definition determinants and application in Cystic Fibrosis

DONEGA', STEFANO
2021

Abstract

Splicing defects can be the direct cause or contribute to the severity of several diseases and exon skipping represents the most frequent pathological event. Splicing mutations that induce exon skipping can affect different cis-acting elements including the splice site consensus sequences (3’ss and 5’ss), the polypyrimidine tract or exonic/ intronic regulatory elements. Modified U1snRNA represents an interesting and promising tool to rescue exon skipping mutations for therapeutic purposes. Modified U1s, named Exon Specific U1s (ExSpeU1s), are based on the genetic engineering of their 5’tail that bind by complementarity to intronic sequences downstream the donor site. This loading on defective exons has been shown to promote their definition but the determinants that regulate this rescue are not known. In this study, using Factor IX and FANCA as models, I explored the elements present in defective exons that negatively affect their response to ExSpeU1s. In addition, I evaluated in detail the therapeutic potential of ExSpeU1s in Cystic Fibrosis (CF) studying a representative panel of splicing mutations. To study the determinants involved in ExSpeU1-mediated rescue of defective exons I focussed on Factor IX (FIX) exon 5 and FANCA (FAN) exon 8 that differ in their ExSpeU1 rescue efficiency. Through the analysis of a series of chimeric FIX-FANCA minigenes and site-directed mutagenesis experiments I have identified a critical cis- acting exonic elements, named ExSpeU1 Inhibitory Element (ExSpeU1-IE) present in the FANCA context that inhibits the ExSpeU1 response. In addition, co- transfection experiments with splicing factors suggested that this element has a context-dependent splicing regulatory mechanism: depending on the exon it might either counteract the function of enhancing splicing factors or promote the activity of silencing factors. To provide a useful rescuing approach in a specific disease, I focussed on Cystic Fibrosis (CF). In the last few years, CF research has been very successful in developing personalised therapies based on the correction of the underlying basic mutations at the protein level. This led to the discovery of drugs that act on specific CFTR protein mutants either as potentiators or correctors. However, CF individuals with mutations that disrupt CFTR RNA processing cannot benefit of these personalized therapies. To prove the potential therapeutic activity of ExSpeU1 on CFTR and to establish a useful platform that can be applied to this disease, I focussed on ten relatively frequent splicing mutations that cause skipping of corresponding exons 5, 10, 13, 16 and 18. Splicing mutations 711+3A>C/G and 711+5G>A are located in the 5’ss consensus of exon 5; 1863C>T (p.Y577Y) and 1898+3A>G in an exonic regulatory element and in the 5’ss consensus of exon 13, respectively; 2789+5G>A and 3120G>A are located at the 5’ss consensus of exon 16 and 18 respectively, whereas TG13T3, TG13T5, TG12T5 are variants at the polypyrimidine tract of exon 10. Using minigene splicing assay, I show that ExSpeU1s can efficiently correct the splicing defects in all these mutations, restoring the CFTR protein levels. This result represents the first step towards the development of a personalized approach based on the ExSpeU1 strategy for rescuing CFTR splicing mutations. With ten mutations and five exons analysed, it is the first time that an exon rescue strategy is shown to work efficiently in a single gene on several exon skipping mutations and different exons.
PAGANI, Franco
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/2488140
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