Molecular basis and therapeutic strategies to rescue pre-mRNA splicing defects in Haemophilia A, Familial Dysautonomia and Spinal Muscular Atrophy

Lo splicing dei pre-mRNA è un punto essenziale della regolazione dell’espressione genica il quale, controllato da complesse interazioni tra elementi in cis- ed in trans- che variano con le specie, tipi cellulari e durante lo sviluppo, può comportare la produzione di un grandissima varietà di isoforme proteiche a partire da un unico trascritto. Mutazioni o variazioni naturali all’interno delle sequenze codificanti e non-codificanti dei trascritti possono compromettere questo processo cellulare, con implicazione patologiche. Exon specific U1 snRNAs (ExSpeU1s) sono molecole di U1 snRNAs modificate per riconoscere in modo specifico le sequenze introniche che si trovano a valle di esoni negativamente influenzati da mutazioni. Gli ExSpeU1s sono stati recentemente proposti come molecole in grado di modificare lo splicing aberrante, correggendo i fenotipi patologici [1]. In questa tesi, per dimostrare l’applicabilità degli ExSpeU1s partendo da una caratterizzazione molecolare in-vitro alla valutazione della loro efficacia in-vivo, ho studiato lo splicing aberrante in: emofilia A, Disautonomia familiare ed atrofia muscolare spinale. L’emofilia A (HA) è una patologia della coagulazione causato da una deficienza del fattore della coagulazione VIII (FVIII). Tra le oltre 1300 mutazioni identificate in emofilia A, il cambio c.6046C>T (p.R2016W) rappresenta la seconda mutazione più frequente nel Nord Italia. Attraverso un sistema di minigeni ed una piattaforma lentivirale per l’espressione di FVIII ricombinante, ho dimostrato che la mutazione c.6046C>T ha un effetto sia a livello del processamento dell’RNA che su quello proteico del FVIII, rispettivamente inducendo un salto dell’esone 19 (≈30%) e riducendo sia la secrezione (11.0±0.4%) che l’attività (6.0±2.9%) del FVIII. Qui, ho caratterizzato un elemento regolatorio di splicing esonico (ESRE) all’interno dell’esone 19 del F8, identificando tre mutazioni missenso (c.6037G>A, p.G2013R; c.6053A>G, p.E2018G; c.6113A>G, p.N2038S) con un diverso impatto sulla secrezione/attività del FVIII e con un severo effetto sullo splicing dell’esone 19, inducendo un salto dell’esone superiore al 60%. Una combinazione di ridotta secrezione ed attività proteica con un’alterazione dello splicing ha prodotto un gradiente di deficit del FVIII, da una forma moderata ad una severa di emofilia A, anche per mutazioni raggruppate nell’esone 19. Interessatemene, ho dimostrato che lo splicing aberrante dell’esone 19 causato da queste mutazioni missenso può essere corretto da uno U1 snRNA modificato, stimolando l’inclusione dell’esone ed aumentando la quantità di trascritti completi. La Disautonomia familiare (FD) è una patologia caratterizzata da un difetto del sistema nervoso autonomo causato nella maggior parte dei casi (>99%) da una mutazione intronica (IVS20+6T>C) nel gene IKBKAP che induce un salto dell’esone 20 attraverso una riduzione dell’affinità tra lo U1 snRNA cellulare ed il sito donatore. Mediante l’uso di sistemi di minigeni, ho creato diversi ExSpeU1s che, riconoscendo la regione intronica a valle dell’esone 20, hanno efficientemente corretto lo splicing aberrante di IKBKAP in-vitro. Qui, ho inoltre dimostrato che questi ExSpeU1s promuovono una inclusione dell’esone 20 in modo attivo e non attraverso meccanismi antisenso. In aggiunta, sfruttando un sistema lentivirale per introdurre la molecola all’interno dei fibroblasti di pazienti FD, ho dimostrato l’efficacia di un ExSpeU1 in exvivo, fornendo una nuova opportunità terapeutica per il trattamento di questa patologia alla quale tuttora non esistono alternative. L’atrofia muscolare spinale (SMA) è una patologia che colpisce principalmente gli α-motoneuroni ed è causata da mutazioni nel gene di sopravvivenza dei motoneuroni 1 (SMN1) che codifica per la proteina SMN. Nello stesso cromosoma si trovano una o più copie del gene parologo (SMN2) che, tuttavia, contiene una mutazione sinonima (c.840C>T) che induce il salto dell’esone 7 di SMN2, riducendo l’espressione della proteina. In questa tesi, ho valutato l’efficacia in-vivo di due ExSpeU1s già caratterizzati per questa patologia [2]. Attraverso un virus adeno-associato 9, ho somministrato queste molecole per via intraperitoneale in modelli animali di SMA con fenotipo lieve e severo. Nel modello di topo SMA lieve, questi ExSpeU1s hanno efficientemente corretto lo splicing aberrante di SMN2, migliorando il fenotipo attraverso il recupero della lunghezza della coda. Nel modello di topo SMA severo, che normalmente muore dopo 10-12 giorni, ho dimostrato che il trattamento con ExSpeU1 estende significativamente la sopravvivenza con un 40% degli animali ancora vivi dopo 250 giorni. Complessivamente, questi risultati ottenuti in-vitro, ex-vivo ed in-vivo forniscono nuove conoscenze riguardanti le potenzialità terapeutiche basate su molecole di U1 snRNA modificate e forniscono le basi per ulteriori studi volti allo sviluppo di nuove terapie per patologie genetiche.Pre-mRNA splicing is an essential step of gene expression regulation, supervised by complex interactions between cis- and trans-acting factors that vary with species, celltypes and during development, which might dictate the synthesis of a multiplicity of different protein isoforms. Mutations or natural variations within both coding and noncoding sequences can affect this process with pathophysiological implications. Exon Specific U1 snRNAs (ExSpeU1s), modified U1 snRNAs that specifically bind to intronic sequences downstream affected exons, have been recently described as splicing-switching molecules able to revert pathological phenotypes [1]. In this thesis, in order to demonstrate the applicability of ExSpeU1s from the molecular characterization in-vitro to the evaluation of the efficacy in-vivo, I studied the misregulation of splicing in Hemophilia A, in Familial Dysautonomia, and in Spinal Muscular Atrophy. Hemophilia A (HA) is a bleeding disorder due to the deficiency of the blood coagulation factor VIII (FVIII). Among the over 1300 point mutations found in HA, the c.6046C>T (p.R2016W) substitution represents the second most frequent mutation in Northern Italy. Through a hybrid minigene system and a lentiviral platform to express recombinant FVIII variants, I demonstrated that the c.6046C>T mutation impacts both the RNA and protein FVIII biology , respectively promoting skipping of exon (≈30%) and reducing both the FVIII secretion (11.0±0.4%) and activity (6.0±2.9%). I characterized the presence of an exonic splicing regulatory element (ESRE) within the F8 exon 19, identifying three missense mutations (c.6037G>A, p.G2013R; c.6053A>G, p.E2018G; c.6113A>G, p.N2038S) with differential impact on FVIII secretion/function and a severe impact on F8 exon 19 splicing, inducing >60% of exon skipping. Combination of reduced protein secretion and activity with splicing alteration produced a gradient of FVIII deficiency, from mild to severe HA, even for mutations clustered in exon 19. Interestingly, I demonstrated that aberrant F8 exon 19 splicing caused by missense mutations can be improved by a modified U1 snRNA, promoting exon inclusion and increasing the amount of full length transcripts. Familial Dysautonomia (FD), characterized by impairment of the autonomous nervous system, is mainly caused (>99% of cases) by the intronic mutation IVS20+6T>C in the IKBKAP gene that promotes skipping of exon 20 through a reduced affinity between the wild type U1 snRNA and the donor site. With minigene assays I developed several ExSpeU1s that, targeting the intronic region downstream of the exon 20 donor site, efficiently correct the aberrant IKBKAP splicing in-vitro. Here, I also demonstrated that ExSpeU1s promote exon 20 inclusion in an active manner and not through antisense mechanisms. Moreover, taking advantage of a lentiviral delivery in FD patients’ fibroblasts, I demonstrated the ex-vivo efficacy of one ExSpeU1, providing a novel therapeutic opportunity to treat a disease for which there are no currently alternatives. Spinal Muscular Atrophy (SMA) primarily affects α-motor neurons and is caused by mutations in the Survival Motor Neuron 1 (SMN1) gene encoding the SMN protein. Interestingly on the same chromosome is located one or more copies of its paralog (SMN2) that however carries a synonymous mutation (c.840C>T) promoting skipping of SMN2 exon 7, which reduces the SMN expression. In this thesis, I challenged the in-vivo efficacy of two already characterized ExSpeU1s for this disease [2]. Through an Adeno-associated virus 9, I intraperitoneally delivered these molecules in SMA animal models with a mild and a severe phenotype. In the mild SMA mouse the ExSpeU1s efficiently corrected the aberrant SMN2 splicing and improved the phenotype that consist of a recover of the tail lenght. In the severe SMA mouse, that normally die at 10-12 days, I demonstrated that treatment with ExSpeU1 significantly extend the survival (40% of animals alive after 250 days). Altogether these in-vitro, ex-vivo and in-vivo data provide novel insights into the potential of the RNA therapeutics based on modified U1 snRNAs and lay the foundation for further studies aimed at developing novel therapies for genetic disorders

Clustered F8 missense mutations cause hemophilia A by combined alteration of splicing and protein biosynthesis and activity

Dissection of pleiotropic effects of missense mutations, rarely investigated in inherited diseases, is fundamental to understanding genotype-phenotype relationships. Missense mutati ons might impair mRNA processing in addition to protein properties. As a model for hemophilia A, we investigated the highly prevalent F8 c.6046c>t/p.R2016W (exon 19) mutation. In expression studies exploiting lentiviral vectors, we demonstrated that the amino acid change impairs both Factor VIII (FVIII) secretion (antigen 11.0±0.4% of wildtype) and activity (6.0±2.9%). Investigations in patientsâ\u80\u99 ectopic F8 mRNA and with minigenes showed that the corresponding nucleotide change also decreases correct splicing to 70±5%, which is predicted to lower further FVIII activity (4.2±2%), consistently with patientsâ\u80\u99 levels (a (p.G2013R) reduced exon inclusion to 41±3% and the c.6053a>g (p.E2018G) to 28±2%, similarly to a variant affecting the 5â\u80\u99 splice site (c.6113a>g, p.N2038S, 26±2%), which displayed normal protein features upon recombinant expression. The p.G2013R reduced both antigen (7.0±0.9%) and activity (8.4±0.8%), while the p.E2018G produced a dysfunctional molecule (antigen: 69.0±18.1%; activity: 19.4±2.3%). In conclusion, differentially altered mRNA and protein patterns produce a gradient of residual activity, and clarify genotype-phenotype relationships. Data detail pathogenic mechanisms that, only in combination, account for moderate/severe disease forms, which in turn determine the mutation profile. Taken together we provide a clear example of interplay between mRNA and protein mechanisms of disease that operate in shaping many other inherited disorders

Exon-specific U1 snRNAs improve ELP1 exon 20 definition and rescue ELP1 protein expression in a familial dysautonomia mouse model

Familial dysautonomia (FD) is a rare genetic disease with no treatment, caused by an intronic point mutation (c.2204+6T > C) that negatively affects the definition of exon 20 in the elongator complex protein 1 gene (ELP1 also known as IKBKAP). This substitution modifies the 50 splice site and, in combination with regulatory splicing factors, induces different levels of exon 20 skipping, in various tissues. Here, we evaluated the therapeutic potential of a novel class of U1 snRNA molecules, exonspecific U1s (ExSpeU1s), in correcting ELP1 exon 20 recognition. Lentivirus-mediated expression of ELP1-ExSpeU1 in FD fibroblasts improved ELP1 splicing and protein levels. We next focused on a transgenic mouse model that recapitulates the same tissue-specific mis-splicing seen in FD patients. Intraperitoneal delivery of ELP1-ExSpeU1s-adeno-associated virus particles successfully increased the production of full-length human ELP1 transcript and protein. This splice-switching class of molecules is the first to specifically correct the ELP1 exon 20 splicing defect. Our data provide proof of principle of ExSpeU1sadeno- associated virus particles as a novel therapeutic strategy for FD

Rescue of spinal muscular atrophy mouse models with AAV9-Exon-specific U1 snRNA

Spinal Muscular Atrophy results from loss-of-function mutations in SMN1 but correcting aberrant splicing of SMN2 offers hope of a cure. However, current splice therapy requires repeated infusions and is expensive. We previously rescued SMA mice by promoting the inclusion of a defective exon in SMN2 with germline expression of Exon-Specific U1 snRNAs (ExspeU1). Here we tested viral delivery of SMN2 ExspeU1s encoded by adeno-associated virus AAV9. Strikingly the virus increased SMN2 exon 7 inclusion and SMN protein levels and rescued the phenotype of mild and severe SMA mice. In the severe mouse, the treatment improved the neuromuscular function and increased the life span from 10 to 219 days. ExspeU1 expression persisted for 1 month and was effective at around one five-hundredth of the concentration of the endogenous U1snRNA. RNA-seq analysis revealed our potential drug rescues aberrant SMA expression and splicing profiles, which are mostly related to DNA damage, cell-cycle control and acute phase response. Vastly overexpressing ExspeU1 more than 100-fold above the therapeutic level in human cells did not significantly alter global gene expression or splicing. These results indicate that AAV-mediated delivery of a modified U1snRNP particle may be a novel therapeutic option against SMA