Background: Limitations of replacement therapy for coagulation deficiencies encourage research toward alternative strategies. The spliceosomal U1snRNA, having key role in pre-mRNA processing, represents an attractive molecule because of its ability to rescue splicing impaired by mutations, a frequent cause of coagulation factor defects (~15%). Though an engineered U1snRNA we have previously rescued factor VII function impaired by the IVS7+5G/A mutation in cellular models. Here, we exploited this approach to correct factor IX (FIX) mutations either at the donor (IVS5-2A/T, IVS5-2A/C, IVS5-2A/G) or acceptor (IVS5-8G,IVS5-9G) splice sites, and causing severe hemophilia B. Materials and Methods: Transfection of BHK cells with splicing-competent FIX cDNA constructs and modified U1snRNA. Evaluation of FIX mRNA (RT-PCR) and protein (ELISA. aPTT, WB) levels. Results: All mutations induced exon 5 skipping from the FIX mRNA, thus resulting in secretion of unactive FIX molecules lacking the EGF2 domain. A single U1snRNA designed to bind by complementarity to the normal FIX IVS5 donor splice site (5’ss) was able to restore exon 5 inclusion and secretion of functional FIX in the presence of all mutations. To improve specificity for F9 gene we subsequently tested a panel of U1snRNAs (Exon Specific U1snRNA) targeting the non-conserved intronic sequences instead of the 5’ss. Intriguingly, we found a gradient of rescue efficacy, which decreased with the distance from the 5’ss. The best ExSpeU1, targeting intronic positions +9 through +18, rescued exon 5 inclusion (from undetectable to 70-80%) and resulted in increased secretion (~2-3 fold) of FIX molecules with procoagulant activity (from undetectable to ~100%). Conclusions: These results demonstrate for the first time that a unique ExSpeU1 is able to restore gene expression impaired by different splicing mutants. The extent of rescue of the procoagulant function, if achieved in patients, would be beyond the therapeutic threshold.

AN EXON-SPECIFIC U1 SMALL NUCLEAR RNA (snRNA) STRATEGY TO CORRECT SPLICING MUTATIONS ASSOCIATED TO HEMOPHILIA B

BALESTRA, Dario;CAVALLARI, Nicola;PAGANI, Franco;BERNARDI, Francesco;PINOTTI, Mirko
2012

Abstract

Background: Limitations of replacement therapy for coagulation deficiencies encourage research toward alternative strategies. The spliceosomal U1snRNA, having key role in pre-mRNA processing, represents an attractive molecule because of its ability to rescue splicing impaired by mutations, a frequent cause of coagulation factor defects (~15%). Though an engineered U1snRNA we have previously rescued factor VII function impaired by the IVS7+5G/A mutation in cellular models. Here, we exploited this approach to correct factor IX (FIX) mutations either at the donor (IVS5-2A/T, IVS5-2A/C, IVS5-2A/G) or acceptor (IVS5-8G,IVS5-9G) splice sites, and causing severe hemophilia B. Materials and Methods: Transfection of BHK cells with splicing-competent FIX cDNA constructs and modified U1snRNA. Evaluation of FIX mRNA (RT-PCR) and protein (ELISA. aPTT, WB) levels. Results: All mutations induced exon 5 skipping from the FIX mRNA, thus resulting in secretion of unactive FIX molecules lacking the EGF2 domain. A single U1snRNA designed to bind by complementarity to the normal FIX IVS5 donor splice site (5’ss) was able to restore exon 5 inclusion and secretion of functional FIX in the presence of all mutations. To improve specificity for F9 gene we subsequently tested a panel of U1snRNAs (Exon Specific U1snRNA) targeting the non-conserved intronic sequences instead of the 5’ss. Intriguingly, we found a gradient of rescue efficacy, which decreased with the distance from the 5’ss. The best ExSpeU1, targeting intronic positions +9 through +18, rescued exon 5 inclusion (from undetectable to 70-80%) and resulted in increased secretion (~2-3 fold) of FIX molecules with procoagulant activity (from undetectable to ~100%). Conclusions: These results demonstrate for the first time that a unique ExSpeU1 is able to restore gene expression impaired by different splicing mutants. The extent of rescue of the procoagulant function, if achieved in patients, would be beyond the therapeutic threshold.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/1728524
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