Background- The ability of the spliceosomal small nuclear RNA U1 (U1snRNA) to rescue pre-mRNA splicing impaired by mutations makes it an attractive therapeutic molecule. However, U1snRNAs have been exploited in cellular models only. Coagulation factor deficiencies, in which splicing mutations are relatively frequent, would strongly benefit from this strategy since even a modest increase of functional protein levels would ameliorate clinical phenotypes. Objectives- To assess the correction of the coagulation F7 c.840+5G>A mutation by the modified U1snRNA+5a (U1+5a) in vivo, previously demonstrated by us in cellular models. Methods- Creation of plasmids and adeno-associated viral [AAV] vectors expressing the human FVII (hFVII) splicing-competent cassette or the U1+5a. Injection in C57BL/6 mice and evaluation of hFVII expression through human-specific assays. Results- We created a novel mouse model of hFVII deficiency by liver-restricted expression, either transient (by hydrodynamic injection of plasmids) or prolonged (by AAV), of the mutated hFVII cassette (FVII+5A) that, at variance from FVIIwt, did not produce detectable hFVII levels. Hydrodynamic co-injection of pU1+5a produced appreciable circulating hFVII levels (up to 367ng/ml, ~17% of hFVIIwt). To assess long-term effects, mice were injected with 1.2x1012 vector genomes (vg)/mouse of AAV2-FVII+5A and of AAV8-U1+5a (1.2x1011 or 6x1011 vg/mouse), which resulted in dose-dependent rescue (3.9±0.8 ng/ml or 23.3±5.1 ng/ml, respectively) at two weeks post-injection. In both experimental systems, we also detected correctly spliced hFVII transcripts and hFVII-positive cells in liver. Conclusions- We provide the first in vivo proof-of-principle of the rescue of gene expression by U1snRNAs, thus highlighting their therapeutic potential in coagulation disorders.

An engineered U1 small nuclear RNA rescues splicing-defective coagulationF7gene expression in mice

BALESTRA, Dario;BERNARDI, Francesco;PINOTTI, Mirko
2014

Abstract

Background- The ability of the spliceosomal small nuclear RNA U1 (U1snRNA) to rescue pre-mRNA splicing impaired by mutations makes it an attractive therapeutic molecule. However, U1snRNAs have been exploited in cellular models only. Coagulation factor deficiencies, in which splicing mutations are relatively frequent, would strongly benefit from this strategy since even a modest increase of functional protein levels would ameliorate clinical phenotypes. Objectives- To assess the correction of the coagulation F7 c.840+5G>A mutation by the modified U1snRNA+5a (U1+5a) in vivo, previously demonstrated by us in cellular models. Methods- Creation of plasmids and adeno-associated viral [AAV] vectors expressing the human FVII (hFVII) splicing-competent cassette or the U1+5a. Injection in C57BL/6 mice and evaluation of hFVII expression through human-specific assays. Results- We created a novel mouse model of hFVII deficiency by liver-restricted expression, either transient (by hydrodynamic injection of plasmids) or prolonged (by AAV), of the mutated hFVII cassette (FVII+5A) that, at variance from FVIIwt, did not produce detectable hFVII levels. Hydrodynamic co-injection of pU1+5a produced appreciable circulating hFVII levels (up to 367ng/ml, ~17% of hFVIIwt). To assess long-term effects, mice were injected with 1.2x1012 vector genomes (vg)/mouse of AAV2-FVII+5A and of AAV8-U1+5a (1.2x1011 or 6x1011 vg/mouse), which resulted in dose-dependent rescue (3.9±0.8 ng/ml or 23.3±5.1 ng/ml, respectively) at two weeks post-injection. In both experimental systems, we also detected correctly spliced hFVII transcripts and hFVII-positive cells in liver. Conclusions- We provide the first in vivo proof-of-principle of the rescue of gene expression by U1snRNAs, thus highlighting their therapeutic potential in coagulation disorders.
2014
Balestra, Dario; A., Faella; P., Margaritis; N., Cavallari; F., Pagani; Bernardi, Francesco; V. R., Arruda; Pinotti, Mirko
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/1941214
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