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An engineered U 1 small nuclear RNA rescues splicing‐defective coagulation F 7 gene expression in mice
Author(s) -
Balestra D.,
Faella A.,
Margaritis P.,
Cavallari N.,
Pagani F.,
Bernardi F.,
Arruda V. R.,
Pinotti M.
Publication year - 2014
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/jth.12471
Subject(s) - rna splicing , mutant , plasmid , biology , microbiology and biotechnology , mutation , rna , gene , messenger rna , recombinant dna , genetics
Summary Background The ability of the spliceosomal small nuclear RNA U 1 ( U 1sn RNA ) to rescue pre‐ mRNA splicing impaired by mutations makes it an attractive therapeutic molecule. Coagulation factor deficiencies due to splicing mutations are relatively frequent and could therefore benefit from this strategy. However, the effects of U 1sn RNA s in vivo remain unknown. Objectives To assess the rescue of the F 7 c.859+5 G > A splicing mutation ( FVII +5 A ), causing severe human factor VII (h FVII ) deficiency, by the modified U 1sn RNA +5a ( U 1+5a) in a murine model. Methods Mice expressing the human F 7 c.859+5 G > A mutant were generated following liver‐directed expression by plasmid or recombinant adeno‐associated viral ( AAV ) vector administration. The rescue of the splice‐site defective pre‐m RNA by U 1+5a was monitored in liver and plasma through h FVII ‐specific assays. Results Injection of plasmids encoding the U 1+5a rescued plasma h FVII levels, which increased from undetectable to ~8.5% of those obtained with the wild‐type h FVII plasmid control. To assess long‐term effects, mice were injected with low and high doses of two AAV vectors encoding the FVII +5 A splice site mutant as template to be corrected by U 1+5a. This strategy resulted in h FVII plasma levels of 3.9 ± 0.8 or 23.3 ± 5.1 ng mL −1 in a dose‐dependent manner, corresponding in patients to circulating FVII levels of ~1–4.5% of normal. Moreover, in both experimental models, we also detected correctly spliced h FVII transcripts and h FVII ‐positive cells in liver cells. Conclusions Here we provide the first in vivo proof‐of‐principle of the rescue of the expression of a splicing‐defective F 7 mutant by U 1sn RNA s, thus highlighting their therapeutic potential in coagulation disorders.