Abstract 754


The small nuclear RNA U1 (U1snRNA), the component of the U1snRNP with a key role in pre-mRNA splicing, is an attractive therapeutic molecule because it is able to rescue splicing impaired by mutations, often associated to human disorders. U1snRNA-mediated rescue of gene expression has been demonstrated in various cellular models of disease, including coagulation factor deficiencies. This approach maintains gene regulation in physiological tissues and overcomes limitations related to vector-mediated delivery of large genes. Although the U1snRNA-based strategy can be particularly beneficial for coagulation deficiencies in which even modest increase of functional protein levels would result in improvement of clinical phenotypes, no studies have been conducted in vivo.

Here, to demonstrate the in vivo efficacy, we chose the F7 c.840+5G>A splicing mutation causing severe human factor VII (hFVII) deficiency and previously shown by us to be efficiently rescued in vitro by a modified U1snRNA variant, the U1+5a.


Due to the absence of mouse models of FVII deficiency caused by splicing mutations, we created a novel in vivo model of human FVII deficiency by liver-restricted expression of the mutated hFVII splicing-competent cassette harboring the F7 c.840+5G>A mutation (FVII+5A). This allowed us to test whether co-expression of U1+5a, under the control of its endogenous promoter, can alleviate the splicing defect. The U1+5a-mediated rescue was assessed in C57BL/6 mice, either by transient (by hydrodynamic injection of plasmids) or prolonged (by adeno-associated viral [AAV] vectors) co-expression of the mutated hFVII minigene FVII+5A and of the U1+5a, driven by separate vectors. To avoid competition for AAV receptor binding, the FVII+5A and U1+5a expression cassettes were packaged into serotype 2 (AAV2-FVII+5A) and 8 (AAV8-U1+5a) vectors. The hFVII expression levels in mice were evaluated by human-specific RT-PCR and immunologic assays.


Co-delivery of plasmid pAAV-FVII+5A (1 or 2 μg/g of mouse body weight) with a molar excess (1.5X) of pAAV-U1+5a resulted in a dose-dependent increase of circulating hFVII levels (50.6±16.0 ng/ml or 178±126 ng/ml, respectively), with a peak of 367ng/ml, corresponding to 16.7% of those observed in mice injected with the pAAV-FVIIwt. In contrast, the pAAV-FVII+5A alone did not produce detectable hFVII protein levels. Immunohistochemical analysis of livers of mice receiving both pAAV-FVII+5A and pAAV-U1+5a clearly showed hFVII-positive cells. Moreover, we demonstrated restoration of hFVII splicing (26±10% of total transcripts) in hepatocytes, to indicate that the U1+5a efficiently re-directed usage of the mutated hFVII splicing site in vivo.

To assess long-term effects of the U1+5a, mice were injected with 1.2 × 1012 vector genomes (vg)/mouse of AAV2-FVII+5A alone or with the AAV8-U1+5a (1.2×1011 or 6×1011 vg/mouse). In all co-transduced mice, we detected hFVII antigen levels in plasma, whereas delivery of AAV2-FVII+5A alone was ineffective. The U1+5a-mediated effect was dose-dependent, as measured by circulating hFVII levels of 3.9±0.8 ng/ml (1.2×1011 vg/mouse) or 23.3±5.1 ng/ml (6×1011 vg/mouse) at two weeks post-injection. These findings were corroborated by the appearance of correctly spliced hFVII transcripts (4±0.5% or 16±3% of the total transcripts, respectively) and of hFVII-positive staining cells in liver histological sections. However, the highest AAV8-U1+5a dose (6×1011 vg/mouse) resulted in mortality within two weeks, an observation that warrants further studies to define the underlying mechanism. It is worth noticing that the lowest AAV8-U1+5a dose (1.2×1011 vg/mouse) resulted in low but detectable and persistent circulating hFVII levels. Moreover, its correction effect was even more appreciable (6.9±1.7 ng/ml) in mice injected with an increased dose of template AAV2-FVII+5A (6×1012 vg/mouse), thus suggesting that in FVII deficient patients bearing splicing defects, expressing the target F7 pre-mRNA in all hepatocytes, the rescue would be likely more robust.


We utilized a novel methodology to model human FVII deficiency caused by a splicing defect and to evaluate correction approaches in vivo. Our data provide the first in vivo proof-of-principle of the U1snRNA-mediated rescue of gene expression, and highlight its therapeutic potential in coagulation factor disorders.


Pagani:RareSplice: co-founder of RareSplice, a start-up company of the University of Ferrara Other. Bernardi:RareSplice: Co-founder of RareSplice, a start-up company of the University of Ferrara Other. Pinotti:RareSplice: Co-founder of RareSplice, a start-up company of the University of Ferrara Other.

Author notes


Asterisk with author names denotes non-ASH members.