Antisense-based RNA therapy of factor V deficiency: in vitro and ex vivo rescue of a F5 deep-intronic splicing mutation

Abstract

Antisense molecules are emerging as a powerful tool to correct splicing defects. Recently, we identified a homozygous deep-intronic mutation (F5 c.1296+268A>G) activating a cryptic donor splice site in a patient with severe coagulation factor V (FV) deficiency and life-threatening bleeding episodes. Here, we assessed the ability of 2 mutation-specific antisense molecules (a morpholino oligonucleotide [MO] and an engineered U7 small nuclear RNA [snRNA]) to correct this splicing defect. COS-1 and HepG2 cells transfected with a F5 minigene construct containing the patient's mutation expressed aberrant messenger RNA (mRNA) in excess of normal mRNA. Treatment with mutation-specific antisense MO (1-5 µM) or a construct expressing antisense U7snRNA (0.25-2 µg) dose-dependently increased the relative amount of correctly spliced mRNA by 1 to 2 orders of magnitude, whereas control MO and U7snRNA were ineffective. Patient-derived megakaryocytes obtained by differentiation of circulating progenitor cells did not express FV, but became positive for FV at immunofluorescence staining after administration of antisense MO or U7snRNA. However, treatment adversely affected cell viability, mainly because of the transfection reagents used to deliver the antisense molecules. Our data provide in vitro and ex vivo proof of principle for the efficacy of RNA therapy in severe FV deficiency, but additional cytotoxicity studies are warranted.

Defects of splicing in antithrombin deficiency

“…As a final consideration; the elucidation of the pathological mechanism associated to one mutation is not only important from a basic point of view. It is also relevant for establishing the most appropriate and personalized therapeutic strategy at the molecular level, particularly with the promising results obtained to rescue mutations causing aberrant splicing …”

“…It is also relevant for establishing the most appropriate and personalized therapeutic strategy at the molecular level, particularly with the promising results obtained to rescue mutations causing aberrant splicing. [25][26][27][28]…”

Small-scale high-throughput sequencing–based identification of new therapeutic tools in cystic fibrosis

“…The development of oligonucleotides that block access to a target site (TSBs) offers new treatment opportunities for other genetic disorders. 32,33 Here, we used this approach to correct the aberrant splicing caused by deep intronic mutations in the CFTR gene (c.1680-883A>G and c.1680-886A>G). The effect of TSBs on aberrant splicing correction in bronchial BEAS-2B cells was rapid and maintained over time, suggesting that TSBs could be a therapeutic tool in patients with CF who have deep intronic mutations in the CFTR gene because TSBs restore normal transcripts.…”

Endocytosis of exogenous factor V by ex‐vivo differentiated megakaryocytes from patients with severe parahaemophilia

“…Blood samples were collected from healthy donors and from three previously described patients with severe FV deficiency (Duckers et al, 2010;Castoldi et al, 2011), namely PD-III (doubly heterozygous for p.Trp255Arg and p.Tyr1623Asp) (Duckers et al, 2010), PD-VI (homozygous for the c.1296 + 268A>G deep-intronic mutation) (Castoldi et al, 2011;Nuzzo et al, 2013) and PD-VII (homozygous for p.Gly2140Asp) (Duckers et al, 2010). None of the patients had received replacement therapy with blood components or concentrates in the 3 months before blood sampling.…”