Evaluation of factor V mRNA to define the residual factor V expression levels in severe factor V deficiency

Lunghi, Barbara; Pinotti, Mirko; Maestri, Iva; Bátorová, Angelika; Bernardi, Francesco

doi:10.3324/haematol.11952

Cited by 18 publications

(17 citation statements)

References 8 publications

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“…However, since point mutations and small insertions/deletions can be overcome by an occasional somatic reversion or rare mistakes during translation ( e.g. ribosome slippage), these defects may be actually compatible with the expression of traces of FV sufficient for minimal haemostasis (Yang et al , 2000; Lunghi et al , 2008). In contrast, truly null FV defects, such as large F5 deletions or chromosomal rearrangements involving the F5 gene, would be incompatible with life if present in the homozygous or doubly heterozygous state, as suggested by the fact that gross F5 gene deletions have never been found in FV‐deficient patients.…”

Section: Congenital Fv Deficiency (Owren Parahaemophilia)mentioning

confidence: 99%

Advances in understanding the bleeding diathesis in factor V deficiency

et al. 2009

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SummaryCoagulation factor V (FV), present in plasma and platelets, is an indispensable clotting factor, as demonstrated by the uniform lethality of FV knock-out mice. Surprisingly, however, severe FV deficiency is rarely fatal in humans. In fact, although several cases of life-threatening intracranial haemorrhage have been reported in FV-deficient newborns, many patients with undetectable FV levels experience only mild to moderate bleeding and do not require routine prophylaxis. While the reasons for this variable phenotypic expression are largely unknown, several observations from different laboratories indicate platelets as crucial players in FV deficiency. Moreover, we have recently shown that plasma levels of tissue factor pathway inhibitor are considerably reduced in FV-deficient plasma, which results in enhanced thrombin generation especially at very low FV levels (<2%). The present review discusses and integrates these findings in the context of the biology of FV and the clinical features of FV deficiency.

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Section: Congenital Fv Deficiency (Owren Parahaemophilia)mentioning

confidence: 99%

Advances in understanding the bleeding diathesis in factor V deficiency

et al. 2009

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“…19,25,26 Since the FV requirement for minimal thrombin generation is well below 1%, [26][27][28][29] traces of FV would already be sufficient to guarantee thrombin formation and to rescue FV-deficient persons from fatal bleeding. However, in vitro experiments have failed to detect any thrombin generation in plasma from patients with undetectable FV, 29,30 although FV-deficient patients have low plasma levels of the anticoagulant protein tissue factor pathway inhibitor (TFPI), 29 which considerably reduces the FV requirement for thrombin generation.…”

Section: Introductionmentioning

confidence: 99%

Residual platelet factor V ensures thrombin generation in patients with severe congenital factor V deficiency and mild bleeding symptoms

Duckers

Simioni

Spiezia

et al. 2010

Blood

123

134

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“…25 Among all coagulation factor deficiencies, FV deficiency represents a particularly suitable target for RNA therapy, because (1) no FV concentrate or recombinant FV preparation is available for substitutive therapy; (2) the FV requirement for adequate hemostasis is extremely low, making a few percentages of FV sufficient to prevent life-threatening bleeding 19,[35][36][37] ; and (3) the large size of the F5 cDNA makes conventional gene therapy particularly challenging. Here, we present the first application of RNA therapy to severe FV deficiency and we provide in vitro and ex vivo evidence that mutation-specific antisense molecules can effectively rescue a F5 splicing mutation.…”

Section: Discussionmentioning

confidence: 99%

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

Nuzzo

Radu

Baralle

et al. 2013

Blood

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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.

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Evaluation of factor V mRNA to define the residual factor V expression levels in severe factor V deficiency

Cited by 18 publications

References 8 publications

Advances in understanding the bleeding diathesis in factor V deficiency

Advances in understanding the bleeding diathesis in factor V deficiency

Residual platelet factor V ensures thrombin generation in patients with severe congenital factor V deficiency and mild bleeding symptoms

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

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