Molecular mechanism and RNAi correction of a dominantnegative von Willebrand Factor gene deletion.

Understanding molecular mechanisms leading to the dominant inheritance of von Willebrand disease (VWD) would improve our knowledge on pathophysiological aspects underlying its high prevalence. We produced a cellular model of severe type 2 VWD, caused by an heterozygous deletion in the VWF gene, to investigate the altered biosynthesis. Co-expression of the VWF inframe deleted cDNA (p.P1105_C1926delinsR) impaired wild type vector-driven protein secretion and function (VWF collagen-binding 1.9 ± 0,5% of wt), which mimicked the patient’s phenotype. Protein studies and cell immunostaining delineated the highly efficient dominant-negative mechanism. The deleted VWF was synthesized in large amounts and preferentially processed, and through a correctly encoded cysteine knot domain formed heterodimers and heterotetramers with wild type VWF. Impaired multimerization was associated with reduced amounts of VWF in late endosomes. The key role of heterodimers as multimer terminators was further supported by introduction of the dimerization mutation C2773R in the deleted construct, which resulted in a quantitative defect with normal multimer size. Targeting the mRNA breakpoint by siRNA selectively inhibited the in-frame deleted VWF expression, and restored secretion of functional VWF (28.0 ± 3.3% of wt). This provided a novel tool to explore mutation-specific gene therapy in a severe form of dominant VWD.