A RNA Interference-based Approach to Autosomal Dominant Glanzmann Thrombasthenia: A Step towards Personalized Treatment
ISTH Academy. Bury L. Jul 10, 2019; 274096; OC 72.1 Topic: Platelet Disorders, Hereditary
Dr. Loredana Bury
Dr. Loredana Bury
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OC 72.1

A RNA Interference-based Approach to Autosomal Dominant Glanzmann Thrombasthenia: A Step towards Personalized Treatment

L. Bury, M. Borghi, E. Piselli, P. Gresele
University of Perugia, Department of Medicine, Perugia, Italy

Main Topic: Platelet Disorders
Category: Platelet Disorders, Hereditary

Background: RNA interference (RNAi) is emerging as a promising precision medicine strategy for gene-therapy of dominant negative hereditary disorders acting by suppressing the expression of the dominant mutant gene.
Autosomal dominant Glanzmann Thrombasthenia (GT) is caused by gain-of-function variants of ITGA2B or ITGB3 which generate a constitutively activated αIIbβ3 leading to platelet dysfunction and defective proplatelet formation.
We previously characterized an autosomal-dominant GT with a heterozygous c.2134+1G>C ITGB3 variant leading to the in-frame deletion of exon 13 and to the generation of a c.2015-2134del transcript deriving from exon12-exon14 fusion. The resulting mutant αIIbβ3 exerts a dominant negative effect on normal αIIbβ3, making this gene variant an ideal candidate for a RNAi-based therapeutic approach.
Aims: Aim of this work was to assess whether an allele-specific siRNA can block selectively the synthesis of the dominant negative β3 protein thus restoring the expression and function of αIIbβ3.
Methods: A siRNA molecule (ITGB3bkp12-14 siRNA) targeting the breakpoint region exon12-exon14 was designed. CHO cells expressing wild-type αIIb were transfected using expression vectors with either wild-type or mutant β3 and treated with the ITGB3bkp12-14 siRNA (1-25 nM). The expression of normal and mutant ITGB3 transcripts was assessed by real-time PCR, while expression and function of αIIbβ3 were assessed by flow cytometry.
Results: A ITGB3bkp12-14 siRNA concentration of 7nM efficiently silenced mutant β3 (by 76±17%) without silencing the wild-type transcript (9±2%). CHO cells expressing both normal and mutant αIIbβ3 constitutively bound PAC-1 (which recognizes active αIIbβ3). Treatment with the ITGB3bkp12-14 siRNA abrogated constitutive binding of PAC-1 and restored normal αIIbβ3 activation triggered by DTT, reestablishing normal phenotype.
Conclusions: Our data show that a RNAi-based strategy for gene therapy of autosomal dominant GT is feasible and represents a first step to personalized treatment for this rare disorder.

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