Development of a dual hybrid AAV vector for endothelial-targeted expression of von Willebrand factor

Abstract

Von Willebrand disease (VWD), the most common inherited bleeding disorder in humans, is caused by quantitative or qualitative defects in von Willebrand factor (VWF). VWD represents a potential target for gene therapy applications, as a single treatment could potentially result in a long-term correction of the disease. In recent years, several liver-directed gene therapy approaches have been exploited for VWD, but their efficacy was generally limited by the large size of the VWF transgene and the reduced hemostatic activity of the protein produced from hepatocytes. In this context, we aimed at developing a gene therapy strategy for gene delivery into endothelial cells, the natural site of biosynthesis of VWF. We optimized an endothelial-specific dual hybrid AAV vector, in which the large VWF cDNA was put under the control of an endothelial promoter and correctly reconstituted upon cell transduction by a combination of trans-splicing and homologous recombination mechanisms. In addition, we modified the AAV vector capsid by introducing an endothelial-targeting peptide to improve the efficiency for endothelial-directed gene transfer. This vector platform allowed the reconstitution of full-length VWF transgene both in vitro in human umbilical vein endothelial cells and in vivo in VWD mice, resulting in long-term expression of VWF.

Structural basis of von Willebrand factor multimerization and tubular storage

“…As the VWD is an ideal target for gene therapy where a single treatment could potentially result in long term correction of the disease and even partial correction could have benefits in lowering the bleeding risks, like in the case of hemophilia 44 . The preliminary study on VWD gene therapy showed limited yet promising prospect [45][46][47][48] , however, one of the main obstacles of gene therapy approach for VWD treatment is the 8kb size of VWF gene. This exceeds the packaging limit (5kb) of the recombinant adeno-associated virus vectors (rAAV) that has been successfully used in gene therapy of hemophilia A, B, and multiple other congenital disease [49][50][51] .…”

In vivo Dominant-Negative Effect of an SCN5A Brugada Syndrome Variant

“…This strategy proof-of-principle was established by Dr. Duan's group in 2007 in a study designed to assess mice whole-body transduction using trans-splicing AAVs (Ghosh et al, 2007(Ghosh et al, , 2008. Then, dual AAV hybrid vectors have been exploited in human gene therapy in the past years, demonstrating the interest of this technique for in vivo efficient gene transfer (Koo et al, 2014;Trapani et al, 2014;Trapani, 2018;Barbon et al, 2021). To the best of our knowledge, we present here, for the first time, overexpression of a cardiac large gene in ∼75% of mice cardiomyocytes.…”

Towards novel treatment options in von Willebrand disease

“…With genetic approaches, one should think of siRNA-based techniques to silence mutant alleles or even gene therapy. [48][49][50] However, many hurdles are there to take in order to make these approaches clinically viable.…”

“…These innovative paths are not only limited to protein‐based strategies but also genetic approaches are in early stage preclinical development. With genetic approaches, one should think of siRNA‐based techniques to silence mutant alleles or even gene therapy 48–50 . However, many hurdles are there to take in order to make these approaches clinically viable.…”