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

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          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.

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          Most cited references45

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          Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection.

          This study examines transgene expression and biodistribution of adeno-associated virus (AAV) pseudotyped 1-9 after tail vein (TV) injection in male mice. Using a cytomegalovirus (CMV)-luciferase transgene, the time-course of expression in each animal was tracked throughout the experiment. The animals were imaged at 7, 14, 29, 56, and 100 days after the TV injection. The total number of photons emitted from each animal was recorded, allowing examination of expression level and kinetics for each pseudotyped virus. The bioluminescence imaging revealed three expression levels (i) low-expression group, AAV2, 3, 4, and 5; (ii) moderate-expression group, AAV1, 6, and 8; and (iii) high-expression group, AAV7 and 9. In addition, imaging revealed two classes of kinetics (i) rapid-onset, for AAV1, 6, 7, 8, and 9; and (ii) slow-onset, for AAV2, 3, 4, and 5. We next evaluated protein expression and viral genome copy numbers in dissected tissues. AAV9 had the best viral genome distribution and highest protein levels. The AAV7 protein and genome copy numbers were comparable to those of AAV9 in the liver. Most surprisingly, AAV4 showed the greatest number of genome copies in lung and kidney, and a high copy number in the heart. AAV6 expression was observed in the heart, liver, and skeletal muscle, and the genome distribution corroborated these observations.
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            Biochemistry and genetics of von Willebrand factor.

            J Sadler (1998)
            Von Willebrand factor (VWF) is a blood glycoprotein that is required for normal hemostasis, and deficiency of VWF, or von Willebrand disease (VWD), is the most common inherited bleeding disorder. VWF mediates the adhesion of platelets to sites of vascular damage by binding to specific platelet membrane glycoproteins and to constituents of exposed connective tissue. These activities appear to be regulated by allosteric mechanisms and possibly by hydrodynamic shear forces. VWF also is a carrier protein for blood clotting factor VIII, and this interaction is required for normal factor VIII survival in the circulation. VWF is assembled from identical approximately 250 kDa subunits into disulfide-linked multimers that may be > 20,000 kDa. Mutations in VWD can disrupt this complex biosynthetic process at several steps to impair the assembly, intracellular targeting, or secretion of VWF multimers. Other VWD mutations impair the survival of VWF in plasma or the function of specific ligand binding sites. This growing body of information about VWF synthesis, structure, and function has allowed the reclassification of VWD based upon distinct pathophysiologic mechanisms that appear to correlate with clinical symptoms and the response to therapy.
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              Update on the pathophysiology and classification of von Willebrand disease: a report of the Subcommittee on von Willebrand Factor.

              von Willebrand disease (VWD) is a bleeding disorder caused by inherited defects in the concentration, structure, or function of von Willebrand factor (VWF). VWD is classified into three primary categories. Type 1 includes partial quantitative deficiency, type 2 includes qualitative defects, and type 3 includes virtually complete deficiency of VWF. VWD type 2 is divided into four secondary categories. Type 2A includes variants with decreased platelet adhesion caused by selective deficiency of high-molecular-weight VWF multimers. Type 2B includes variants with increased affinity for platelet glycoprotein Ib. Type 2M includes variants with markedly defective platelet adhesion despite a relatively normal size distribution of VWF multimers. Type 2N includes variants with markedly decreased affinity for factor VIII. These six categories of VWD correlate with important clinical features and therapeutic requirements. Some VWF gene mutations, alone or in combination, have complex effects and give rise to mixed VWD phenotypes. Certain VWD types, especially type 1 and type 2A, encompass several pathophysiologic mechanisms that sometimes can be distinguished by appropriate laboratory studies. The clinical significance of this heterogeneity is under investigation, which may support further subdivision of VWD type 1 or type 2A in the future.
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                Author and article information

                Contributors
                federico.mingozzi@sparktx.com
                Journal
                Gene Ther
                Gene Ther
                Gene Therapy
                Nature Publishing Group UK (London )
                0969-7128
                1476-5462
                17 January 2021
                17 January 2021
                2023
                : 30
                : 3-4
                : 245-254
                Affiliations
                [1 ]GRID grid.419946.7, ISNI 0000 0004 0641 2700, Généthon, ; 91000 Evry, France
                [2 ]GRID grid.8390.2, ISNI 0000 0001 2180 5818, Université Paris-Saclay, Université Evry, INSERM, Généthon, Integrare research unit UMR_S951, ; 91000 Evry, France
                [3 ]GRID grid.460789.4, ISNI 0000 0004 4910 6535, Laboratory of Hemostasis, Inflammation and Thrombosis, Institut National de la Santé et de la Recherche Médicale UMR_1176, , Université Paris-Saclay, ; 94276 Le Kremlin-Bicêtre, France
                [4 ]GRID grid.476706.4, ISNI 0000 0004 7647 0615, Present Address: Spark Therapeutics, ; Philadelphia, PA 19103 USA
                Author information
                http://orcid.org/0000-0001-5216-817X
                http://orcid.org/0000-0002-8271-6795
                http://orcid.org/0000-0001-5152-9156
                http://orcid.org/0000-0003-0685-3752
                Article
                218
                10.1038/s41434-020-00218-6
                10113149
                33456057
                fee1f4f0-97ac-42fa-b148-510baca976a8
                © The Author(s), under exclusive licence to Springer Nature Limited 2021

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 23 June 2020
                : 6 November 2020
                : 30 December 2020
                Funding
                Funded by: FundRef https://doi.org/10.13039/100010663, EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council);
                Award ID: 617432
                Award Recipient :
                Categories
                Brief Communication
                Custom metadata
                © Springer Nature Limited 2023

                Molecular medicine
                molecular biology,diseases
                Molecular medicine
                molecular biology, diseases

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