Altered cleavage of human factor VIII at the B-domain and acidic region 3 interface enhances expression after gene therapy in hemophilia A mice

Giang N Nguyen; Jonathan R Lindgren; Maria C Seleme; Samita Kafle; Catherine B Zander; X Long Zheng; Denise E Sabatino

doi:10.1016/j.jtha.2023.04.012

Altered cleavage of human factor VIII at the B-domain and acidic region 3 interface enhances expression after gene therapy in hemophilia A mice

J Thromb Haemost. 2023 Aug;21(8):2101-2113. doi: 10.1016/j.jtha.2023.04.012. Epub 2023 Apr 18.

Authors

Giang N Nguyen¹, Jonathan R Lindgren¹, Maria C Seleme¹, Samita Kafle¹, Catherine B Zander², X Long Zheng³, Denise E Sabatino⁴

Affiliations

¹ The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
² Department of Pathology, University of Alabama at Birmingham School of Medicine, University of Alabama, Birmingham, Alabama, USA.
³ Department of Pathology and Laboratory Medicine, The University of Kansas Medical Center, Kansas City, Kansas, USA; Institute of Reproductive Medicine and Developmental Science, The University of Kansas Medical Center, Kansas City, Kansas, USA.
⁴ The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA; Department of Pediatrics, Division of Hematology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA. Electronic address: dsabatin@pennmedicine.upenn.edu.

PMID: 37080538
DOI: 10.1016/j.jtha.2023.04.012

Abstract

Background: Variants of human factor VIII (hFVIII) have been developed to further understand the structure and function of hFVIII and improve gene-based therapeutics. We have previously characterized several hFVIII variants of the furin cleavage site (1645-1648) with improved secretion. We have also identified a second cleavage site in the acidic region 3 (a3) (1657-1658) that becomes the primary hFVIII intracellular cleavage position in the absence of the furin site. We tested a hypothesis that modification of this site may confer additional functional advantages to hFVIII.

Objectives: The aim of this study was to conduct the biochemical and functional characterization of hFVIII variants of the furin cleavage site, the a3 cleavage site, or in combination, both in vitro and in vivo after AAV mediated gene therapy.

Methods: Recombinant hFVIII variants of the furin cleavage site (hFVIII-Δ3), the a3 cleavage site (hFVIII-S1657P/D1658E [SP/DE]), or in combination (hFVIII-Δ3-SP/DE) were purified and characterized in vitro and in vivo.

Results: Recombinant hFVIII-Δ3, hFVIII-SP/DE, and hFVIII-Δ3-SP/DE variants all had comparable specific activity to B-domain deleted (BDD) hFVIII. Hemophilia A mice tolerant to hFVIII did not develop immune responses to hFVIII after protein challenge with these variants or after adeno-associated virus (AAV) delivery. Following AAV delivery, hFVIII-Δ3-SP/DE resulted in expression levels that were 2- to 5-fold higher than those with hFVIII-BDD in hemophilia A mice.

Conclusion: The novel hFVIII-Δ3-SP/DE variant of the furin and a3 cleavage sites significantly improved secretion compared with hFVIII-BDD. This key feature of the Δ3-SP/DE variant provides a unique strategy that can be combined with other approaches to further improve factor VIII expression to achieve superior efficacy in AAV-based gene therapy for hemophilia A.

Keywords: adeno-associated viral vector; blood coagulation factors; factor VIII; genetic therapy; hemophilia.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Animals
Factor VIII* / metabolism
Furin / genetics
Genetic Therapy / methods
Genetic Vectors
Hemophilia A* / genetics
Hemophilia A* / therapy
Humans
Mice
Protein Domains

Substances

Factor VIII
Furin

Abstract

Publication types

MeSH terms

Substances

Grants and funding