Recombinant Adeno-Associated Viral Vectors Expressing Human Coagulation FIX-E456H Variant in Hemophilia B Mice

Sandra Le Quellec; Allison P. Dane; Elena Barbon; Jean-Claude Bordet; Federico Mingozzi; Yesim Dargaud; Thibaut Marais; Maria-Grazia Biferi; Claude Négrier; Amit C. Nathawani; Nathalie Enjolras

doi:10.1055/s-0039-1697658

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2019; 119(12): 1956-1967
DOI: 10.1055/s-0039-1697658

Coagulation and Fibrinolysis

Georg Thieme Verlag KG Stuttgart · New York

Recombinant Adeno-Associated Viral Vectors Expressing Human Coagulation FIX-E456H Variant in Hemophilia B Mice

Sandra Le Quellec

¹EA 4609-Hémostase et Cancer, SFR Lyon Est, Université Claude Bernard Lyon I, Lyon, France

²Laboratoire d'hématologie, Hopital Louis Pradel, Hospices Civils de Lyon, Bron, France

,

Allison P. Dane

³Research Department of Haematology, University College of London Cancer Institute, London, United Kingdom

,

Elena Barbon

⁴Genethon, UMR_S951 INSERM, Univ Evry, Université Paris Saclay, EPHE, Evry, France

,

Jean-Claude Bordet

¹EA 4609-Hémostase et Cancer, SFR Lyon Est, Université Claude Bernard Lyon I, Lyon, France

²Laboratoire d'hématologie, Hopital Louis Pradel, Hospices Civils de Lyon, Bron, France

,

Federico Mingozzi

⁴Genethon, UMR_S951 INSERM, Univ Evry, Université Paris Saclay, EPHE, Evry, France

,

Yesim Dargaud

¹EA 4609-Hémostase et Cancer, SFR Lyon Est, Université Claude Bernard Lyon I, Lyon, France

²Laboratoire d'hématologie, Hopital Louis Pradel, Hospices Civils de Lyon, Bron, France

,

Thibaut Marais

⁵UPMC Paris 06, INSERM UMRS974, Center of Research in Myology, Sorbonne Université, Paris, France

,

Maria-Grazia Biferi

⁵UPMC Paris 06, INSERM UMRS974, Center of Research in Myology, Sorbonne Université, Paris, France

,

Claude Négrier

¹EA 4609-Hémostase et Cancer, SFR Lyon Est, Université Claude Bernard Lyon I, Lyon, France

²Laboratoire d'hématologie, Hopital Louis Pradel, Hospices Civils de Lyon, Bron, France

,

Amit C. Nathawani

³Research Department of Haematology, University College of London Cancer Institute, London, United Kingdom

,

Nathalie Enjolras

¹EA 4609-Hémostase et Cancer, SFR Lyon Est, Université Claude Bernard Lyon I, Lyon, France

²Laboratoire d'hématologie, Hopital Louis Pradel, Hospices Civils de Lyon, Bron, France

› Author Affiliations

Abstract

Gene therapy using recombinant adeno-associated virus (AAV) has induced sustained long-term coagulation human factor IX (hFIX) levels in hemophilia B (HB) patients. However, asymptomatic transient liver toxicity was observed at high vector doses, highlighting the need to improve the potency of these vectors. We report the generation of an AAV transgene cassette containing the hyperfunctional hFIX-E456H variant showing improved binding to platelets, with a comparison to wild-type hFIX (hFIX-WT) and hFIX-R384L variant (Padua) transgenes, containing F9 truncated-intron 1 (I1). In vitro specific activity was increased by 3.2- and 4.2-fold with hFIX-E456H and hFIX-R384L variants compared with hFIX-WT, using chromogenic assay, and by 7-and 8.6-fold with hFIX-E456H and hFIX-R384L variants compared with hFIX-WT, using one-stage assay. The transgenes were packaged into single-stranded AAV2/8 vectors that were tail vein injected at 5 × 10⁹, 2 × 10¹⁰, and 5 × 10¹⁰ vg per mouse in HB mice. Plasma FIX activity level, assessed by chromogenic assay, was up to fourfold higher for hFIX-E456H compared with hFIX-WT and was not different compared with hFIX-R384L, among the three dose cohorts. Overall, the in vivo specific activity was increased by threefold for hFIX-E456H and 4.9-fold for hFIX-R384L compared with hFIX-WT. At the lower dose of 5 × 10⁹ vg, the blood loss was significantly lower for hFIX-E456H compared with hFIX-WT, but did not differ compared with hFIX-R384L. The results found for the hFIX-E456H variant indicate that it might be a suitable alternative for gene therapy of HB.

Keywords

gene therapy - hemophilia B - factor IX - platelets - factor IX Padua

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References

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Supplementary Material

Supplementary Material