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Ferrata Storti Foundation
Coagulation & its Disorders
Deciphering the Ets-1/2-mediated transcriptional regulation of F8 gene identifies a minimal F8 promoter for hemophilia A gene therapy
*RF and EB contributed equally as co-first authors
ABSTRACT
Amajor challenge in the development of a gene therapy for hemophilia A is the selection of cell type- or tissue-specific promoters to ensure factor VIII (FVIII) expression without eliciting an immune response. As liver sinusoidal endothelial cells are the major FVIII source, understand- ing the transcriptional F8 regulation in these cells would help to optimize the minimal F8 promoter (pF8) to efficiently drive FVIII expression. In silico analyses predicted several binding sites (BS) for the E26 transformation-spe- cific (Ets) transcription factors Ets-1 and Ets-2 in the pF8. Reporter assays demonstrated a significant up-regulation of pF8 activity by Ets-1 or Ets- 1/Est-2 combination, while Ets-2 alone was ineffective. Moreover, Ets-1/Ets- 2-DNA binding domain mutants (DBD) abolished promoter activation only when the Ets-1 DBD was removed, suggesting that pF8 up-regulation may occur through Ets-1/Ets-2 interaction with Ets-1 bound to DNA. pF8 carry- ing Ets-BS deletions unveiled two Ets-BS essential for pF8 activity and response to Ets overexpression. Lentivirus-mediated delivery of green fluo- rescent protein (GFP) or FVIII cassettes driven by the shortened promoters, led to GFP expression mainly in endothelial cells in the liver and to long- term FVIII activity without inhibitor formation in HA mice. These data strongly support the potential application of these promoters in hemophilia A gene therapy.
Introduction
Hemophilia A (HA) is a recessive X-linked bleeding disorder caused by a number of F8 gene mutations which associate with deficiencies of the coagulation factor VIII (FVIII).1,2 As HA is a monogenic disease with a lifelong elevated bleeding risk with no conclusive therapeutic options, it is an ideal candidate for gene therapy.3 In the last years many forward steps in the development of new therapeutic strate- gies have been made, thus rapidly changing the therapeutic landscape of HA. The introduction of the extended half-life FVIII concentrates, of the bi-specific mono- clonal antibody Emicizumab and the start of gene therapy clinical trials have remarkably contributed to the improvement of patient care.4
The development of effective gene therapeutic approaches for HA, has seen lit- tle progression when compared to hemophilia B5. This is due to the complexity of FVIII synthesis, the size of the FVIII protein (2,351 amino acids) and its high immunogenic properties, with the development of neutralizing antibodies in 25% of patients when FVIII is exogenously administered.6,7 As it stands, the goal is to provide a single dose therapy that is effective and offers a lasting cure with sus- tained FVIII activity to HA patients.
Haematologica 2021 Volume 106(6):1624-1635
Rosella Famà,1* Ester Borroni,1* Simone Merlin,1 Chiara Airoldi,2
Silvia Pignani,1 Alessia Cucci,1 Davide Corà,2,3 Valentina Bruscaggin,1 Sharon Scardellato,1 Stefania Faletti,4 Giuliana Pelicci,2,4 Mirko Pinotti,5 Gillian E. Walker1 and Antonia Follenzi1
Department of Health Sciences, Università del Piemonte Orientale, Novara; 2Department of Translational Medicine, Università del Piemonte Orientale, Novara; 3Center for Translational Research on Autoimmune and Allergic Disease (CAAD), University of Piemonte Orientale, Novara; 4Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milan and 5Department of Life Sciences and Biotechnology, Università di Ferrara, Ferrara, Italy
1
Correspondence:
ANTONIA FOLLENZI
antonia.follenzi@med.uniupo.it
Received: October 3, 2019. Accepted: May 20, 2020. Pre-published: May 28, 2020.
https://doi.org/10.3324/haematol.2019.239202
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