Abstract Number: OC 03.3
Meeting: ISTH 2020 Congress
Background: A major challenge in the development of a gene therapy for hemophilia A (HA) 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 (LSECs) are the major FVIII source, understanding the transcriptional F8 regulation in these cells would help optimize the minimal F8 promoter (pF8) to efficiently drive FVIII expression.
Aims: To investigate the modulation of F8 promoter activity and deciphering the role of Ets-1/2 transcription factors.
Methods: In silico analyses predicted several binding sites (BS) for the E26 transformation-specific (Ets) transcription factors Ets-1 and Ets-2 along the pF8. Ets-1 and Ets-2 or both were transfected with pF8 in ECV or HEK293T cell line and luciferase assays have been performed to study the promoter activity in response to these factors. Ets-1/Ets-2-DNA binding domain mutants (DBD) were generated as well as shortened forms of pF8 carrying Ets-BS deletion, and evaluated in vitro by reporter assays and in vivo by injecting lentiviral vectors (LV) carrying GFP or FVIII cassettes in C57Bl/6 or B6/129 hemophilic (HA) mice.
Results: Reporter assays demonstrated a significant up-regulation of pF8 activity by Ets-1 or Ets-1/Est-2 combination, while Ets2 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. In vivo, LV delivery of GFP or FVIII cassettes driven by these promoters, led to transgene expression mainly in LSECs as well as long-term FVIII activity without inhibitor formation.
Conclusions: This approach revealed the -342bp region, where three Ets-1 and one Ets-2 sites were maintained, as a minimal sequence required to maintain in vitro activity and a regulated FVIII expression restricted to LSECs, representing a potential application in HA gene therapy.
To cite this abstract in AMA style:Famà R, Borroni E, Merlin S, Pignani S, Airoldi C, Cucci A, Bruscaggin V, Scardellato S, Pinotti M, Walker GE, Follenzi A. Deciphering the Ets-1/2-mediated Transcriptional Regulation of F8 Gene Identifies a Minimal F8 Promoter for Hemophilia A Gene Therapy [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/deciphering-the-ets-1-2-mediated-transcriptional-regulation-of-f8-gene-identifies-a-minimal-f8-promoter-for-hemophilia-a-gene-therapy/. Accessed January 24, 2022.
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