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R. Famà et al.
Ets-2 in pF8 regulation offers a new insight into the molec- ular mechanisms promoting FVIII expression in specific cell types. To date, F8 has not been reported to be amongst the large number of genes shown to be direct tar- gets of Ets-1 and Ets-2 regulation, such as KDR, FLT1, ANGPT2, TEK, VWF47 and CDH5.48 Interestingly, all these genes are normally expressed at different stages in EC, suggesting a common regulation with FVIII.
Establishing the minimal promoter sequence required for the maintenance of a regulated FVIII expression restricted to LSEC, offers new perspectives for developing novel approaches to cure HA. For example, it provides the possibility of inserting a pF8 into an AAV-FVIII construct. Currently, these classes of vectors are the safest and most extensively used for HA gene therapy,49 however, they are limited by the size of the expression cassette, making it impossible to introduce both F8 and its full-length pro- moter. The efficient pF8 size reduction (~70%) described herein, opens up the possibility of engineering shortened chimeric pF8 by including sequences enriched in TFBS (like Ets-1) which can enhance the pF8 activity, as has been described for the transthyretin promoter in hepato- cytes.50
Disclosures
AF, RF, SM, DZ are named inventors of the patent “PRO- MOTER FOR CELL-SPECIFIC GENE EXPRESSION AND USES THEREOF” (PCT/IB2017/053460).
Contributions
RF designed and performed most of the experiments; SM injected lentiviral vectors in mice, performed blood coagulation and enzyme-linked immunosorbent assays; EB designed, per- formed and analyzed immunofluorescences stainings; AC and SS helped with cloning experiments and luciferase assays; CA carried out statistical analyses; MP and SP generated and tested the impact of CRISPRa system on pF8; VB produced lentiviral vectors for in vivo experiments; SF and GP helped in planning and performing some in vitro experiments; GEW performed west- ern blotting analysis and DC performed data analysis; AF con- ceived, supervised the study and generated funding; RF, GEW and AF wrote the paper; all authors critically revised the paper and approved the version to be published.
Aknowledgments
The authors thank Dr. Chiara Borsotti for help in revising the manuscript. Dr. Diego Zanolini for the contribution in planning some initial cloning experiments. Dr. Diego Cotella for providing the promoterless pNL1.1 plasmid and suggestions for luciferase exper- iments setting. Dr. Daniela Capello and Prof. Fabrizio Faggiano for contribution in luciferase assays data analysis. Dr. Silvia Buzzi for performing mice maintenance and coagulation assays.
Funding
This work was supported partly by European Research Council (ERC) #261178 and Horizon 2020 (HemAcure project #66742) to AF.
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