OC 01.1 - Base and Prime editing of DNA as a new therapeutic option for Hemophilia A

Saturday, July 9, 2022

1:00 PM – 1:15 PM

Location: S11A

Presentation Format: In-Person

Presenting Author(s)

Elena Tonetto

PhD student
Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy
Ferrara, Emilia-Romagna, Italy

Co-Author(s)

SP

Silvia Pignani

PhD
Department of Health Sciences, University of Piemonte Orientale, Novara, Italy
Novara, Piemonte, Italy
GR

Giacomo Roman

PhD student
Department of Health Sciences, University of Piemonte Orientale, Novara, Italy
Novara, Piemonte, Italy
Antonia Follenzi, MD, PhD

Full professor
Department of Health Sciences, University of Piemonte Orientale, Novara, Italy
Novara, Piemonte, Italy
FB

Francesco Bernardi

Professor of Biochemistry
Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology, Ferrara University, Ferrara (Italy)
Verona, Veneto, Italy
DL

David Liu

PhD
Howard Hughes Medical Institute, Harvard University, Cambridge, USA
Cambridge, Massachusetts, United States
MP

Mirko Pinotti

Professor
Department of Life Sciences and Biotechnology and LTTA Center, University of Ferrara, Ferrara 44121, Italy
Ferrara, Emilia-Romagna, Italy
DB

Dario Balestra

PhD
Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy
Ferrara, Emilia-Romagna, Italy

Background: Replacement therapy as well as non-replacement therapy, albeit significantly improved Hemophilia A (HA) treatment, do not provide a definitive cure and still have limitations. This led to exploitation of gene therapy and genome editing approaches based on homologous recombination (HSR), which are not optimized yet. Recently, DNA base (BE) and prime (PE) editing approaches non-relying on HSR have been developed to cleanly install or revert point mutations which, in HA, are the most represented ones when excluded the IVS22 inversion.

Aims: To exploit BE and PE approaches and correct representative F8 missense and nonsense variants at DNA level and rescue FVIII expression.

Methods: Cellular models created by expression of recombinant FVIII variants; design and delivery of BE and PE components and evaluation of secreted FVIII levels (ELISA, coagulation/chromogenic assays).

Results: In transient and stable expression systems, the c.6046C>T(p.R2016W), c.6496C>T(p.R2166*), c.6545G>A(p.R2182H), c.6682C>T(p.R2228*), c.6683G>A(p.R2228Q) led to reduced secreted FVIII levels (p.R2166*>p.R2228Q>p.R2016W>p.R2182H) while the p.R2228* change produced appreciable FVIII protein levels attributable to a truncated isoform. Screening of a large sets of BE/PE variants and guide RNAs identified combinations that rescued FVIII secretion for the p.R2166*, p.R2182H and p.R2228Q mutations. Extensive studies on the p.R2166* and p.R2228Q changes demonstrated that the BE and PE-mediated reversion of mutations, detectable at DNA levels, resulted in appreciable rescue of secreted FVIII protein and activity levels (up to 20% of FVIII-WT).

Conclusion(s): For the first time, the BE and PE approach has been applied to Hemophilia A. Data on two relatively frequent missense and nonsense mutations causing severe HA provided the proof-of-principle of efficacy of base editing in cellular models, and studies in HA blood-outgrowth endothelial cells (BOECs) are currently in progress. This lay the foundation for studies with BE/PE in animal models, which might open a new avenue toward an innovative and personalized HA cure.