OC 21.1 - Rescue of an FVIII splicing variant with engineered U1snRNAs

Sunday, July 10, 2022

2:45 PM – 3:00 PM

Location: ExCel Center, S11A

Presentation Format: In-Person

Presenting Author(s)

Laura Peretto

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

Co-Author(s)

RT

Rebecca Tarantino

Master School Student
University of Ferrara
Ferrara, Emilia-Romagna, Italy
AB

Alessandra Borchiellini

Doctor, in charge of regional reference centre of haemorrhagic and thrombotic disorders.
A.O.U. Città della Salute e della Scienza di Torino
Torino, 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
Antonia Follenzi, MD, PhD

Full professor
Department of Health Sciences, University of Piemonte Orientale, Novara, Italy
Novara, Piemonte, Italy
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: Hemophilia A (HA) is an X-linked recessive hemorrhagic disorder caused by coagulation factor VIII (FVIII) deficiency. Among all HA-causing mutations, those affecting splicing are relatively frequent, particularly in severe forms. These mutation types, often leading to exon skipping, can be rescued by RNA therapeutics based on variants of the key U1snRNA spliceosomal component, as shown in several human disease models.

Aims: To dissect the molecular mechanisms of the F8 c.1752+5g>c variant associated with moderate HA (FVIII: C < 5) in two brothers, and assess the U1snRNA-mediated rescue.

Methods: Creation of expression vectors for the wild-type (pIVS11wt) and mutant (pIVS11+5c) F8 minigenes and for the engineered U1 snRNAs designed to base pairs to the mutated 5’ss (compensatory U1snRNA) or to less-conserved downstream intronic sequences (Exon Specific U1snRNA). Transient transfection of various human hepatoma cell lines and RT-PCR to evaluate splicing patterns and their modulation by U1snRNA variants.

Results: Bioinformatic analysis did not predict an impact on splicing for the F8 c.1752+5g>c mutation. However, splicing pattern analysis of the hepatoma cells expressing the pIVS11+5c variant revealed that the change leads to exon 11 skipping, with low levels of correctly spliced transcripts (~10 %). Co-transfection of U1snRNA variants significantly improves FVIII exon 11 definition and thus inclusion, with the compensatory U1snRNA associated with exon 11 inclusion up to 92%.

Conclusion(s): We provided experimental evidence that the c.1752+5g>c mutation impairing F8 exon 11 definition and lead to exon skipping with trace levels of correct transcripts, in accordance with the HA patients’ phenotype. Importantly, exon inclusion of the defective exon can be efficiently restored by the use of appropriately designed U1snRNA variants, which are currently under investigation through lentiviral-mediated delivery in Blood-Born Endothelial Cells (BOECs) isolated from HA patients.