OR01
Characterization and deployment in vivo of FiCAT genome writer
N Artigas(1) J Martínez-de-Lagos(1,2) J Jaraba-Wallace(1) M Güell(1,2) A Sánchez-Mejías(1)
1:Integra Therapeutics S.L.; 2:Universitat Pompeu Fabra
Find Cut-and-Transfer (FiCAT) is a robust gene writing platform, combining the precision of CRISPR-Cas9 systems, with an engineered piggyBac transposase with donor DNA processing and high transfer capacity. Here, with the aim to demonstrate FiCAT activity in vivo, we performed precise gene delivery to mouse models liver cells using viral-free delivery vectors via systemic administration. We were able to efficiently deliver FiCAT to mice liver targeting genomic safe harbors, together with a reporter transposon in DNA form. Stable transduction was confirmed by qPCR analysis of the inserted transgene and at 6 weeks after transduction. Lipid nanoparticles (LNPs) have been extensively validated both pre-clinically and clinically for the delivery of different nucleic acid cargos. Once optimized FiCAT mRNA-based LNP formulation, specific cut capacity in mice liver was analyzed by targeted NGS. In this work, we were able to successfully deliver mRNA cargos via LNPs in vivo, and optimize DNA cargos delivery for codelivery with FiCAT machinery; for enabling gene writing in vivo. Our work validates the feasibility of doing viral free stable gene transfer in vivo; which is one of the challenges in our desire to deliver safely and efficiently therapeutic genes into the target cells and achieve precise and efficient in vivo targeted insertion of DNA fragments in mammalian genomes.
