Abstract 14946: Robust Genome Editing of Endothelial Hif-2α in Adult Mice by Ec-Targeted Nanoparticle Delivery of Prime Editing System Inhibits Pulmonary Hypertension

Introduction: Prime Editing (PE) is a site-specific mutagenesis approach for highly precise genome editing, thus has the potential for safer editing to treat human diseases. However, the genome editing efficiency has been shown only 5% in adult mice. The in vivo efficacy of prim editing remains unclear. Hypothesis: Endothelial cell-targeted nanoparticle delivery of the all-in-one plasmid DNA (15kb) expressing the prime editing under the control of CDH5 promoter will induce robust genome editing of Hif2a selectively in endothelial cells in adult mice and inhibit PH. Methods: To target endothelium for robust genome editing, we employed EndoNP1 nanoparticles to deliver the all-in-one prime editing plasmid DNA under the control of CDH5 promoter to adult WT and Egln1 Tie2Cre mice. The prime editing was designed to change 1-2 nucleotides to introduce a STOP codon in mouse Hif2a coding region. Hypoxia-induced PH in mice and Egln1 Tie2Cre mouse model of severe PAH were employed to determine the therapeutic potential of prime editing in PH treatment. Results: Single administration of mixture of nanoparticles and prime editing plasmid DNA caused 30% genome editing efficacy selectively in endothelial cells of WT mice and also Egln1 Tie2Cre mice. The genome editing efficiency was increased to approximately 50% with 2 administrations of the nanoparticle:plasmid DNA mixture in a 1 week interval, resulting in >70% reduction of HIF-2a protein. One week after the 2 nd administration, WT mice were subjected to hypoxia (10%) for 3 weeks. Pulmonary hypertension was attenuated in the prime edited mice with endothelial Hif2a early stop codon. Conclusions: These findings for the first time demonstrate unprecedented efficacy of genome editing of prime editing selectively in ECs of adult mice which exhibit phenotype similar to genetic knockout mice. Thus, EC-targeted nanoparticle delivery of the prime editing system may have great potential for treatment of human diseases associated with endothelial cell dysfunction, such as pulmonary arterial hypertension.