Abstract 17091: High Efficient Therapeutic Genome Editing in Cardiovascular Endotheliumby Nanoparticle Delivery of Crispr Plasmid
Introduction: Therapeutic delivery of CRISPR system components to induce in vivo genome editing in postnatal life has great translational potential. Recent studies employing non-viral delivery of small guide RNA (gRNA) and Cas9 mRNA have achieved efficient in vivo genome editing in adult mice. However, as often seen in other RNA therapeutic studies with non-viral delivery of antisense and siRNA, the efficiency is limited to the liver. Hypothesis: Novel nanoparticle can deliver CRISPR system components in vivo to selectively target cardiovascular endothelium in adult mice. Methods: We developed a novel nanoparticles. Mixture of the nanoparticle:plasmid DNA expressing Cas9 under the control of the human CDH5 promoter and gRNA driven by the U6 promoter was administered i.v. to adult mice. Seven to ten days later, various organ tissues were collected for analysis of the efficiency of genomic editing and knockout of protein expression. The phenotype of CRISPR-mediated in vivo knockout of Pik3cg which encodes the G protein-coupled receptor-activated p110gamma isoform of PI3K was compared to Pik3cg null mice in response to sepsis challenge. Results: Therapeutic delivery of nanoparticles loaded with the all-in-one CRISPR plasmid DNA induced highly efficient genome editing in endothelial cells (ECs) of the cardiovascular system including lung, heart, aorta, and peripheral blood vessels in adult mice. The Indel rate was as great as 50% in ECs isolated from these vascular beds. Immunostaining and Western blotting demonstrated greater than 70% decrease of protein expression in cardiovascular endothelial cells. Pik3cg -gRNA-induced genome editing diminished p110γPI3K expression in pulmonary vascular ECs, which led to impaired vascular repair and resolution of inflammation after sepsis challenge as seen in Pik3cg -/- mice. Conclusion: We have developed a simple and highly efficient method for in vivo genome editing selectively in the cardiovascular endothelium. This strategy will greatly facilitate cardiovascular research and may enable therapeutic genome editing for prevention and treatment of cardiovascular diseases.