Researchers have developed a nanoparticle to deliver genome editing technology, including CRISPR/Cas9, to endothelial cells. This is the first time that vascular endothelial cells have been edited.
The usual way to deliver CRISPR/Cas9 – through a virus – does not work for this cell type and the technique could have major implications for the treatment of coronary artery disease, stroke, pulmonary artery hypertension, and other conditions. This study was done in a mouse model.
The researchers report that “nanoparticle delivery of plasmid DNA is a powerful tool to rapidly and efficiently alter expression of gene/s in ECs for cardiovascular research and potential gene therapy.”
Their findings were published in the journal Cell Reports. The research was done by the lab of Youyang Zhao of Stanley Manne Children’s Research Institute at Ann & Robert H. Lurie Children’s Hospital of Chicago.
The vascular endothelium is a monolayer of endothelial cells (ECs) lining the luminal surface of blood vessels and plays a crucial role in vascular homeostasis and maintenance of tissue fluid balance.
The researchers report that a single intravenous administration of mixture of nanoparticles and plasmid DNA expressing Cas9 controlled by CDH5 promoter and guide RNA (U6 promoter) induced highly efficient genome editing in ECs of the vasculatures, including lung, heart, aorta, and peripheral vessels in adult mice. The nanoparticle carrying CRISPR/Cas9 plasmid DNA required a few days to be effective.
Western blotting and immunofluorescent staining showed there was an approximately 80% decrease of protein expression selectively in ECs, resulting in a phenotype similar to that of genetic knockout mice
“The nanoparticle we developed is a powerful new delivery system for genome editing in vascular endothelial cells, and could be used to treat many diseases, including acute respiratory distress syndrome from severe COVID-19,” said Zhao, who is senior author of the paper.
They reported that their technique can induce robust genome editing of at least two genes and also introduce transgene expression in the same cells. “Such a strategy,” they wrote, “should greatly facilitate the delineation of gene functions in the vascular system with potential for non-viral gene therapy of vascular diseases.”
Zhao noted that, “With this nanoparticle we can introduce genes to inhibit vascular injury and/or promote vascular repair, correct gene mutations and turn genes on or off to restore normal function. It also allows us to edit multiple genes at the same time. This is an important advance for treating any disease caused by endothelial dysfunction.”
They used the biodegradable material poly(ethylene glycol) methyl ether-block-poly(lactide-coglycolide), or PEG-b-PLGA, copolymer-based nanoparticle formulated with polyethyleneiminel as a starting material to generate the nanoparticles. Nanoparticles were highly enriched in mouse liver and found in heart and lung at 8 hours post administration.
The researchers reported that PEG5000 Da-b-PLGA (designated as PP) nanoparticles achieved the best tissue distribution in heart and lung. PP nanoparticles exhibited an even, whole-body distribution without specific enrichment in the liver at 5 hours post administration, as revealed by IVIS tomography.
Endothelial dysfunction is at the root of many diseases, including coronary artery disease, stroke, bronchopulmonary dysplasia and pulmonary artery hypertension. Zhao explained that genome editing in endothelial cells could even treat cancers by cutting off the blood supply to the tumor or blocking cancer metastasis.
