Abstract
SUMMARYVascular endothelium plays a crucial role in vascular homeostasis and tissue fluid balance. To target endothelium for robust genome editing, we developed poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) (PEG-b-PLGA) copolymer-based nanoparticle formulated with polyethyleneimine. A single i.v. 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 endothelial cells (ECs) of the vasculatures, including lung, heart, aorta, and peripheral vessels in adult mice. Western blotting and immunofluorescent staining demonstrated an ~80% decrease of protein expression selectively in ECs, resulting in a phenotype similar to that of genetic knockout mice. Nanoparticle delivery of plasmid DNA could induce genome editing of two genes or genome editing and transgene expression in ECs simultaneously. Thus, 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.
Highlights
The vascular endothelium is a monolayer of endothelial cells (ECs) lining the luminal surface of blood vessels
We describe the development of poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) (PEG-b-PLGA; PP)-based nanoparticles with excellent biodistribution for vascular delivery and show that polyethyleneimine (PEI)-formulated PP nanoparticle-mediated delivery of the all-in-one-CRISPR plasmid DNA expressing Cas9 under the control of human CDH5 promoter and guide RNA driven by the U6 promoter results in highly efficient genome editing in ECs of various vascular beds including lung, heart, aorta, and peripheral vasculature in adult mice with a single administration, which leads to disruption of gene expression ($80% decrease of protein expression in ECs) and a phenotype mimicking that of genetic knockout mice
The size of the PP/ PEI nanoparticles complexed with the all-in-one CRISPRCAG plasmid DNA (Ran et al, 2013) (Figure S2A) expressing Cas9 under the control of the chicken Actb promoter with a CMV enhancer (CAG) and guide RNA (gRNA) driven by the U6 promoter were less than 300 nm with 90% of them in the range of 80 to 250 nm (Figure 1E)
Summary
The vascular endothelium is a monolayer of endothelial cells (ECs) lining the luminal surface of blood vessels. The endothelial monolayer plays a crucial role in vascular homeostasis and maintenance of tissue fluid balance (Aird, 2008; Carmeliet, 2000; Cines et al, 1998; Rafii et al, 2016). It helps to maintain an anti-thrombotic and anti-inflammatory state of the microvascular bed, and control the tone and proliferative state of the underlying vascular smooth muscle cells (Aird, 2008; Cines et al, 1998; Owens et al, 2004). Endothelial dysfunction figures prominently in the etiologies of many diseases such as atherosclerosis, the pathological process underlying the major cardiovascular diseases (myocardial infarction, stroke, coronary artery disease, and peripheral artery disease) (Hansson, 2005; Libby et al, 2002; Ross, 1999), sepsis, acute respiratory distress syndrome (Aird, 2003; Lee and Slutsky, 2010; Matthay et al, 2012), and COVID-19 respiratory distress (Ackermann et al, 2020; Marini and Gattinoni, 2020; Varga et al, 2020)
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