CRISPR/Cas9 Delivery Mediated with Hydroxyl-Rich Nanosystems for Gene Editing in Aorta

Abstract

A CRISPR/Cas9 system has emerged as a powerful tool for gene editing to treat genetic mutation related diseases. Due to the complete endothelial barrier, effective delivery of the CRISPR/Cas9 system to vasculatures remains a challenge for in vivo gene editing of genetic vascular diseases especially in aorta. Herein, it is reported that CHO-PGEA (cholesterol (CHO)-terminated ethanolamine-aminated poly(glycidyl methacrylate)) with rich hydroxyl groups can deliver a plasmid based pCas9-sgFbn1 system for the knockout of exon 10 in Fbn1 gene. This is the first report of a polycation-mediated CRISPR/Cas9 system for gene editing in aorta of adult mice. CHO-PGEA/pCas9-sgFbn1 nanosystems can effectively contribute to the knockout of exon 10 in Fbn1 in vascular smooth muscle cells in vitro, which leads to the change of the phosphorylation of Smad2/3 and the increased expression of two downstream signals of Fbn1: Mmp-2 and Ctgf. For in vivo application, the aortic enrichment of CHO-PGEA/Cas9-sgFbn1 is achieved by administering a pressor dose of angiotensin II (Ang II). The effects of the pCas9-sgFbn1 system targeting Fbn1 demonstrate an increase in the expression of Mmp-2 and Ctgf in aorta. Thus, the combination of CHO-PGEA/pCas9-sgFbn1 nanosystems with Ang II infusion can provide the possibility for in vivo gene editing in aorta.

Keywords: CRISPR‐associated nuclease 9 delivery; aorta disease; cationic carriers; genome editing; hydroxyl‐rich.

Figure 1

Schematic illustration of the formation of CHO‐PGEA/pCas9 nanoparticles and the angiotensin II (Ang II)‐assisted in vivo delivery for efficient gene editing in adult mouse aorta.

Figure 2

a) Schematic diagram of pCas9‐sgFbn1 plasmid. b) Agarose gel electrophoresis. c) Particle sizes and d) zeta potentials of polycation/pCas9‐sgFbn1 complexes at various N/P ratios. e) Morphologies of CHO‐PGEA/pCas9‐sgFbn1 nanoparticles at N/P = 15 measured by atomic force microscope. f) Protein assay of polycation/pCas9‐sgFbn1 complexes (at their respective N/P ratio) treated with excess BSA. g) Hemolysis ratio of red blood cells (RBCs) treated with different polycaiton/pCas9‐sgFbn1 complexes at the concentration of 0.1 and 1 mg mL⁻¹, where deionized water and PBS were used as positive and negative controls. h) Morphologies of RBCs treated with PBS, PEI/pCas9‐sgFbn1, and CHO‐PGEA/pCas9‐sgFbn1 (*P < 0.05; data are from three independent experiments).

Figure 3

a) Fluorescence images of GFP expression mediated by CHO‐PGEA at different N/P ratios and PEI (at the optimal N/P ratio of 10) in mouse vascular smooth muscle cells (VSMCs). b) Flow cytometry analysis for cellular uptake efficiency of VSMCs incubated with CHO‐PGEA/pCas9‐sgFbn1 and PEI/pCas9‐sgFbn1 at their optimal N/P ratios. c) Fluorescence images of VSMCs treated with CHO‐PGEA/pCas9‐sgFbn1 at 0.5, 2, and 6 h respectively. Green: YOYO‐1‐labeled pCas9‐sgFbn1; Red: Lyso Tracker Red‐labeled the endosomes and lysosomes; Blue: DAPI‐labeled nuclei. The scale bar indicates 10 µm. ***P < 0.001.

Figure 4

a) Real‐time PCR analysis for Cas9 mRNA expression in VSMCs treated as indicated. b,c) Western blot analysis of Cas9 protein expression in VSMCs incubated with nanoparticles with an anti‐Flag antibody. d) Sanger sequencing of PCR amplicon of the targeted Fbn1 locus in CHO‐PGEA/pCas9‐sgFbn1 treated VSMCs. e) Western blot analysis of Fbn1 induced phosphorylation of Smad2/3 (p‐Smad2/3) in VSMCs after treatment with CHO‐PGEA/pCas9‐sgFbn1 or CHO‐PGEA/pCas9‐sgnull. f) The mRNA levels of Fbn1 targeted genes Mmp‐2 and Ctgf in VSMCs after the treatments with CHO‐PGEA/pCas9‐sgFbn1 and CHO‐PGEA/pCas9‐sgnull. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 5

a) Cardiovascular image with the detecting area. b) Representative images and c) relative ROI of Ang II‐mediated or saline‐mediated mouse aorta after the accumulation of pCas9‐Cy5 and polycation/pCas9‐Cy5 nanoparticles determined by Xenogen IVIS imaging system. d) Fluorescence images of Ang II‐mediated or saline‐mediated mouse aortic sections at various treatments (*P < 0.05).

Figure 6

a) Key time points of different treatments in mouse. b) Real‐time PCR analysis for in vivo Cas9 levels in adult mice aortic tissues with or without Ang II treatment followed by intravenous injection of CHO‐PGEA/pCas9‐sgFbn1. c) Relative expression of Fbn1 targeted genes Mmp‐2 and Ctgf mRNAs in mice aortic tissues. d,e) The aortic diameters were measured by H&E staining in the CHO‐PGEA/pCas9‐sgnull and CHO‐PGEA/pCas9‐sgFbn1 groups. *P < 0.05, **P < 0.01, ***P < 0.001, and ns, no significant difference.

Figure 7

a) H&E staining of liver and kidney after Ang II infusion followed by CHO‐PGEA/pCas9‐sgnull and CHO‐PGEA/pCas9‐Fbn1 injection. The scale bar indicates 50 µm. b) Plasma chemistry profile analysis of alanine transaminase (ALT), aspartate transaminase (AST), total bilirubin (TBIL), albumin (ALB), Creatinine (CREA), and urea.