Leverage of nuclease-deficient CasX for preventing pathological angiogenesis

Abstract

Gene editing with a CRISPR/Cas system is a novel potential strategy for treating human diseases. Pharmacological inhibition of phosphoinositide 3-kinase (PI3K) δ suppresses retinal angiogenesis in a mouse model of oxygen-induced retinopathy. Here we show that an innovative system of adeno-associated virus (AAV)-mediated CRISPR/nuclease-deficient (d)CasX fused with the Krueppel-associated box (KRAB) domain is leveraged to block (81.2% ± 6.5%) in vitro expression of p110δ, the catalytic subunit of PI3Kδ, encoded by Pik3cd. This CRISPR/dCasX-KRAB (4, 269 bp) system is small enough to be fit into a single AAV vector. We then document that recombinant AAV serotype (rAAV)1 efficiently transduces vascular endothelial cells from pathologic retinal vessels, which show high expression of p110δ; furthermore, we demonstrate that blockade of retinal p110δ expression by intravitreally injected rAAV1-CRISPR/dCasX-KRAB targeting the Pik3cd promoter prevents (32.1% ± 5.3%) retinal p110δ expression as well as pathological retinal angiogenesis in a mouse model of oxygen-induced retinopathy. These data establish a strong foundation for treating pathological angiogenesis by AAV-mediated CRISPR interference with p110δ expression.

Keywords: CRISPR/dCasX-KRAB; MT: RNA/DNA editing; PI3Kδ; Pik3cd; angiogenesis; oxygen-induced retinopathy; rAAV1.

Graphical abstract

Figure 1

Establishment of an AAV vector expressing CRISPR/dCasX-KRAB (A) Schematic diagram of constructing an AAV-CRISPR/dCasX-KRAB (dCasX) vector. The pAAV-U6-sgRNA/RSV-SpCas9 (V1) vector was originated from pAAV-RSV-SpCas9 (Addgene, 85450). The pAAV-U6-sgRNA/RSV-CasX_gene1 (V2) was derived from V1 by replacement of the Cas9 with CasX_gene1. The pAAV-U6-sgRNA/RSV-dCasX (V3) was originated from V2. ITR, inverted terminal repeat; U6, a promoter of polymerase III; RSV, a promoter of RSV. (B) HEK293T cells were transfected with the plasmid of pAAV-dCasX/sgRNA-lacZ. 48 h later, the transfected cells were immunostained with a primary antibody against CasX. Green signals indicate dCasX expression. Scale bar, 100 μm. (C). Lysates of transfected HEK293T cells were subjected to Western blot analysis using indicated antibodies. A representative of at least three independent experiments is shown. (D) The bar graphs are mean ± SD of three independent experiments. The data was analyzed using one-way ANOVA followed by the Tukey honest significant difference post hoc test. ∗∗p < 0.01.

Figure 2

CRISPR/dCasX-KRAB-mediated blockage of p110δ expression in vitro (A) HRECs, ARPE-19 cells and RAW264.7 were infected with rAAV1 virus carrying dCasX/sgRNA-lacZ. After 24, 48, and 72 h, the infected cells were immunostained with a primary antibody against dCasX. Green signals indicate dCasX expression. Scale bar, 20 μm. (B) Graphical schematic of a mouse Pik3cd locus. mPK5 sgRNA was originated from the Pik3cd promoter. The PAM is marked in blue. (C and D) Raw264.7 cells were infected with rAAV1-dCasX/sgRNA-lacZ and dCasX/sgRNA-mPK5, respectively. The transfected cells were subjected to western blotting analyses 48 h later (C and D). The bar graphs are mean ± SD of three independent experiments. The data was analyzed using one-way ANOVA followed by the Tukey honest significant difference post hoc test. ∗∗p < 0.01. (E) DNA samples from a ChIP assay with RAW264.7 cells, which were infected with rAAV1-dCasX/sgRNA-lacZ and dCasX/sgRNA-mPK5, respectively, were subjected to PCR and agarose gel electrophoresis analysis. Non-immune IgG served a negative control.

Figure 3

rAAV1-delivered dCasX expression in vivo (A and B) Normal P12 mice were injected intravitreally with rAAV1 carrying dCasX/sgRNA-lacZ (1μL, 1.62 × 10E13 vg/mL). After injection for 1, 3, and 5 days, frozen eyeball sections were stained with a primary antibody against dCasX. Red signals indicate dCasX expression, and green indicate Panck expression. Scale bar, 20 μm. (C and D) Whole-mount retinas from the intravitreally injected in mice were stained with IB4 (red) and antibodies against dCasX and CD11β (green) and then with a fluorescent-labeled secondary antibody. Images were obtained in the immunofluorescence confocal microscope. Scale bars, 10 or 20 μm. (E–P) Co-localization analysis of dCasX expression in vivo was performed by ImageJ. Two regions were selected for each image.

Figure 4

rAAV1 infects pathological vascular ECs with high efficiency (A) P12 mice that had been exposed to 75% oxygen for 5 days were injected intravitreally with rAAV1-dCasX/sgRNA-lacZ (1μL, 1.62 × 10E13 vg/mL). Whole-mount retinas from the P17 mice in a model of OIR were stained with IB4 (red) and a primary antibody against dCasX and then a fluorescent-labeled secondary antibody. Scale bar, 20 μm. (B–E) Co-localization analysis of dCasx expression with tufts in vivo was performed by ImageJ. Two regions were selected for each image.

Figure 5

CRISPR/dCasX-KRAB-mediated blockade of p110δ expression suppresses pathological angiogenesis in a mouse model of OIR (A) Schematic graph of the OIR model. Litters of P12 mice that had been exposed to 75% oxygen for 5 days were injected intravitreally with rAAV1 (1 μL, 1.62 × 10E13 vg/mL) carrying dCasX/sgRNA-lacZ or dCasX/sgRNA-mPK5. (B) The whole-mount retinas from the injected P17 mice were stained with IB4. Arrows indicate tufts. AIR indicates mice that were kept in room air without exposure to 70% oxygen. L, left eye of the mice; R, right eye of the mice. Scale bar, 100 μm. (C–E) Quantification of tufts and avascular areas as described previously. The bar graphs are mean ± SD (n = 6). The data were analyzed using one-way ANOVA followed by the Tukey honest significant difference post hoc test. ∗∗∗p < 0.01. NS, not significant.

Figure 6

dCasX-mediated p110δ attenuation suppresses Akt activation and VEGF production in the mouse model of OIR (A and B) Retinal mRNA and proteins from P17 mice (n = 6) subjected to qPCR (A) and western blotting analyses with indicated antibodies (B), respectively. (C) The intensity of bands. The bar graphs are mean ± SD of three independent experiments. The data were analyzed using one-way ANOVA followed by the Tukey honest significant difference (HSD) post hoc test. ∗p < 0.05, ∗∗p < 0.01. (D) Clarified vitreous (5 μL) from the P17 mice with or without experiencing OIR was subjected to ELISA analysis by following the instructions of a Quantikine Mouse VEGF ELISA Kit. The bar graphs are mean ± SD of six mice. The data were analyzed using one-way ANOVA followed by the Tukey HSD post hoc test. NS, not significant. ∗p < 0.05; ∗∗p < 0.01.