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Genome Editing of Pik3cd Impedes Abnormal Retinal Angiogenesis
Wenyi Wu1,2,3, Gaoen Ma4, Hui Qi5
1Department of Ophthalmology, Hunan Key Laboratory of Ophthalmology, National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, China.
Abstract:
Abnormal angiogenesis is associated with myriad human diseases, including proliferative diabetic retinopathy (PDR). Signaling transduction through phosphoinositide 3-kinases (PI3Ks) plays a critical role in angiogenesis. Herein, we showed that p110δ, the catalytic subunit of PI3Kδ, was highly expressed in pathological retinal vascular endothelial cells (ECs) in a mouse model of oxygen-induced retinopathy (OIR) and in fibrovascular membranes from patients with PDR. To explore novel intervention with PI3Kδ expression, we developed a recombinant dual adeno-associated viral (rAAV) system for delivering CRISPR/Cas9 in which Streptococcus pyogenes (Sp) Cas9 expression was driven by an endothelial specific promoter of the intercellular adhesion molecule 2 (pICAM2) to edit genomic Pik3cd, the gene encoding p110δ. We then demonstrated that infection of cultured mouse vascular ECs with the dual rAAV1s of rAAV1-pICAM2-SpCas9 and rAAV1-SpGuide targeting genomic Pik3cd resulted in 80% DNA insertion/deletion in the locus of genomic Pik3cd and 70% depletion of p110δ expression. Furthermore, we showed that in the mouse model of OIR editing retinal Pik3cd with the dual rAAV1s resulted in not only a significant decrease in p110δ expression, and Akt activation, but also a dramatic reduction in pathological retinal angiogenesis. These findings reveal that Pik3cd editing is a novel approach to treating abnormal retinal angiogenesis.
Insights
Gene editing of PI3Kδ (phosphoinositide 3-kinase delta) effectively reduced pathological retinal angiogenesis in a mouse model. This novel approach targets abnormal blood vessel growth implicated in diseases like PDR.
Area of Science:
- Ophthalmology
- Molecular Biology
- Genetics
Background:
- Abnormal angiogenesis, or new blood vessel formation, is a hallmark of diseases like proliferative diabetic retinopathy (PDR).
- Signaling pathways involving phosphoinositide 3-kinases (PI3Ks) are crucial regulators of angiogenesis.
- The p110δ catalytic subunit of PI3Kδ is upregulated in pathological retinal vessels.
Purpose of the Study:
- To investigate the therapeutic potential of targeting PI3Kδ for abnormal retinal angiogenesis.
- To develop and validate a CRISPR/Cas9 gene editing system to reduce PI3Kδ expression in endothelial cells.
Main Methods:
- Development of a dual recombinant adeno-associated viral (rAAV) system for delivering CRISPR/Cas9.
- Endothelial-specific expression of Streptococcus pyogenes (Sp) Cas9 using the intercellular adhesion molecule 2 promoter (pICAM2).
- Targeted genomic editing of Pik3cd, the gene encoding p110δ, in cultured endothelial cells and a mouse model of oxygen-induced retinopathy (OIR).
Main Results:
- The rAAV system achieved 80% DNA insertion/deletion in the Pik3cd locus and 70% depletion of p110δ expression in vitro.
- In the OIR mouse model, Pik3cd editing significantly decreased p110δ expression and Akt activation.
- CRISPR/Cas9-mediated Pik3cd editing markedly reduced pathological retinal angiogenesis in OIR mice.
Conclusions:
- Genomic editing of Pik3cd represents a novel strategy for treating abnormal retinal angiogenesis.
- Targeting PI3Kδ pathway via gene editing offers a potential therapeutic avenue for PDR and related vascular diseases.
