Exosomes are small biological vesicles with the characteristics of low immunogenicity and directional movement to target cells, and are considered to be an ideal transport carrier. CRISPR interference (CRISPRi) technology is the latest endogenous gene regulation technology developed based on the CRISPR/Cas9 system in recent years. It can inhibit the expression of target genes without changing the genome sequence. As a potential target of diabetic ED, JAK2 has received increasing attention. This study planned to combine exosomes and CRISPRi technology to achieve the regulation of PDE5 and JAK2, and explore the impact on the activity and function of the corpus cavernosum smooth muscle cells (CCSMCs) in high glucose environment.

1. 293T cells overexpressing CD9-HuR were constructed. The above 293T cells were transfected with lentivirus containing dCas9-ARE sequence (HuR protein binding sequence) and sgRNA (target JAK2 and/or PDE5 gene). The exosomes extracted from the above 293T cells were co-incubated with CCSMCs to verify the knockdown of PDE5 and JAK2 genes. 2. Then, CCSMCs co-incubated with or without engineered exosomes were cultured in medium with normal concentration of glucose (5 mmol/L) or high glucose (30 mmol/L) respectively. Control group, high glucose group, PDE5-CRISPRi group, JAK2-CRISPRi group, and PDE5+JAK2-CRISPRi group under high glucose conditions were set up. The levels of oxidative stress, apoptosis and RhoA/ROCK pathway in each group were detected by methods such as WB and immunofluorescence.

1. Compared with normal CCSMCs, the expression of PDE5 and/or JAK2 was significantly reduced in CCSMCs co-incubated with engineered exosomes. 2. Under the high glucose environment, the levels of oxidative stress, apoptosis and Ca2+ concentration in CCSMCs increased significantly, accompanied by the activation of RhoA/ROCK pathway, while the above changes were significantly reduced in PDE5 and/or JAK2 knockdown CCSMCs. What's more, the double gene knockdown group had the most significant effect.

Exosomes delivering CRISPRi technology could down-regulate the expression of PDE5 and JAK2 in CCSMCs under high glucose environment, and alleviate the increased levels of oxidative stress, apoptosis and RhoA/ROCK pathway. Our results provide new ideas and theoretical basis for CRISPRi gene therapy in diabetic ED.