(220) Exosomes Delivering CRISPRi Technology Ameliorate Erectile Dysfunction by Targeting PDE5 and JAK2 in Diabetic ED Rats

Introduction 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 could inhibit the expression of target genes without changing the genome sequence. As a potential target of diabetic erectile dysfunction (DMED), JAK2 has received increasing attention. Objective This study planned to combine exosomes and CRISPRi technology to achieve the regulation of PDE5 and JAK2, and explore its therapeutic effect and potential mechanism on DMED rats. Methods 1. 293T cells overexpressing CD9-HuR were constructed. The above 293T cells were transfected with lentivirus containing dCas9-ARE sequence (ARE: 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 high glucose (30 mmol/L) environment. The changes in cellular function and related molecules in each group were detected by methods such as WB and immunofluorescence. 3. The above engineered exosomes were injected into the corpus cavernosum of DMED rats. The erectile function was evaluated by electrical stimulation, and the corpus cavernosum tissue was collected for subsequent testing. Results 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. 3. Compared with normal rats, the erectile function of DEMD rats was severely impaired, PDE5 levels and JAK2 phosphorylation in the corpus cavernosum increased, the levels of tissular oxidative stress, apoptosis and fibrosis increased, and the dysfunction of dilatation and contraction in blood vessels occurred. Exosome delivering CRISPRi technology downregulates PDE5 levels and JAK2 phosphorylation, improving erectile function and alleviating these pathological changes. Conclusions This project confirmed that PDE5 and JAK2 were involved in the pathogenesis of DMED. Exosomes delivering CRISPRi technology could play a therapeutic role in DMED rats by down-regulating PDE5 and JAK2. This discovery provides a new idea for exosomes-mediated gene therapy, and provides a new method and theoretical basis for the safe and efficient treatment of DMED. Disclosure No