Depletion of GSDMD by AAV-Mediated Crispr/SaCas9 Rescues Hyperoxia-induced Cell Death in Vitro

Abstract

Purpose : Gasdermin D (GSDMD) is a key executor of inflammasome-mediated pyroptotic cell death and inflammation. Previously we demonstrated that global knockout of the GSDMD gene in vivo prevents hyperoxia-induced retinopathy in a neonatal murine model. Here we investigated if depletion of GSDMD expression with single strand AAV9 vector-mediated Crispr/SaCas9 gene editing rescues hyperoxia-induced cell death in murine retinal ganglion cell-5 (RGC-5) and primary retinal microvascular endothelial cells (RMVECs) in vitro.

Methods : Three editing vectors (AAV9-SagRNA-SaCas9-HA) each containing a guide RNA (SagRNA) targeting GSDMD exons 2, 3 or 4 were constructed with a HA tag fused to SaCas9 C-terminal plus a control vector with a scrambled SagRNA. Each of these vectors was transduced into RGC-5 and RMVECs with the helper adenovirus 5. Immunofluorescence and Western blotting for the HA tag were used to confirm transduction. Western blot was used to examine the efficiency of depletion of GSDMD expression. To examine the effects of these vectors on preventing hyperoxia-induced cell death, transduced RGC-5 and RMVECs were incubated in room air (21% O₂) or hyperoxia (95% O₂) for 64 h. Cell survival was detected by cell counting and pyroptotic cell death was assessed by LDH assays.

Results : All GSDMD gene-editing vectors as well as the control vector were effectively transduced in RGC-5 and RMVECs, as evidenced by immunofluorescence and Western blotting. Western blots showed that GSDMD expression was drastically decreased in RGC-5 and RMVECs by the vector containing the guide RNA targeting exon 4. Moreover, DNA sequencing revealed that this vector effectively edited a GSDMD genomic mutation. We found this vector significantly attenuated hyperoxia-reduced cell survival by 40% in RGC-5 compared to controls (p<0.001). Similarly, depletion of GSDMD in RMVECs significantly increased cell numbers under hyperoxia compared to controls by 4-fold (p<0.005). Furthermore, LDH assay showed that hyperoxia-induced cell death in RMVECs transduced with this vector was significantly decreased by 3-fold compared to controls (p<0.001).

Conclusions : Depletion of GSDMD expression by AAV9-mediated Crispr/SaCa9 gene editing markedly rescued hyperoxia-induced cell death in RGC-5 and RMVECs. Thus, this strategy has potential as a novel treatment to prevent oxygen-induced retinopathy in neonatal rodent models as well as in premature infants.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.