Abstract Number: Esoc2026a706 a Vnn2 Knockout Ipsc-Derived Model to Explore Its Role as a Potential Therapeutic Target in Post-Stroke Recovery

Abstract Background and aims VNN2 has emerged as a molecule potentially associated with an improved outcome after both ischemic and hemorrhagic stroke, supported by recent findings from genetic and protein-level studies. VNN2 is mainly expressed in cells involved in inflammation, including neutrophils. Its role in inflammation could be related to its pantetheinase activity (related to oxidative stress) or its potential role in neutrophil transmigration. However, although this protein has been known for over a decade, its functions remain poorly defined. Thus, this study focuses on exploring the contribution of VNN2 to the variability in stroke outcome by conducting functional experiments in cellular models. Methods VNN2 knockout isogenic iPSCs lines were generated by CRISPR/Cas9 genome editing. The generated lines were karyotyped, and their pluripotency capability was confirmed by immunostaining for pluripotency markers (SOX2, SSEA4, TRA1-81, NANOG, OCT3/4) and by differentiation into three germ layers followed by immunostaining (α-FP and SOX17 for endoderm; α-SMA and Brachyury for mesoderm; PAX6 and TUJ1 for ectoderm). Results We obtained two isogenic lines carrying heterozygous (KO+/-) VNN2 alleles and one compound heterozygous (KO-/-) line with truncating deletions and insertions, and we verified the absence of undesired off-targets. Finally, these isogenic lines were characterized by confirming their pluripotency. Conclusions We have generated several VNN2 KO isogenic iPSCs lines with the aim of differentiating them into neutrophils and investigating VNN2-related functions, including transendothelial migration, adhesion and pantetheinase activity, all of which are relevant to stroke outcome. Conflict of interest Nothing to disclose.