• Francesca A Voza, Barry J Byrne, Yulexi Y Ortiz, Yan Li, Nga Le, Lucy Osafo, Antoine C Ribieras, Hongwei Shao, Carlos Theodore Huerta, Yuntao Wei, Gustavo Falero-Diaz, Andres Franco-Bravo, Roberta M Lassance-Soares, Roberto I Vazquez-Padron, Zhao-Jun Liu, and Omaida C Velazquez.
• DeWitt Daughtry Family Department of Surgery, University of Miami School of Medicine, Miami, FL.
• Ann. Surg. 2024 Oct 1; 280 (4): 570583570-583.

ObjectiveThis study focuses on dose-response investigation using a codon-optimized and de novo-synthesized E-Selectin/AAV2 (E-Sel/AAV2) vector in preparation for Investigational New Drug enabling of subsequent clinical studies.BackgroundGene therapy is a potential solution for patients suffering from chronic limb-threatening ischemia. Understanding the dose for effective gene delivery is crucial for future Investigational New Drug-enabling studies.MethodsExpression of the codon-optimized E-Selectin gene was assessed by flow cytometry following in vitro cell transfection assay and RT-qPCR for murine limbs injected in vivo with AAV-m-E-Selectin (E-Sel/AAV2). Dose-response studies involved 3 cohorts of FVB/NJ mice (n=6/group) with escalating log doses of E-Selectin/AAV2 injected intramuscularly in divided aliquots, ranging from 2 × 10 9 VG to 2 × 10 11 VG, into ischemic limbs created by left femoral artery/vein ligation/excision and administration of nitric oxide synthase inhibitor, L-NAME. Limb perfusion, extent of gangrene free limb, functional limb recovery, and therapeutic angiogenesis were assessed.ResultsCodon-optimized E-Sel/AAV2 gene therapy exhibits a superior expression level than WT E-Sel/AAV2 gene therapy both in vitro and in vivo. Mice treated with a high dose (2 × 10 11 VG) of E-Sel/AAV2 showed significantly improved perfusion indices, lower Faber scores, increased running stamina, and neovascularization compared with lower doses tested with control groups, indicating a distinct dose-dependent response. No toxicity was detected in any of the animal groups studied.ConclusionsE-Sel/AAV2 Vascular Regeneration Gene Therapy holds promise for enhancing the recovery of ischemic hindlimb perfusion and function, with the effective dose identified in this study as 2 × 10 11 VG aliquots injected intramuscularly.Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.

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