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, Omaida C Velazquez
{"title":"Codon-Optimized and de novo-Synthesized E-Selectin/AAV2 Dose-Response Study for Vascular Regeneration Gene Therapy.","authors":"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, Omaida C Velazquez","doi":"10.1097/SLA.0000000000006436","DOIUrl":null,"url":null,"abstract":"<p><strong>Objective: </strong>This 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.</p><p><strong>Background: </strong>Gene 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.</p><p><strong>Methods: </strong>Expression 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.</p><p><strong>Results: </strong>Codon-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.</p><p><strong>Conclusions: </strong>E-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.</p>","PeriodicalId":8017,"journal":{"name":"Annals of surgery","volume":null,"pages":null},"PeriodicalIF":7.5000,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11379359/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Annals of surgery","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1097/SLA.0000000000006436","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/7/8 0:00:00","PubModel":"Epub","JCR":"Q1","JCRName":"SURGERY","Score":null,"Total":0}
引用次数: 0
Abstract
Objective: This 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.
Background: Gene 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.
Methods: Expression 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.
Results: Codon-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.
Conclusions: E-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.
期刊介绍:
The Annals of Surgery is a renowned surgery journal, recognized globally for its extensive scholarly references. It serves as a valuable resource for the international medical community by disseminating knowledge regarding important developments in surgical science and practice. Surgeons regularly turn to the Annals of Surgery to stay updated on innovative practices and techniques. The journal also offers special editorial features such as "Advances in Surgical Technique," offering timely coverage of ongoing clinical issues. Additionally, the journal publishes monthly review articles that address the latest concerns in surgical practice.