Codon-Optimized and de novo-Synthesized E-Selectin/AAV2 Dose-Response Study for Vascular Regeneration Gene Therapy.

IF 7.5 1区 医学 Q1 SURGERY
Annals of surgery Pub Date : 2024-10-01 Epub Date: 2024-07-08 DOI:10.1097/SLA.0000000000006436
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
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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.

用于血管再生基因疗法的编码优化和新合成 E-选择素/AAV2剂量反应研究
研究目的本研究的重点是使用经过密码子优化和新合成的 E-选择素/AAV2(E-Sel/AAV2)载体进行剂量反应调查,为后续临床研究的新药研究(IND)做准备:背景:基因疗法是慢性肢体缺血(CLTI)患者的一种潜在解决方案。背景:基因疗法是慢性肢体缺血(CLTI)患者的潜在解决方案,了解基因有效传递的剂量对于未来的新药临床试验至关重要:方法:对体内注射 AAV-m-E-Selectin (E-Sel/AAV2)的小鼠肢体进行体外细胞转染试验和 RT-qPCR 试验后,通过流式细胞术评估了经过密码子优化的 E-Selectin 基因的表达情况。剂量反应研究涉及三组 FVB/NJ 小鼠(n=6/组),将对数剂量递增的 E-Selectin/AAV2 分成 2×109 VG 至 2×1011 VG 的等分注射到通过左股动脉/静脉结扎/切除术和一氧化氮合酶抑制剂 L-NAME 造成的缺血肢体中。对肢体灌注、无坏疽肢体范围、肢体功能恢复和治疗性血管生成进行了评估:结果:编码优化的 E-Sel/AAV2 基因疗法在体外和体内的表达水平均优于 WT E-Sel/AAV2 基因疗法。使用高剂量(2×1011 VG)E-Sel/AAV2治疗的小鼠与低剂量对照组相比,灌注指数明显改善,法布尔评分降低,跑步耐力增强,新生血管增多,表明存在明显的剂量依赖性反应。研究的所有动物组均未发现毒性:结论:E-Sel/AAV2 血管再生基因疗法(VRGT)有望促进缺血后肢灌注和功能的恢复,本研究确定的有效剂量为 IM 注射 2×1011 VG 等分注射液。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Annals of surgery
Annals of surgery 医学-外科
CiteScore
14.40
自引率
4.40%
发文量
687
审稿时长
4 months
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.
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