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

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.
Competing Interests: Z.J.L. and O.C.V. declare the following potential conflicts of interest with respect to the research, authorship, and/or presentation and/or publication of some aspects of this work: the E-Selectin gene modification technologies were developed in our research laboratory and patented/licensed by the University of Miami. O.C.V. and Z.J.L. are co-inventors of this technology. This technology is fully owned by University of Miami and is Licensed with exclusivity to Ambulero Inc . This technology is currently under preclinical development by Ambulero Inc., a new incubator company spin out from the University of Miami that focuses on developing new vascular treatments for ischemic tissue conditions, wound healing, and limb salvage. Co-authors, Z.J.L. and O.C.V., serve as consultants and chief scientific and medical advisory officers to Ambulero Inc. ; are co-inventors of the technologies; and are minority shareholders in Ambulero Inc. Co-authors, Z.J.L. and O.C.V. are also funded for this work by the NIH/NHLBI and Philanthropy (Eloise & David Kimmelman Foundation) in ongoing preclinical investigations of these technologies. The remaining authors report no conflicts of interest.
(Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)