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Optimal Dosage for Vascular Regeneration Gene Therapy Utilizing GLP-Grade E-Selectin/AAV2
*Francesca A. Voza, *Nga T. Le, *Yulexi Ortiz, *Antoine Ribieras, *Carlos T. Huerta, *Yan Li, *Lucy Osafo, *Zhao-Jun Liu, Omaida C. Velazquez
Surgery, University of Miami/ Jackson Health System, Miami, FL

Objective: In the United States, a need for innovative vascular regeneration therapies has prompted the exploration of gene therapy to alleviate the substantial risk of major limb amputation in millions of patients with chronic limb-threatening ischemia (CLTI). Utilization of adeno-associated virus (AAV) holds promising therapeutic potential. Therefore, determining the optimal dosage for effective gene delivery is crucial to balance therapeutic efficacy with potential adverse effects. After showing the pro-angiogenic effects of a research-grade E-Selectin/AAV gene therapy in a gangrene mouse model, we have conducted a dosage study using GLP grade codon-optimized and de novo-synthesized E-selectin/AAV2 with the aim to identify the optimal dosage and determine the necessary scale-up for the design of subsequent clinical trials.

Methods: Proprietary human and murine E-selectin AAV vectors were produced under GLP conditions for pre-clinical vascular regeneration gene therapy (VRGT). Transduction efficiency of this codon-optimized and de novo synthesized AAV was evaluated by in vitro cell transduction assays. For our in vivo studies, three cohorts of FVB/NJ mice (9-12 weeks old, N=6/group) were first injected intramuscular with E-selectin/AAV2 in escalating log doses ranging from 2x10^9 VG to 2x10^11 VG, with control groups receiving GFP/AAV2 injections. Hindlimb ischemia and gangrene were then induced by left femoral artery/vein ligation/excision (FAVLE) along with increased oxidative stress using nitric oxide synthase inhibitor, L-NAME. Recovery of limb perfusion and function were weekly assessed postoperatively by Laser Doppler imaging (LDI), Faber hindlimb ischemia scores, treadmill exhaustion running distance up to POD 21. Limb/foot neovascularization was evaluated by confocal microscopy after whole-body Dil perfusion.

Results: We demonstrated that VRGT effectively transduced human cells in vitro and displayed high transcript fold post injection in ischemic limb tissues in vivo. Mice treated with a high dose (2x10^11 VG) of E-selectin/AAV2 demonstrated less severe limb ischemia, and significantly improved perfusion indices compared to control (p=0.0019, N=6/group) and lower doses groups (p=0.0006 and p=0.0082, N=6/group). A significant increase in neovascularization was also observed in the high dose group compared to GFP control (0.35 ±0.20 to 0.84±0.26; p=0.0080, N=5/group). Running stamina significantly improved with VRGT at 2x10^11 VG dosage compared to the GFP control (p=0.0047), not seen with other dosages, establishing a clear dose-dependent response.

Conclusion: VRGT shows promises in enhancing the recovery of ischemic hindlimb perfusion and function, with the optimal dose identified as 2x10^11 VG. This pre-clinical study sets the stage for further studies using the same AAV construct manufactured under cGMP conditions, and subsequent Investigational New Drug (IND) process leading to initiation of a first-in-man clinical pilot study.

Dose Effect of Vascular Regeneration Gene Therapy E-Sel/AAV2
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