Abstract
Abstract only Introduction: Chronic Limb Threatening Ischemia (CLTI) represents a major public health burden with with an estimated 15 to 20% CLTI patients undergoing major limb amputation facing a 50% mortality risk at 5 years post amputation. We previously demonstrated the pro-angiogenic actions of a research-grade E-Selectin/AAV gene therapy in a hindlimb gangrene murine model. In this study, a dosage study utilizing GLP grade codon-optimized and DeNovo-synthesized E-selectin/AAV2 was conducted dosage optimization and determination of the necessary scale-up for subsequent clinical trials. Methods: codon-optimized and de novo synthesized human and murine E-selectin AAV vectors under GLP conditions was evaluated in vitro by cell transduction assays. E-selectin/AAV2 i.m. injection in three cohorts of FVB/NJ mice (9-12 weeks old, N=6/group) with GFP/AAV2 (9-12 weeks old, N=6/group) groups, was performed in vivo in escalating log doses ranging from 2x10^9 VG to 2x10^11 VG. Hindlimb ischemia and gangrene were subsequently induced through left femoral artery/vein ligation/excision (FAVLE). Limb perfusion recovery and function was assessed postoperatively by Laser Doppler Imaging (LDI), Faber hindlimb ischemia scores, treadmill exhaustion test, whole-body Dil perfusion Results: Treatment with high dose (2x10^11 VG) of E-selectin/AAV2 significantly improve perfusion indices compared to control groups (p=.0019, N=6/group) and lower doses (p=.0006 and p=.0082, N=6/group) with decreased Faber’s scores. A significant increase in neovascularization was also observed compared to GFP control (0.41 ±0.14 to 0.95±0.18; p=.0080, N=5/group) and lower doses (p=.0026 and p=.009, N=5/group). Running stamina was also significantly improve by VRGT with a 2x10^11 VG dosage treatment compared to GFP (p=0.0047) control and lower doses, establishing a clear dose-dependent response. Conclusion: VGRT improves recovery of ischemic hindlimb perfusion and function at an optimal dose of 2x10 11 VG. This pre-clinical study paves the way towards further studies using the same AAV construct manufactured under cGMP conditions and subsequent Investigational New Drug (IND) process for clinical pilot studies and disease burden relief.