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 (IND)-enabling of subsequent clinical studies.

BACKGROUND

Gene therapy is a potential solution for patients suffering from chronic limb-threatening ischemia (CLTI). Understanding the dose for effective gene delivery is crucial for future IND-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 three cohorts of FVB/NJ mice (n=6/group) with escalating log doses of E-Selectin/AAV2 injected intramuscularly (IM) in divided aliquots, ranging from 2×109 VG to 2×1011 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 superior expression level than WT E-Sel/AAV2 gene therapy both in vitro and in vivo. Mice treated with a high dose (2×1011 VG) of E-Sel/AAV2 showed significantly improved perfusion indices, lower Faber's 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.

CONCLUSION

E-Sel/AAV2 Vascular Regeneration Gene Therapy (VRGT) holds promise for enhancing the recovery of ischemic hindlimb perfusion and function, with the effective dose identified in this study as 2×1011 VG aliquots injected IM.

Informations de copyright

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Déclaration de conflit d'intérêts

Conflict of interest statement: Barry J. Byrne, Yulexi Y. Ortiz, Yan Li, Nga Le, Lucy Osafo, Antoine C. Ribieras, , Hongwei Shao, Carlos Theodore Huerta , Yuntao Wei, Andres Franco Bravo, Gustavo Falero-Diaz1, Roberta M Lassance-Soares, Roberto I Vazquez-Padron and Francesca Voza have no commercial or financial relationships that could be construed as a potential conflict of interest. Dr. Zhao-Jun Liu (Z.J.L.) and Dr. Omaida C. Velazquez (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 pre-clinical 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, for wound healing, and for 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.