In this study, the effects of different combinations of the genes Vegf, Ang, and Gdnf injected both using direct virus-mediated injection (adenovirus, Ad5) and umbilical cord blood mononuclear cells (UCBCs) on the processes of stimulation of post-ischemic innervation, angiogenesis, and regeneration in skeletal muscle were investigated in a rat hindlimb chronic ischemia model. It was shown that more pronounced stimulation of angiogenesis and restoration of post-ischemic innervation were achieved both in the early (28 days post-ischemia, dpi) and late (42 dpi) terms of the experiment in the calf muscle when UCBCs delivered the combination of Ad5-Vegf and Ad5-Ang compared to the direct injection of the same vector combination into the area of ischemia. At the same time, the inclusion of Ad5-Gdnf in the combination of Ad5-Vegf and Ad5-Ang directly injected or administered by UCBCs provided a significant increase in the number of centronuclear muscle fibers, indicating stimulation of post-ischemic reparative myogenesis. This study allowed us to determine the most effective gene combinations for angiogenesis and neurogenesis, which, in the future, may serve as the basis for the development of gene and gene cell products for the treatment of chronic lower limb ischemia.

Chronic lower limb ischemia is a debilitating condition, particularly prevalent among elderly patients and individuals ineligible for revascularization procedures. Gene therapy aimed at promoting therapeutic angiogenesis presents a promising alternative treatment strategy. Objectives: This study evaluated the preclinical safety of a gene therapy drug composed of the plasmids pBudK-coVEGF-coANG and pBudK-coGDNF in laboratory animals. Safety assessment followed a single intramuscular injection at a dose 30 times higher than the proposed therapeutic level. Methods: Acute toxicity was monitored over a 24-h period. Genotoxicity was assessed using the micronucleus test at doses of 200, 1000, and 5000 μg/kg. Bone marrow cytology was analyzed to detect hematopoietic toxicity. Delayed toxicity was evaluated over a two-week recovery period. Results: No signs of acute toxicity were observed, even at the highest dose. The micronucleus test revealed no genotoxic effects, with no significant increase in micronucleated polychromatic erythrocytes compared to control groups. Bone marrow erythroblast parameters remained within normal physiological ranges. Additionally, no delayed adverse effects were detected during the recovery period. Conclusions: The gene therapy drug demonstrated a favorable preclinical safety profile, exhibiting no evidence of toxicity or genotoxicity, even at substantially elevated doses. These findings support the continued development of this therapy as a potential treatment for chronic lower limb ischemia in patients who are not candidates for surgical intervention.

Peripheral arterial disease and critical limb-threatening ischemia remain major clinical challenges, particularly in patients who are not candidates for surgical or endovascular revascularization. These limitations have stimulated extensive investigation into therapeutic angiogenesis using gene therapy approaches. This review summarizes experimental and clinical studies employing non-viral and viral gene delivery systems for the transfer of angiogenic factors, including VEGF, FGF-2, HGF, HIF-1α, and SDF-1. Particular attention is given to vector platforms, study design, patient populations, clinical endpoints, and safety outcomes reported in preclinical investigations and clinical trials. Although several studies demonstrated biological activity and favorable safety profiles, randomized trials such as RAVE and TAMARIS failed to demonstrate consistent and significant clinical efficacy. These findings emphasize the translational challenges associated with therapeutic angiogenesis and highlight the persistent gap between promising preclinical data and clinical outcomes in humans. Future progress in the field will likely depend on improved vector engineering, tissue-specific and regulated gene expression systems, optimized delivery strategies, and the integration of gene therapy with emerging regenerative technologies.