Gene therapy is one of the promising approaches for regenerative medicine. Local and long-term expression of essential growth factors allows to achieve the desired therapeutic effect. However, some aspects of prolonged usage of genetic constructs encoding growth factors, such as toxicity, mutagenicity, genotoxicity, and ability to disseminate from the injection site and mediate ectopic expression of therapeutic proteins, are poorly investigated. These aspects of gene therapy drugs' usage became the subject of this study. To study plasmid biodistribution, toxicity, mutagenicity, and genotoxicity, we used previously described bicistronic genetic construct encoding human brain-derived neurotrophic factor (hBDNF) and human urokinase plasminogen activator (huPA) for nerve repair. Biodistribution studies were conducted in mice: a course of intramuscular plasmid injections was followed by the study of the content of the plasmid (real-time polymerase chain reaction) and recombinant proteins (enzyme-linked immunosorbent assay) in murine organs and tissues. The study of the plasmid chronic toxicity was carried out on rats with registration of their weight dynamics, neurological status, emotional state, and blood test parameters. The mutagenicity of the plasmid was studied in an in vivo DNA comet test in mice. Plasmid genotoxicity was investigated in the model of somatic recombination in Drosophila females. We have shown that plasmids can disseminate from the injection site, but do not mediate ectopic expression of growth factors upon repeated intramuscular injections. The studied plasmid also does not reveal toxic, mutagenic, or genotoxic effects. During the toxicological study on rats, we have shown that daily injections of this genetic construct, despite its ability to disseminate from the injection site, do not affect the physical, cognitive, and emotional state of experimental animals. We have demonstrated the safety of the bicistronic plasmid, encoding hBDNF and huPA, upon its repeated administration. The properties of genetic constructs strongly depend on their sequence and delivery approach, which requires conducting of their safety studies in each specific case. Impact statement Gene therapy is one of the promising approaches for regenerative medicine. Local and long-term expression of essential growth factors allows to achieve the desired therapeutic effect. However, some aspects of prolonged usage of genetic constructs encoding growth factors, such as toxicity, mutagenicity, genotoxicity, and ability to disseminate from the injection site and mediate ectopic expression of therapeutic proteins, are poorly investigated. These aspects of gene therapy became the subject of this study. To our knowledge, this is a unique study that provides a thorough safety investigation of a bicistronic plasmid after its readministration.