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Biodistribution and Safety Studies of a Bicistronic Plasmid for Nerve Repair
Tissue Engineering, Part C: Methods ( IF 2.7 ) Pub Date : 2021-06-16 , DOI: 10.1089/ten.tec.2021.0033
Maxim Karagyaur 1, 2 , Alexandra Rostovtseva 2 , Stalik Dzhauari 2 , Eugene Kozlov 3 , Lyubov Lebedeva 3 , Polina Klimovich 2 , Vadim Balabanyan 2 , Ekaterina Semina 2, 4 , Veronika Sysoeva 2 , Yulii Shidlovskii 3, 5 , Vladimir Popov 2 , Dmitry Stambolsky 6
Affiliation  

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.

中文翻译:

用于神经修复的双顺反子质粒的生物分布和安全性研究

基因治疗是再生医学有前景的方法之一。必需生长因子的局部和长期表达可以实现所需的治疗效果。然而,长期使用编码生长因子的遗传构建体的某些方面,例如毒性、致突变性、基因毒性以及从注射部位传播和介导治疗性蛋白质的异位表达的能力,研究得很少。基因治疗药物使用的这些方面成为本研究的主题。为了研究质粒的​​生物分布、毒性、致突变性和基因毒性,我们使用先前描述的双顺反子基因构建体编码人脑源性神经营养因子 (hBDNF) 和人尿激酶纤溶酶原激活剂 (huPA) 进行神经修复。在小鼠中进行了生物分布研究:肌肉注射质粒后,研究了小鼠器官和组织中质粒(实时聚合酶链反应)和重组蛋白(酶联免疫吸附试验)的含量。对大鼠进行了质粒慢性毒性研究,记录了大鼠的体重动态、神经状态、情绪状态和血液测试参数。质粒的致突变性在体内小鼠 DNA 彗星试验。在果蝇雌性体细胞重组模型中研究了质粒的遗传毒性。我们已经表明质粒可以从注射部位传播,但在重复肌肉注射时不会介导生长因子的异位表达。研究的质粒也没有揭示毒性、诱变或基因毒性作用。在对大鼠的毒理学研究中,我们已经表明,尽管这种基因构建体能够从注射部位传播,但每天注射它不会影响实验动物的身体、认知和情绪状态。我们已经证明了编码 hBDNF 和 huPA 的双顺反子质粒在重复给药后的安全性。遗传构建体的特性在很大程度上取决于它们的序列和传递方法,
更新日期:2021-06-24
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