Recovering and standardizing of the original genotherapeutic construction for stimulation of nervous tissue regeneration

Traumatic nerve injury is one of the most common cause of permanent disability. The problem of nerve regeneration is based on the slow growth of nerve fibers and short-term production of neutrophic factors (NGF, BDNF, NT-3, GDNF, etc.), which stimulate the survival of damaged neurons and growth of nerve fibers. In most cases, the intrinsic regenerative potential is insufficient to restore innervation, which requires the replacement therapy with neurotrophins.

One of the most promising therapeutic approaches for regenerative medicine is gene therapy that allows to prolong the local production of neurotrophic factors. Previously, we created a bicistronic gene therapy construct pNCure (a plasmid for the treatment of nerves), based on the modified pVax1 plasmid construct approved by the FDA (U.S. Food and Drug Administration), and encoding the cDNA sequences of brain-derived neurotrophic factor (BDNF) and urokinase-type plasminogen activator (uPA). It revealed a prominent therapeutic activity in a model of traumatic nerve injury in mice.

Gene therapy products is a particular class of drugs that need particular approaches to production, purification and standartization. There is practically no information on the purification and standardization of gene therapy drugs in Russian, and this is the topic of our manuscript. Here, we have elucidated the issues of plasmid production in E. coli cells with subsequent multistage purification, as well as the issues of evaluation the identity and quantity of the plasmid (using PCR and UV spectrometry methods), and determination of pH and quantity of related and secondary impurities within the substance. The results of the study have demonstrated that the proposed approach to plasmid production, purification and standartization allows to obtain the desired high-quality gene therapy drug that meets all the requirements of the XIV State Pharmacopia.