Нажмите на эту строку чтобы перейти к Новостям сайта "Русский врач"

Перейти
на сайт
журнала
"Врач"
Перейти на сайт журнала "Медицинская сестра"
Перейти на сайт журнала "Фармация"
Перейти на сайт журнала "Молекулярная медицина"
Перейти на сайт журнала "Вопросы биологической, медицинской и фармацевтической химии"
Журнал включен в российские и международные библиотечные и реферативные базы данных

ВАК (Россия)
РИНЦ (Россия)
Эко-Вектор (Россия)

RECOVERING AND STANDARDIZING OF THE ORIGINAL GENOTHERAPEUTIC CONSTRUCTION FOR STIMULATION OF NERVOUS TISSUE REGENERATION

DOI: https://doi.org/10.29296/25877313-2023-02-05
Issue: 
2
Year: 
2023

M.N. Karagyaur
Ph.D (Biol.), Senior Research Scientist, Institute for Regenerative Medicine, Medical Research and Education Center;
Faculty of Medicine, Lomonosov Moscow State University (Moscow, Russia)
E-mail: m.karagyaur@mail.ru
A.I. Rostovtseva
Post-graduate Student, Faculty of Medicine, Lomonosov Moscow State University (Moscow, Russia)
E-mail: s.rostov94@mail.ru
P.S. Klimovich
Ph.D (Biol.), Senior Research Scientist, Faculty of Medicine, Lomonosov Moscow State University (Moscow, Russia)
E-mail: lex2050@mail.ru
V.Yu. Balabanyan
Dr.Sc. (Pharm.), Leading Research Scientist, Institute for Regenerative Medicine, Medical Research and Education Center;
Faculty of Medicine, Lomonosov Moscow State University (Moscow, Russia)
E-mail: bal.pharm@mail.ru
D.V. Stambolsky
Ph.D (Biol.), Leading Research Scientist, Medical Research and Education Center,
Lomonosov Moscow State University (Moscow, Russia)
E-mail: dstambolsky@gmail.com

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 elucidat-ed 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 sub-stance. 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.

Keywords: 
gene therapy
plasmid
brain-derived neurotrophic factor
urokinase-type plasminogen activator
microbiological production
standardization
authenticity
bacterial endotoxins

References: 
  1. Rasulić L., Savić A., Vitošević F., et al. Iatrogenic peripheral nerve injuries-surgical treatment and outcome: 10 Years' Experience. World Neurosurg. 2017; 103: 841–851.e6. DOI: 10.1016/j.wneu.2017.04.099.
  2. Björkman M.A., Björkman-Burtscher I.M., Mannfolk P., et al. Re-construction of sciatic nerve after traumatic injury in humans – fac-tors influencing outcome as related to neurobiological knowledge from animal. J Brachial Plex Peripher Nerve Inj. 2012; 7(1): 7. DOI: 10.1186/1749-7221-7-7.
  3. Gordon B. Neurotrophic factors and their receptors in axonal re-generation and functional recovery after peripheral nerve injury. Molecular Neurobiology. 2003; 27(3): 277–323.
  4. Frostick S.P., Yin Q., Kemp G.J. Schwann cells, neurotrophic fac-tors, and peripheral nerve regeneration. Microsurgery. 1998. 18: 397–405.
  5. Gu X., Ding F., Yang Y., et al. Construction of tissue engineered nerve grafts and their application in peripheral nerve regeneration. Prog Neurobiol. 2011; 93: 204–230.
  6. Karagyaur M., Dyikanov D., Makarevich P., et al. Non-viral trans-fer of BDNF and uPA stimulates peripheral nerve regeneration. Bi-omed Pharmacother. 2015; 74: 6370.
  7. Lykissas M.G., Batistatou A.K., Charalabopoulos K.A., et al. The role of neurotrophins in axonal growth, guidance, and regeneration. Curr Neurovasc Res. 2007; 4: 143-151.
  8. Zhang J.Y., Luo X.G., Xian C.J., et al. Endogenous BDNF is re-quired for myelination and regeneration of injured sciatic nerve in rodents. Eur. J. Neurosci. 2000; 12: 41714180.
  9. Karagyaur M., Rostovtseva A., Semina E. et al. A bicistronic plas-mid encoding brain-derived neurotrophic factor and urokinase plasminogen activatorstimulates peripheral nerve regeneration after injury. J Pharm Exp Therapeutics. 2020; 372(3): 248–255. DOI: 10.1124/jpet.119.261594.
  10. Государственная фармакопея Российской Федерации. В 4-х томах. Изд. XIV. М. 2018 (Gosudarstvennaja farmakopeja Ros-sijskoj Federacii. V 4-h tomah. Izd. XIV. M. 2018).