Developing a species-specific genetic agent for treatment of skin defects in dogs

E.Yu. Zakirova a*, A.M. Aimaletdinov a**, N.M. Alexandrova a***, I.M. Ganiev a****, S.A. Sofronova a*****, A.N. Valeeva b******, E.E. Garanina a*******, A.A. Rizvanov a********

aKazan Federal University, Kazan, 420008 Russia

bKazan State Academy of Veterinary Medicine, Kazan, 420074 Russia

E-mail: *lenahamzina@yandex.ru, **allekss1982@mail.ru, ***natalya5566@yandex.ru, ****ilnurgm-vgora@mail.ru, *****svetaaleta@mail.ru, ******anastasya.74@mail.ru, *******kathryn.cherenkova@gmail.com, ********rizvanov@gmail.com

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DOI: 10.26907/2542-064X.2020.3.361-380

For citation: Zakirova E.Yu., Aimaletdinov A.M., Alexandrova N.M., Ganiev I.M.,  Sofro­nova S.A., Valeeva A.N., Garanina E.E., Rizvanov A.A. Developing a species-specific genetic agent for treatment of skin defects in dogs. Uchenye Zapiski Kazanskogo Universiteta. Seriya Estestvennye Nauki, 2020, vol. 162, no. 3, pp. 361–380. doi: 10.26907/2542-064X.2020.3.361-380. (In Russian)

Received April 17, 2020

Abstract

Gene therapy is important in veterinary medicine due to the current need for more species-specific drugs, because they have proven to prevent an allergic response to heterologous proteins and other problems with the recipient immunity. In this study, we developed species-specific genetically engineered plasmid constructs based on the pBUDK – clVECF164 – clFGF2 plasmid DNA, all encoding genes of the canine vascular endothelial and fibroblast growth factors, which can be used to treat skin, muscle, and ligament injuries in dogs. In vitro studies of these constructs demonstrated that they induce angiogenesis in mesenchymal stem cells. In vivo studies of the plasmid DNA revealed stimulation of skin regeneration in rats and dogs without affecting the general state of animals. No complications were observed after the subcutaneous injection of the plasmid DNA. The results obtained offer a tremendous potential for further advance in veterinary medicine.

Keywords: species-specific gene therapy, vascular endothelial growth factor, fibroblast growth factor, dog, skin damage

Acknowledgments. The study was supported by the Kazan Federal University Strategic Academic Leadership Program.

Figure Captions

Fig. 1. a) Physical map of the pBUDK-clVEGF164-clFGF2 plasmid DNA; b) electrophoresis of the plasmid DNA in 8% agarose gel: 1 – preparation of the pBUDK-clVEGF164-clFGF2 plasmid DNA (upper band – relaxed circle, lower band – supercoil); 2 – plasmid DNA after the SacI and Mlul restriction; 3 – DNA marker (ThermoScientific Inc.) (number of base pairs in the DNA strand per band marker, on the right side of each band).

Fig. 2. Relative level of the fgf2 and vegf164 expression in the MSC of a dog after the transfection with the pBUDК-clVEGF164-clFGF2 plasmid DNA. Note: * р ≤ 0.005 compared with the intact MSC (n = 3).

Fig. 3. Histological slides of the rat skin from the control (a, b, c) and experimental (d, e, f) groups stained for CD34 (green), cell nuclei are blue (DAPI).

Fig. 4. Histological slides of the rat skin in the area of complete reepithelialization on the 28th day after the skin injury, hematoxylin and eosin staining: a) control group; b) experimental group. Note: I – epidermis; II – dermis; 1 – basal epidermal layer; 2 – papillary layer; 3 – microvasculature; 4 – erythrocyte diapedesis.

Fig. 5. Dynamics of changes in the area of skin injuries in dogs. Note: the arrow shows the day of subcutaneous injection of the pBUDK-clVEGF164-clFGF2 plasmid DNA to dog no. 1.

Fig. 6. Histological slides of the dog skin in the area of complete reepithelialization on the 70th day after the skin injury, hematoxylin and eosin staining: a) dog no. 1, b) dog no. 2. Note: I – epidermis; II – dermis; 1 – basal epidermal layer; 2 – microvasculature.

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