| clinical trials |

| BIOSCIENCE TODAY SEPTEMBER•OCTOBER 2018 |

Gene therapy applications in medicine Over the last few decades advances in genetics and more recently gene therapy have accelerated. Not only have they been used in vitro and animal models but now the initial studies in humans and larger mammals are starting to be published. These applications present many complex ideas which must be solved before successful use. The decisions of which genes to use or target, the delivery methods, fully understanding the disorders, disease or injuries and working out the mechanisms of action of the therapy itself once trials are underway. In addition to understanding the background science the clinical trials must be carefully applied and compared to the most recent treatments available.

tissue in the horse. Their approach was design a dual expression cassette plasmid DNA (pDNA) containing equine vascular endothelial growth factor A (VEGFA164) and basic fibroblast growth factor (FGF2) sequences. These were under the control of the eukaryotic promoters EF-1alpha and CMV. VEGF is known to stimulate DNA synthesis and cell proliferation, is involved in angiogenesis and attracts endothelial progenitor cells in addition to stabilising blood vessels. VEGF also attracts macrophages, monocytes, smooth muscle cells and granulocytes which are necessary for wound healing and increases vascular permeability following wounding. In turn FGF2 is mitogenic and is also involved in angiogenesis, helps to develop connective tissue and is involved with wound healing and stimulates cell proliferation. The authors concluded that all of these factors would assist with tissue regeneration and repair in cases of torn ligaments and tendons in equine lameness.

Lameness in horses, and other animals, presents a significant problem. Not only is it a very commonly occurring problem, it also causes significant pain, is presently difficult to treat and even successful treatment can be short lived with high levels of relapse. This was what Professor Albert Rizvanov set out to investigate when starting the gene therapy programme at Kazan Federal University. The type of lameness was related to torn tendons and ligaments, which is a problem seen in many conditions in humans and animals throughout the body. In addition his work set out to investigate a type of gene therapy which could be adapted to help conditions in all parts of the body ranging from reproductive issues through to spinal problems.

The plasmid construct pBUDK-ecVEGF164-ecFGF2 was generated on the base of pBudCE4.1 in line with recommendations given by the US Food and Drug Administration (FDA), the European Medicine Agency (EMA) and the ‘Content and Review of Chemistry, Manufacturing, and Control (CMC) gene therapy documents. Their initial paper describes not only how this was designed and created but also how it was tested in vitro (Litvin et al., 2016). The recombinant plasmid was sequenced, underwent restriction analysis and gel electrophoresis of the restriction fragments. HEK293FT cells were transfected. Fluorescence immunohistochemistry and western blot analysis showed expression of VEGFA164 and FGF2.

Professor Rizvanov, his research team and collaborators started their work by developing a plasmid gene delivery system which would help regenerate ligament and tendon

Once this plasmid construct had been created the team worked to start using it in naturally occurring equine cases of tendon and ligament injuries. Their results represented

Catrin S Rutland, Albert A Rizvanov and Milomir Kovac

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VEGF IS KNOWN TO STIMULATE DNA SYNTHESIS AND CELL PROLIFERATION, IS INVOLVED IN ANGIOGENESIS AND ATTRACTS ENDOTHELIAL PROGENITOR CELLS IN ADDITION TO STABILISING BLOOD VESSELS.