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.
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.
Professor Rizvanov, his research team and collaborators started their work by developing a plasmid gene delivery system which would help regenerate ligament and tendon 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.
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.
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
the first successful trial of gene therapy in equine lameness. The plasmid DNA was injected directly into the torn ligament/tendon tissue. In this case the suspensory ligament branch and the superficial digital flexor tendon as these represent the most common and serious injuries in horses. Ligament healing is also made more complex by the formation of scar tissue, in these cases type III collagen formation is problematic. In normal tendons type III collagen comprises around 5% of the tissue but in scar tissue this can increase to 30% and in turn reduce elasticity and strength which means that the injury is more likely to reoccur.
Alongside an appropriate exercise rehabilitation plan the results from the trial in injured horses (Kovac et al., 2017) showed that tissue regeneration occurred. The treatment also worked quickly (2-3 months) with complete functionality restored to the animals. Throughout the follow up period of 12 months both horses returned to their pre-injury exercise levels and neither suffered from relapse. It was also noted that scar tissue did not form at the sites of injury and neither horses had negative side effects from the treatment. Conventional therapies often result in tissue scarring and therefore relapse rates upon resuming exercise can be high (up to 60% in some cases). Regenerative medicine techniques can take 4-6 months for restoration but relapse rates drop to around 20%. It should be noted that many of these studies are in high workload animals such as racehorses, therefore differing conditions and workloads must be considered.
Nearly a year later the collaborative team from Kazan Federal University, Moscow State University and University of Nottingham have published the results from a larger study containing ten injured horses (Kovac et al., 2018) and the results support the first study. Eight of the ten horses made a full clinical recovery. The ninth horse showed full tissue regeneration but sustained an unrelated injury to another limb and therefore could not be described as not lame. The tenth horse the lameness did not significantly improve as the tissue did not fully regenerate back to pre-injury levels. On average the other eight horses saw successful tissue regeneration within just 20 days. The levels of injury and lameness affected the speed of recovery. All of the horses resumed their pre-injury activities and level of fitness/competition. One horse showed a slight reaction (swelling) at the site of infection which subsided quickly and none of the other horses showed negative side effects. A combination of veterinary examinations, ultrasound and colour Doppler ultrasonography was used to assess the outcomes. In addition own opinion on pre-injury and post-treatment fitness was sought.
In addition to looking at tissue repair and lameness levels, the team saw that blood vessels developed following injection with the species-specific plasmid construct but then regressed following tissue regeneration. In addition scar tissue was not present in the treated areas.
This research represents a significant advancement in regenerative medicine and gene therapy. By understanding the mechanisms of action and the process of healing the team have created a gene therapy which is very promising. They recently wrote an editorial on gene therapy, regenerative medicine and some of the factors which must be considered when considering this as a therapeutic medicine (Rizvanov et al., 2018). In addition they have also carried out similar work in a veterinary case in a tear present in a canine cruciate ligament (Zakirova et al., 2014). Whilst this represents exciting research the authors acknowledge that more studies are required before this gene therapy can be used in medical practice. In addition training would be required for clinicians who will use these technologies. It is also hoped that as veterinary and human gene therapy treatments progress, the information can be used to improve clinical efficacy and outcomes.
The work in these studies was funded by a Program of Competitive Growth of Kazan Federal University and subsidy allocated to Kazan Federal University for the state assignment in the sphere of scientific activities 20.5175.2017/6.7, The Ministry of Education (RFMEFI59414X0003), Interdisciplinary Center for Analytical Microscopy, Pharmaceutical Research and Education Center, Kazan.