Diabetes mellitus (DM) is a major risk factor for the development of critical limb ischemia (CLI), a condition whereby the vessels supplying oxygen and nutrient to the lower limbs get blocked with an atherosclerotic plaque. Patients suffering from CLI have a low standard therapy success rate and often require amputation of the limb with a mortality rate of >60% over 5 years (Spreen et al 2016). Endothelial colony-forming cells (ECFCs) are progenitor endothelial cells that produce key angiogenic factors and can form new vessels. ECFCs are a potential therapeutic for patients suffering from DM-CLI as they can facilitate reperfusion of the ischemic limb (Kang et al 2017). However, while results in vitro and in vivo with ECFCs are promising, to date there is no clinical trial using ECFCs. Current ECFCs produce nitric oxide to a lesser extent. By genetically modifying the ECFCs to produce endothelial nitric oxide synthase (eNOS), an enzyme that makes nitric oxide (NO, a potent vasodilator), we can improve the function of these cells and the revascularisation of the ischaemic limb, thereby improving the therapeutic potential of ECFCs for patients with DM-CLI.
In this project, we will transfect umbilical cord (UC) derived ECFCs with a novel safe harbor eNOS plasmid which integrates at specific sites on the genome. We will compare the transfection efficiency of electroporation and lipofection using a GFP plasmid and fluorescence imaging. We will confirm the expression of eNOS using qPCR and assess eNOS function using the Griess assay versus unmodified cells.
This work will establish a novel modified cell therapy, with no random integration, which will promote the revascularisation of the ischaemic limb. This will provide a new therapeutic option for patients with CLI or DM-CLI. Additionally, this therapy can also be trialed for other ischaemic conditions such as coronary artery disease.
