蓝莓视频

Lead Researcher :

Supported by the Cardiovascular Theme

Lymphatic vessels are essential for draining excess fluid from the body. Disruption of lymphatic networks leads to fluid accumulation and swelling in arms and legs, known as lymphoedema, a debilitating condition and considerable disease burden with no effective treatments. By analysing DNA of thousands of NHS patients with rare diseases, studies identified mutations in 40 genes reported to cause an inherited form of this condition, called primary lymphoedema. However, three quarters of primary lymphoedema patients still lack a genetic diagnosis. We recently discovered some patients with primary lymphoedema carrying genetic changes in ERG, an essential protein that controls the activity of genes in the endothelial cells lining both blood and lymphatic vessels. In healthy individuals, ERG plays a crucial role in promoting blood vessel growth and maintaining normal vessel function. However, ERG鈥檚 role in regulating the activity of genes in lymphatic vessels and how this may go wrong in some primary lymphoedema patients is unclear.

To study how the changes in ERG from primary lymphoedema patients causes it to function incorrectly, we used methods to recreate the faulty ERG protein so that it matches the form found in the lymphatic endothelial cells of the patients. Obtaining endothelial cells from patients and healthy donors is difficult, so we used 鈥済ene editing鈥� techniques to create faulty versions of ERG in otherwise healthy cells grown in the laboratory. We tested different methods to 鈥渆dit鈥� the ERG gene so that it matched two different faulty versions of ERG that we previously identified in primary lymphoedema patients. Gene editing of ERG was first carried out in freshly isolated human skin lymphatic endothelial cells (called HDLEC), which were purchased from a commercial supplier. We used different methods to introduce the editing components into HDLEC 鈥� comparing the use of chemical agents or electrical pulses to create holes in the cell membrane to enable the gene editing tools to enter the cell. By changing the concentration of the reagents, we were able to increase DNA editing efficiency. However, the numbers of cells carrying the faulty ERG was very low. To overcome this, we used a method of selecting individual edited cells and then growing them until they multiplied enough times to obtain enough cells for testing. Even though this improved the efficiency of edited ERG, the extended growing time of the HDLECs needed for this method exhausted the ability of the cells to maintain their lymphatic characteristics. Luckily, during the pilot project, we were able to obtain a newly created long-lived lymphatic endothelial cell line which doesn鈥檛 suffer from this problem. Ongoing experiments are now focussed on using the newly established gene editing techniques to create the faulty ERG gene in these immortal cells to study how the ERG mutations affect the endothelial cell鈥檚 ability to grow, migrate and form lymphatic vessels. This will provide novel insights into causes of primary lymphoedema so that future patients can get a genetic diagnosis and the prospect of treatment.