Projects
Name
Gene Therapy as a new therapeutic approach for Diabetic Retinopathy
University
Portugal ( PorMSIC) - Nova University of Lisbon, Lisbon
Domain
Genetics
Departement
Gene Therapy Lab CEDOC Chronic Diseases Research Center Nova Medical School/ Faculdade de Ciencias Medicas
Head
Gabriela Silva
Tutor
Rute Silva Araujo, Daniela Santos
Languages
English, Portuguese
Duration
4 weeks
Availability
Cities/Months Jan Feb Mar Apr May Jun Jul Augt Sep Oct Nov Dec
No No No No No No Yes No No No No No
Type of Research Project
- Basic science
What is the background of the project?
Later stages of diabetic retinopathy (DR) has proliferative phase in which there is neovascularization of the retina, increasing the probability of vision loss. However, aside from pathological damage, common treatments such as photocoagulation and vitrectomy, applied in advanced disease stages, can cause further damage to the visual field. Additionally, anti- vascular endothelial growth factor (VEGF) agents used to prevent and delay progression of neovascularization require frequent local administration, increasing the risk of retinal detachment, vitreous hemorrhage, and cataract formation. The unsatisfactory effect of anti-VEGF therapy suggests the importance of other factors in neovascularization and DR progression. Among them, Placental Growth Factor (PlGF) was found in high levels in the vitreous and the retina of DR patients. Additionally, emerging evidence indicates that Pigment Epithelium-Derived Factor (PEDF), is a multifunctional protein that can target multiple pathways to exert neurotropic, neuroprotective, anti-angiogenic, anti-vasopermeability, anti-inflammation, anti-thrombogenic, and anti-oxidative effects against DR. Herein, restoring PEDF levels can constitute an interesting therapeutic strategy, but requires a therapy that avoids repeated injections and reaches sustained levels of PEDF. These requirements are met by gene therapy, recently shown effective for long-term treatment of several retinal pathologies.
What is the aim of the project?
The aim of this project is to evaluate PEDF, PlGF and VEGF expression in vitrectomy byproducts of type 1 and type 2 diabetes (T1DM and T2DM) patients to correlate its expression with the severity and progression of the disease. It will also evaluate the effect of gene therapy for the long-term expression of PEDF. Additionally, it will be evaluating the impact of a dual gene therapy approach promoting PEDF overexpression and, simultaneously, PlGF suppression in order to establish its therapeutic potential in DR.
What techniques and methods are used?
The impact of the knockdown of PlGF gene expression in human retinal pigment epithelium (RPE) cells will be evaluated and compared with simultaneous PlGF suppression and PEDF overexpression in D407 cells. It will be studying the effect on cell proliferation, apoptosis and angiogenic potential of RPE cells under both conditions. To perform the knockdown of PlGF gene expression we will use a small interfering ribonucleic acid (siRNA) corresponding to PlGF messenger RNA (mRNA) since previous studies have shown the efficiency and potential of siRNA in gene silencing. For PEDF overexpression, a self-replicating episomal vector widely studied in our lab, will be used. The transfection efficiency of siRNA it will be evaluated by flow-cytometry analysis with a scrambled siRNA (scRNA) used as control. To determine if the suppression of PlGF gene and overexpression of PEDF have no deleterious effect on RPE cell survival, we will assay cell proliferation and death. The gene and protein levels of PlGF, PEDF and VEGF in the culture medium of treated RPE cells will be examined by real-time PCR (Polymerase Chain Reaction) and ELISA (Enzyme-Linked Immunosorbent Assay), respectively. To study the angiogenic activity of RPE cells-derived medium after treatment with a dual gene therapy, a tube formation assay using umbilical vein endothelial cells (HUVECS) will be employed.
What is the role of the student?
- The student will observe the practical experiments but will be highly involved in the analysis of the results
What are the tasks expected to be accomplished by the student?
All work will be done, firstly, with supervision in order to give the student enough time to understand the procedures and learn from observation. But after learning the techniques, hopefully the student will easily perform some experiments on his/her own. If so, the student will be able to perform various different techniques, such as flow-cytometry, real-time PCR and ELISA.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
Yes
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student will prepare a presentation
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Responsability, interest and basic lab/molecular biology knowledge.
Are there any legal limitations in the student’s involvement
No
Hours
8
Type of students accepted
This project accepts:
- Medical students
- Graduated students (less than 6 months)
- Pre-Medical students from the American-British system
- Students in biomedical fields
Articles
- Calado; SM; Oliveira; AV; Machado; S; Haase R; Silva; GA. Sustained Gene Expression in the Retina by Improved Episomal Vectors. Tissue Eng. Part A 20; 2692–2698 (2014).
- Calado; S.M; Oliveira; AV; Machado; S; Haase R; Silva; GA pEPito-driven PEDF expression ameliorates Diabetic Retinopathy hallmarks. Hum. Gene Ther. Methods (2016)