Session Title: New Drug Treatment and Technology II
Session Date/Time: Sunday 20/09/2015 | 09:00-10:30
Paper Time: 09:00
First Author: : Z.Soheili IRAN, ISLAMIC REPUBLIC OF
Co Author(s): : E. Ranaei Pirmardan H. Ahmadieh S. Mowla M. Rezaei Kanavi S. Samiei N. Daftarian
PURPOSE:The eye has unique advantages as a target organ for gene therapy of both inherited and acquired ocular impairments and offers a valuable model system for gene delivery. The most widely used vectors for ocular gene delivery are based on adeno-associated virus (AAV), because they elicit minimal immune responses and mediate long-term expression in a variety of non-dividing retinal cell types. The purpose of this work was to establish an efficient method for in vivo retinal gene delivery, using of AAV helper-free system. This was the starting point for our subsequent gene expression studies.
The purpose of this work was to establish an efficient method for in vivo retinal gene delivery, using of AAV helper-free system.
AAV2 system (helper-free, Agilent Tech.) was subjected to cloning of enhanced green fluorescence protein (EGFP) coding sequence. EGFP expression was confirmed by fluorescent microscope following 293T cells transfection. Triple transfection of 293T cells by the main construct, gene of interest containing one, and two other supplementary vectors, pRC and pHelper, was performed using calcium-phosphate method and viral particles were purified and concentrated using heparin sulfate and amicon columns. Virus titre was determined by flow cytometry and quantitative PCR. Viruses were either subretinally and intravitreally injected to mouse retinal using 30µm glass needle. EGFP expression was evaluated by retinal flat-mount and thin sectioning of paraffin-embedded tissues.
Enzymatic digestion and sequencing confirmed vector validity. EGFP expression showed successful transcription of cloned gene and valuable amount of transfection efficiency in experiments. Well defined cytopathic effect (CPE) was sign of effective virus production by the cultures. Viral titration was about 1012 genomic particles/ml and 108 infectious particles/ml. Radiant green cells infected with AAV2-EGFP viruses represented successful infection in experiments.
Construction of the main vector, including EGFP, was validated and expression of gene of interest controlled by the CMV promoter, verified. Viral titration assay showed appreciable efficiency of virus production using HEK293T cells. After purification and concentration, the viral particles were suitable for in vivo expression. We found efficient and sustained retinal cells transduction and the resultant would be applied for in vivo genes expression analysis in mouse model organism.