Session Title: New Drug Treatment and Technology II
Session Date/Time: Sunday 20/09/2015 | 09:00-10:30
Paper Time: 09:16
First Author: : H.Ahmadieh IRAN, ISLAMIC REPUBLIC OF
Co Author(s): : S. Ghaderi S. Ahmadian Z. Soheili S. Samiee
PURPOSE:To generate pure and high titer preparations of infective rAAV introducing desired transgene into human retinal cells
To generate pure and high titer preparations of infective rAAV introducing desired transgene into human retinal cells
For high volume production of rAAV, 80%confluent and evenly distributed HEK293T cells, in five 15cm tissue culture dishes, were triple transfected with the recombinant pAAV-MCS, pAAV-RC and pHelper plasmids, according to calcium–phosphate based protocol recommended by AAV Helper Free System. Three days post transfection, rAAV producer cells were harvested and lysed in lysis buffer including 150Mm NaCl, 20mM Tris and 0.5% sodium deoxycholate. Following lysate centrifugation at 3000×g for 15 minutes, the rAAV containing supernatant was subjected to equilibrated HiTrap heparin column and allowed to flow through. To wash the column, three washing buffers including 20mM Tris and 100,200,300 mM NaCl were used orderly, and to elute the virus from the column, three elution buffers, containing 20mM Tris and 400,450,500 mM NaCl, were applied. Finally, the purified eluate was concentrated by centrifugation through Amicon filter and titer of prepared vector was determined by quantitative real time PCR, a novel genomic method for rAAV titration.
As viral production proceeded, phenotypic changes like detachment and rounding up of some rAAV producing cells from the plate in addition to color change in the medium from red to yellow as signs of virus generation, were successfully observed. Besides, a clean and concentrated stock with a final volume of 700μl and high titer of 1×107 units per microliter was achieved.
In recent years, many gene delivery approaches have focused on recombinant adeno-associated viruses, as useful vectors among vector toolkit for efficient and long-term gene transfer in a variety of tissues including retina. The exponential application of these viral vectors has increased the demand for large-scale production and efficient purification procedure of rAAV. Described method which is easily adapted to most molecular biology laboratories, can serve as a reliable strategy to produce highly purified and concentrated rAAV stocks, ideally suited for in vitro or in vivo investigations.