Generation of High-titer Recombinant Lentiviruses Carrying Nurotrophic Factor GDNF Gene and Their Transfer to Human Astrocytes

Lentiviruses are considered one of the most effective recombinant viruses for gene transfer to mammalian cells and tissues. This study comprises of two essential parts: (1) evaluation of efficiency of protein purification columns in concentration of recombinant lentiviruses, and (2) production of recombinant lentiviruses carrying GDNF coding sequences. In part (1) we co-transfected human embryonic kidney cell line HEK-293T with three lentivirus vectors called transfer (carrying either GFP or Jred), packaging and envelope vectors. After a filtration step, we applied the supernatant from transfected cells to Amicon protein columns for concentration purposes. Centrifugation removed 99% of the supernatant and left behind 500-µl-volume of solution full of virions. We thereby produced a of virus stock. Various dilutions of this stock were added to HEK-293T cells that produced up to 100% infected cells positively expressing transgenes. To examine whether the removed supernatant (overflow) has any trace of infective virus by chance, we also used dilutions of the overflow for infection and observed no sign of eGFP or Jred expression. Given the need for a high-titer virus stock for successful target cell transduction, our results indicate that our filtration method of virus concentration is able to produce high virus titer and is cost-effective and less time consuming than previous methods. In part (2), due to the importance of neurotrophic factor GDNF in differentiation and neuroprotection as well as in therapy of neurodegenerative disorders, we ligated GDNF coding sequence into the lentivirus backbone in the second phase of our study. We applied the same method outlined above to produce high-titer recombinant viruses. Following infection of human astrocytoma cells with this virus stock, we detected 3-fold increase in GDNF mRNA expression using RT-PCR. Lentiviruses carrying GDNF can therefore be generated at high titer using the column method and applied for differentiation and neuroprotection studies.