Adenovirus Production Protocol--Genemedi

1. Adenovirus plasmid construction
The gene of interest is cloned into one of the ITR/MCS-containing adenovirus vectors to generate pAd -GOI. The purity and RNA contaminants of viral plasmid should be taken into consideration.

2. Adenovirus packaging
The recombinant adenovirus viral plasmid pAd-GOI is co-transfected into the 293A with pAd-BHGlox(delta)E1,3, which together supply all the trans-acting factors required for adenovirus replication and packaging in the293A cells.

3. Plaque formation and cell collection
Pick an isolated viral plaque together with surrounding agarose, and transfer into 1 ml fresh medium and incubate overnight. In general, three to six plaques should be picked to compare their virus titer, then the one with the highest titer will be proceeded into subsequent experiments.

4. Virus amplification
On the next day, add virus-containing supernatant into fresh, pre-seeded 293A cells to amplify virus. Collect cells and supernatant when observing formation of plaques, and proceed into a freeze-thaw cycle 3 times before collecting all viruses. The collected virus is recognized as passage 1 (P1 virus). Then, infect fresh 293A cells with P1 virus. Perform infection-collection cycle three times till P4 virus is obtained, and expand virus production into large-scale through P4 virus infection. When formation of plaques is observed, viruses are collected for purification and concentration.

5. Adenovirus purification
The purification process of rAdV is composed of three steps: PEG8000 condensation, CsCl density gradient centrifugation and dialysis. Add 50 ml PEG8000 solution (20% PEG8000 in ultra-pure water with 2.5 M NaCl) per 100ml virus supernatant, placing on ice for 1 hour to pull-down viruses. Centrifuge the mixture, discard the supernatant and resuspend virus pellet in 10 ml CsCl solution at density of 1.10g/ml (the virus-containing CsCl solution should be pink). Then, carry out CsCl density gradient centrifugation with Beckman SW28 rotor at 26,000 rpm, 4℃ for 2 hours. Collect virus band between 1.30g/ml and 1.40g/ml layers with a syringe, transfer into dialysis bag. Put the dialysis bag containing virus in dialysis buffer, and stir at 4℃ overnight. Replace with fresh buffer once during dialysis. Collect virus from the bag, adjust volume to 500 µl with PBS, and determine the titer. Purified rAdV should be kept at 4℃ for no more than a week or at -80℃ for long time storage.

6. Adenovirus titer detection
Viral titer is determined by plaque assay of rAdV. Plate 293A cells in 60-mm dishes 24 hours ahead. When cell confluence reaches approx. 100%, add diluted virus at different concentrations and incubate at 37℃ 4 to 8 hours after infection, then cover cells with 8 ml low-melting-point agarose solution (10% FBS, 1.25% agarose). Calculate the titer of rAdV by counting number of plaques in 9-11 days of culturing.

7. Quality control of adenovirus
After adenovirus titer detection, the infection activity needs to be evaluated before animal experiment by infecting cells such as 293T, CHO to test the gene expression.

Adenovirus - Guidelines

NIH Guidelines for Adeno-Associated (AAV) Lentivirus and Adenovirus	Guidance for Gene Therapy Clinical trials-FDA
Human Gene Therapy for Hemophilia	Human Gene Therapy For Rare Diseases
Human Gene Therapy for Retinal Disorders

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