Project: The Virus

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The AAV Gene Therapy Platform

Gene therapy requires introduction of therapeutic DNA into a patient's somatic cells, typically achieved with a viral vector. For many years medical researchers have been struggling to optimize existing transfection systems to eliminate the risk of random insertions, carcinogenicity, and severe immune responses. Recently, the Adeno-associated virus (AAV) has matured as a gene therapy agent and has won favor for its safety efficiency. In fact, the first gene therapy drug (approved outside China) was

We chose the adeno-associated virus (AAV) as a platform to transfer therapeutic genes into human cells. AAV is known for its safety as a viral vector for the following reasons:

• A single-copy insertion is made at a specific site within an intron on human Chromosome 19. Chance of random-insertion mediated mutagenesis is extremely low.
• AAV infection does not cause severe immune responses as do other vectors such as the adenoviral vector or lentiviral vector. Therefore minimizing the risk of severe complications.

1. Add NotI cleavage sites on the both ends of mCherry gene. (the AAV vector we bought has NotI cleavage sites )
2. Insert mCherry gene into the AAV vector by restriction endonuclease NotI. Then we got the recombinant vector mCherry-AAV.
3. Transfect the mCherry-AAV into 293T cell line through calcium phosphate-based method. During this step the plasmids, pAAV-RC and pHelper, which express the shell of AAV are needed. (this kind of method that divide the virus into more than one expressing plasmids makes AAV safe)
4. Collect the virus through dry ice-ethanol bath and water bath in 37℃, and then add the virus into the 293T cell line.
5. After 48h, we can track the AAV in 293T cell lines by detecting the fluorescence of mCherry.

Figure1. 293T cell under mcherry fluorescence

Figure2. 293T cell under whitefield

Figure3. 293T cell after merge

We can see from the figure that recombinant mcherry-AAV vectors express in the 293T cell successfully, which prove that the AAV system is practicable.
However, the system is not exactly perfect. Although when 293T cells are producing virus we can detect 70%-80% positive rate, after virus infect the 293T cells, less than 1% positive rate can be observed. Another condition can’t be ignored that we use 293T as the virus-producing platform, but in fact, there are various 293 cell lines, such as 293F, 293A and etc. Therefore, 293T cell lines may not be the best cell line to produce the AAV virus.
Then we sort the 293T cells which have mcherry fluorescence, and after incubate the cell as many as enough, we can test the expression of mcherry on the mRNA level by qPCR.

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