Team:Tsinghua/Project/Virus

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Latest revision as of 23:55, 17 October 2014

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.

Constructing and Characterizing the AAV Platform

An AAV platform was set up (with commercially purchased plasmids) in a BSL2 lab with special care. We included an mCherry fluorescent protein encoding gene into the AAV construct to assess transfection efficiency in HEK 293 cells. The full experiment procedures are listed as follows.

  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

A stable but relatively low transfection rate for the AAV vector was observed in most transfection attempts. In the AAV production phase, more than 80% of cells are involved, however, due to low virus concentrations and low transfection rates, we were only able to achieve 1% AAV infection rate in most of our attempts. However, these issues can be compensated by either increasing the viral dosage, or by using alternative cell lines more suitable to AAV production.

In order to confirm that AAV vectors only convey a single-copy insertion, mCherry-positive cells (indicating successful AAV infection) will be amplified via cell sorting, and then subjected to qPCR analysis to assess the relative amount of foreign genes introduced to the cellular genome.

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