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Adeno Associated Virus (AAV)

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Adeno Associated Virus (AAV) Vector System

AAV Vector System
AAV Rep-Cap Plasmids
AAV Expression Vector

a) Introduction of rAAV vector system

Though wild-type AAV is not associated with human disease, it is naturally defective and requiring helper adenovirus or herpes simplex virus (HSV) coinfection for AAV replication, so recombinant AAV (rAAV) has been an attractive vector for gene therapy. In 1984, rAAV was used for the first time by replacing the viral genome with a transgene, which was then transfected into cells that were infected with adenovirus for production of rAAV.

Traditionally, rAAV vectors used in clinical trials were prepared with a plasmid containing the therapeutic gene flanked by AAV-inverted terminal repeats (ITRs), co-transfected with AAV packaging plasmid AAV-RC (AAV replication and AAV capsid) and pHelper (AAV helper plasmid). The adenovirus helper factors, such as E1A, E1B, E2A, E4ORF6 and VA RNAs, would be provided by the third helper plasmid. Given that HEK293 cells, a commonly used AAV production cells, already contain the E1A/E1b gene, only E2A, E4ORF6 and VA RNAs need to be provided by the helper plasmid.

b) Helper-free rAAV system

The current method of the rAAV production is based on the adenovirus-free transient transfection of all elements that are required for AAV production in host cells, such as HEK293 cells. It involves the co-transfection of 3 plasmids into AAV production cells as shown in Figure 3.

1. AAV-GOI: an AAV ITR-containing plasmid carrying the gene of interest (GOI);

2. AAV-RC: an AAV serotype plasmid that carries Rep and Capsid genes;

3. pHelper: an AAV helper plasmid that provides the helper genes isolated from adenovirus.

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