AAV Production and library
AAV Capsid Selection
AAV vectors are powerful tools for gene delivery and gene therapy. Hundreds of identified natural AAV serotypes and increasing number of engineered variants allow us to find better capsids for each cell type and tissue, which is vital for effective gene therapies. However, there has been no good method to achieve that. Currently, the common way to compare AAV variants is to test them side-by-side individually, whereas it is difficult to generate, control and handle hundreds of AAV vectors in every lab or company while limited candidates decrease the chance to find better AAV variants.
To solve this problem, AAVnerGene developed two methods (qPCR-based and NGS-based), which allow us to fastly and semi-high-throughputly compare the AAV variants both in vivo and in vitro.
In the qPCR based methods, each capsid variant packages a DNA barcoded genome carrying a reporter gene(such as EGFP). The unique barcode is located between coding sequence and Poly-A signal. A specific primer pair targeting the unique barcod is used to identify the transduction efficiency of each AAV vector. The capsid/barcoded-transgene pairs are transfected and AAV vectors are purified independently. After titration, equal amount of each AAV vector is pooled together and generate the final pool of AAV vectors. The relative transduction activity of each AAV vector can be obtained by comparing the abundance of barcoded-RNAs in target cells to the barcoded-DNAs of AAV pools before infection by using barcode-specific primer pairs.
In the NGS based methods, each capsid variant packages a DNA barcoded genome carrying a reporter gene(such us EGFP). The unique barcode is located between coding sequence and Poly-A signal, which is the same with the qPCR based methods. The capsid/barcoded-transgene pairs are transfected independently and pooled together during the purification. The relative transduction activity of each AAV vector can be obtained by comparing the abundance of barcoded-RNAs in target cells to the barcoded-DNAs of AAV pools before infection using NGS.
Difference between qPCR-based and NGS based methods