AAV Comparison and Selection Using qPCR Methods

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 provides AAV capsid selection services, which will help customers fastly and semi-high-throughputly compare the AAV variants both in vivo and in vitro.​​
 

Design of qPCR Based Capsid Selection

In order to efficiently compare the transduction efficiency of multiple AAV variants, 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. Then, the capsid/barcoded-transgene pairs were transfected into HEK 293 cells to produce AAV vectors. 

 

For less AAV variants(<24), the capsid/barcoded-transgene pairs are transfected and purified independently. After titration, equal amount of AAV vectors are pooled together and generate the final AAV vectors pool.

 
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qPCR Based Selection Protocol

 

The qPCR based selection can be used easily both in vivo and in vitro. 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. Customers also can compare the entering cell efficiency of AAV variants by comparing barcoded-DNAs in target cells to the barcoded-DNAs of AAV pools before infection.

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qPCR Primer Design

All the AAV variants used the same For primer but each one has its unique Rev primer. The Rev primers target the barcoded sequences.

 

AAV Capsids  

There are hundreds of AAV capsids published, including well known serotypes (AAV1-13), engineered ones and other native capsids isolated from human, monkey or other species. 

  • Native Serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13.

  • Y/F mutants: single-, double-, triple-, quad-, penta-, hexa-, mutation of any native serotypes.

  • Engineered AAV Capsid: AAV-PHP.B, AAV-PHP.eB, AAV-PHP.B2, AAV-PHP.B3, AAV-PHP.S, AAV-retro,  AAV-7M8, AAV-F, AAV-Vec, AAV-Nec, AAV-SCH9, AAV-SCH2, AAV-SHH10, AAV6.1, AAV6.2, AAV-B1, AAV-BP2, etc.

  • AAV from Human: Hu.1, Hu.2, Hu.3, Hu.6, Hu.10, Hu.11, Hu.13, Hu.15, Hu.16, Hu.17, Hu.18, Hu.19, Hu.20,  Hu.37, Hu.45, Hu.47, Hu.48, Hu.49, Hu.52, Hu.58, etc.

  • AAV from Rh: Rh10, Rh.22, Rh.23, Rh.24, Rh.32, Rh.32.33, Rh.33, Rh.34, Rh.35, Rh.39, Rh.43, Rh.47, Rh.49 Rh.52, Rh.74, etc.

  • AAV from Others: AAV-Po.1, AAV-Go.1, AAV-VR355, AAV-Cy.5, etc.

These versatile serotypes provide us different tropism for specific cells in vitro and in vivo. For your specific purpose, the researcher needs to choose the best performer for your projects. We can package most of the serotypes and can help you choose the best one. 

Contact us, our AAV experts will help you choose the potential AAV capsid for your projects. 

 
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Advantages of qPCR Based AAV Selection

  • Presence of reporter gene allows enrichment of transgene-expressing cells and avoids the viral replication and packaging steps.  

  • The DNA barcodes screenings allow to evaluate the disruption of each  capsids for each type of target cell.

  • The transcribed RNA barcodes screenings allow the evaluating of putative capsids for each target cell.

  • More actual results. The efficacy of each AAV candidate is obtained by directly comparing the quantity of barcode before and after infection rather than comparing with other AAV variants.

  • The pre-made barcoded AAV pools allow us to fastly and semi- high-throughputly compare and  select AAV variants both in vivo and in vitro.

 

Got a quote

We provide service to package the customer selected capsids, promoters, reporter genes and generate the AAV vectors accordingly.

 

For more information, please contact us:

AAVnerGene Inc.

customer@aavnergene.com

443-717-3076