NGS Based 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 NGS based AAV capsid selection, which allows us to fastly and semi-high-throughputly compare hundreds of  AAV variants at once both in vivo and in vitro.​​ 
 

Design of NGS Based AAV Capsid Selection 

In the NGS based AAV capsid selection, each capsid variant packages a DNA barcoded  genome carrying a reporter gene(such as EGFP). The unique barcode(25bp random sequence) is located between coding sequence and Poly-A signal. The capsid/barcoded-transgene pairs were transfected independently and pooled together during purification processes, which is different with the qPCR-based methods.

Advance3.png

NGS Based Selection Protocol

NGS-based AAV capsid selection can be performed 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. The results can be obtained by NGS. 

Advance2.png

 Capsids for Selection

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. 

Advantages of NGS 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.

  • High-throughput. Hundreds of AAV vectors can be generated at once, pooled and tested together. The pre-made barcoded AAV pools allow us to fast and semi- high-throughput compare and  select AAV variants both in vivo and in vitro.

 Difference between qPCR and NGS based AAV Capsid Selection

  • The qPCR based AAV selection methods can be only used for less capsids(usually less than 24). however, the capsid for NGS-based selection is unlimited. 

  •  In the qPCR-based selection methed, specific primers are used to detect the abundance of barcodes. The selection can be done in the labs. In the NGS-based selection, DNA libraries are prepapred and sent out for NGS service.

  • The qPCR based AAV selection methods can be used to compare the common used AAV capsids for each type of cells. The NGS-based selection can be further used to discovery new AAV variants for each cell types.

Custom AAVcap-Advance Capsid Selection Kit

We provide service to package the customer selected capsids, promoters, reporter genes and generate the AAVcap selection kit accordingly.

For more information, please contact us:

AAVnerGene Inc.

customer@aavnergene.com

443-717-3076