AAV Capsid Library Production

AAV libraries are typically created by generating a large number of AAV vectors, each with a different DNA sequence in the transgene region, and then pooling these vectors to create a library of diverse sequences. The goal of creating such libraries is to be able to select AAV vectors with specific properties or functions, such as high transduction efficiency or tissue specificity. 

Producing high-quality and diverse AAV libraries can be technically challenging. One challenge in creating AAV libraries is ensuring that each vector in the library has a unique DNA sequence, as even small errors or duplications in the transgene region can lead to biased or incomplete representation of the library. For the majority of libraries, vast population diversity requires multiplexed production, in which a library of ITR-containing plasmid variants is transfected together into cells to generate the viral library. This process has the potential to be confounded by cross-packaging and mosaicism, in which particles are comprised of genomes and capsid monomers derived from different library members. AAV library variants are prone to cross-packaging and capsid mosaic formation when produced at high plasmid levels. Producing an AAV capsid library in low copy number can help reduce the cross packaging and capsid mosaic formation. However, reduce the library plasmid copy number during the transfection would introduce another challenge, generating a large number of AAV vectors efficiently and reproducibly.

By using the mini-pHelper-Rep plasmid and high efficiency suspension HEK 293T cell lines, AAVnerGene was able to make AAV library production more accessible and affordable for researchers and biotech companies. 

Reduce the cross-packaging and capsid mosaicism

Cross-packaging and capsid mosaicism are two major issues that can affect AAV production and the diversity of the resulting library. 

  • Cross-packaging occurs when the AAV genome packaged inside the capsid is not identical to the capsid proteins that form the outside of the viral particle. This can occur when multiple AAV genomes are present in the same cell and the viral packaging machinery assembles capsids with genomes from different AAV variants. 
  • Capsid mosaicism, on the other hand, occurs when a single AAV capsid is composed of subunits from different capsid variants. This can occur due to recombination events during replication or packaging. 

Both cross-packaging and capsid mosaicism can decrease the diversity of the AAV library and complicate downstream analysis. Therefore, it is important to minimize these issues during AAV library production.

Developing novel methods to improve AAV library yield while maintaining low cross-packaging and mosaicism is an important area of research. By increasing AAV library yield, it may be possible to produce larger libraries with greater diversity, which could lead to the identification of more effective AAV capsids for gene therapy applications.

Reduce the cross-packaging and capsid mosaicism

Co-transfection of the rep and helper plasmids is necessary to provide the necessary functions for AAV replication and packaging. At AAVnerGene, We developed the mini-pHelper plasmid to replace the original Ad helper plasmid. Its compact size allows for more efficient transfection and reduces the overall cost of the process. Moreover, mini-pHelper not only increase regular AAV yield, but also increase AAV library production.

The mini-pHelper-Rep plasmid is specifically designed for AAV capsid library production. It contains the Rep gene that has been integrated into the mini-pHelper plasmid backbone, which provides Ad helper functions for efficient packaging of AAV vectors. AAV libraries can be efficiently packaged by co-transfecting the mini-pHelper-Rep plasmid along with the AAV library plasmids into host cells. 

Developing novel methods to improve AAV library yield while maintaining low cross-packaging and mosaicism is an important area of research. By increasing AAV library yield, it may be possible to produce larger libraries with greater diversity, which could lead to the identification of more effective AAV capsids for gene therapy applications. Co-transfection of the rep and helper plasmids is necessary to provide the necessary functions for AAV replication and packaging. At AAVnerGene, We developed the mini-pHelper plasmid to replace the original Ad helper plasmid. Its compact size allows for more efficient transfection and reduces the overall cost of the process. Moreover, mini-pHelper not only increase regular AAV yield, but also increase AAV library production.

The mini-pHelper-Rep plasmid is specifically designed for AAV capsid library production. It contains the Rep gene that has been integrated into the mini-pHelper plasmid backbone, which provides Ad helper functions for efficient packaging of AAV vectors. AAV libraries can be efficiently packaged by co-transfecting the mini-pHelper-Rep plasmid along with the AAV library plasmids into host cells. 

mini-pHelper-Rep for AAV library production

Cross-packaging and capsid mosaicism

How to reduce the Cross-packaging and capsid mosaicism

Limiting the number of input plasmids is one approach to reduce the risk of capsid mosaicism in AAV library production. By reducing the number of input plasmids, the chances of co-infection of a single cell by multiple library members are minimized, which decreases the likelihood of generating chimeric capsids. However, limiting the number of input plasmid also reduce the overall yield of AAV particles. This can be a trade-off that needs to be carefully balanced in order to obtain a high-quality AAV library.

AAV library production

We offer packaging of random peptide libraries from modified AAV capsid plasmid using our customer provide helper plasmids or our mini-pHelper-Rep library production system. Our pricing is dependent on quantity and transfection conditions. Pricing increases with lower copies per cell.

Scale

Packaging Conditions

Price

Turnaround

2.00E+12

1000 copies/cell

$11,800

2-3 weeks

 

200 copies/cell

$19,800

2-3 weeks

 

100 copies/cell

$29,800

2-3 weeks

 

<50 copies/cell

Please call to discuss

2.00E+13

1000 copies/cell

$48,800

3-4 weeks

 

200 copies/cell

$98,000

3-4 weeks

 

100 copies/cell

$148,800

3-4 weeks

 

<50 copies/cell

Please call to discuss

2.00E+14

Please call to discuss

Please call to discuss

AAV Library QC Services

Quality control is an important step in the production of AAV libraries to ensure that the library is of high quality and suitable for downstream applications. AAVnerGene provide

Catalog QC Method Turnaround Time Price Sample Requirement
AQC001 Genome Titration ddPCR/qPCR 3-5 day $200/sample *Primer/probe sequences; ~20μl of samples
AQC002 Capsid Titration ELISA 3-5 day $400/sample *Serotypes and buffers; ~20μl of samples
AQC003 Purity Analysis SDS-PAGE 3-5 day $200/sample ~20μl of sample with titer >1E+12vg/ml
AQC004 Full/Empty Ratio AUC 3-5 day $2000/sample ~400μl of sample with titer >1E+12vg/ml
AQC005 Endotoxin LAL assay 3-5 day $200/sample ~20μl of samples
AQC006 Endotoxin Removal Resin-PMB 3-5 day $200/sample ~20μl of samples
AQC007 Bioburden Testing Direct plating 3-5 day $200/sample ~20μl of samples
AQC008 Vector Genome Identity PacBio Seq 4 weeks Request ~100μl of sample with titer >1E+12vg/ml
AQC009 Mycoplasma Testing PCR 3-5 day $200/sample ~20μl of samples
AQC010 Library Complexity NGS 3-4 weeks Request ~20μl of samples