ATHENA-II Capsid Evolution Platform
ATHENA-II is a Random Peptide Insertion AAV Capsid Library Platform, specifically designed to develop novel AAV capsids with tissue-specific targeting capabilities. This platform achieves this by incorporating random peptide (RP) sequences into the variable regions (VRs) of the AAV capsid protein. These peptide insertions can alter key capsid properties, including tropism, transduction efficiency, and immunogenicity. By screening these libraries, researchers can identify and isolate novel AAV capsids with improved characteristics, enabling the selection of optimal variants for targeted gene therapy applications.
AAVnerGene offers end-to-end services for AAV Capsid Libraries, encompassing design/construction, production, and evolution.
AAVnerGene offers AAV Capsid Library Design/Construction services, allowing customers to select their preferred promoter, capsid template, insertion site, and insertion length. For example, our premade AAV capsid libraries use a CAG/P40 hybrid promoter: CAG promoter drives cap expression during evolution and P40 promoter regulates cap expression during AAV production. Customers only need to replace the CAG promoter with their chosen promoter. For this service, we only charge a cloning fee ($300/clone + gene synthesis fee) and/or plasmid preparation fee.
The insertion sites, peptide length, and the number of unique variants determine the properties and diversity of the capsids available for selection. AAV VR-VIII and VR-IV are the most commonly used variable regions for engineering. The insertion of 7 amino acids offers a good balance between AAV packaging efficiency and library diversity. Higher complexity increases the likelihood of discovering rare, high-performing variants. We offer the generation of random peptide insertion libraries from any modified AAV capsids, targeting any variable regions or sites with any desired length of random peptide. Pricing is primarily based on the complexity of the requested library. Each library includes the amplification of 1 mg of endotoxin-free plasmid.
| Library complexity | Price | Turnaround Time |
| 1.00E+07 | $12,800 | 2-4 weeks |
| 1.00E+08 | $21,800 | 2-4 weeks |
| 1.00E+09 | $68,000 | 3-5 weeks |
| Higher | Please contact us to discuss | |
Note: Complexity is calculated based on the number of clones obtained after transformation. We also provide NGS (Next-Generation Sequencing) services to verify library diversity, available for an additional fee.
Producing high-quality and diverse AAV capsid libraries, can be technically challenging. 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 capsid libraries production more accessible and affordable for researchers and biotech companies.
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Production Scale |
Packaging Conditions | Price |
Turnaround |
| 2.00E+12 VG | 1000 copies/cell | $11,800 |
2-3 weeks |
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200 copies/cell |
$19,800 | 2-3 weeks | |
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100 copies/cell |
$29,800 |
2-3 weeks |
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<50 copies/cell |
Please call to discuss |
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2.00E+13 VG |
1000 copies/cell | $48,800 |
3-4 weeks |
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200 copies/cell |
$98,000 |
3-4 weeks |
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100 copies/cell |
$148,800 |
3-4 weeks |
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<50 copies/cell |
Please call to discuss |
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>2.00E+13 VG |
Please call to discuss |
Please call to discuss |
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AAVnerGene offers comprehensive AAV capsid screening services both in vivo and in vitro. In vitro, we utilize human and animal cell lines, primary cells, iPSC-derived cells and organoids, to provide controlled assessments. In vivo, we screen AAV capsids using animal models such as mice, rats, non-human primates, pigs, dogs and other large animals. These screening services are designed to support AAV-based research and therapeutic development, offering insights into vector performance and AAV evolution, and suitability for different applications. Our standard evolution process consists of four rounds of enrichment and verification. In Round-I, the top 1e5 variants from a high-complexity initial library are corrected. Round-II narrows down the selection to the top 100 variants. During Round-III, the selected 100 capsids are recorded and verified using NGS to identify the top 10 candidates. Finally, Round-IV involves individually testing the top 10 candidates.
| Model | Tissue | Evolution Process | Turnaround | Price |
| Cell line iPSC Organoid Mouse Rat NHP Others | Brain Liver Lung Heart Muscle Retina Others | Round-I Round-II Round-III Round-IV | TBD | Request a Quote |
Pre-made AAV Capsid Library
In the premade AAV capsid libraries, the Cap gene with random peptides is driven by a CAG/P40 hybrid promoter, with the CAG promoter used for Cap expression during evolution and the AAV2 P40 promoter for Cap production.
| Library Name | AAV Capsid Library |
| Library type | ATHENA-II random peptide insertion |
| Purpose | Create and evolve novel AAV capsids |
| Complexity | High (>1e9 variants) |
| Expression Cassette | AAV-CAG/P40-Cap-NNKs |
| Barcodes | Random peptides serve as barcodes |
| Production Condition | Library plasmid + mini-Helper-Rep plasmid |
| Selection Method | NGS (>100 M reads) |
| Premade AAV Capsid Libraries | Random Peptide insertion at VR-IV and VR-VIII of AAV1, AAV2, AAV3B, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.10, AAVrh74 |