AAV Capsid Library Design/Construction

AAV Capsid Structure

AAV capsids possess nine surface-exposed VRs, VR-I to VR-IX, essential for determining tropism and receptor binding. Current directed evolution efforts primarily focus on VR-VIII and VR-IV.  VR-VIII is particularly the spike positions around the 3-fold axis (N587/R588 of AAV2 or Q588/A589 of AAV9), which serve as key engineering sites. This focus has led to well-known evolved capsids like AAV2-7M8, AAV-PhP.eB, AAV-Cap.Mac, and MyoAAVs. VR-IV, the most radially protruding loop, plays a role in neutralizing antibody and surface receptor binding due to its proximity to the 3-fold symmetry axis. Compared to VR-VIII, VR-IV is less permissive for peptide inserts. However, enhancing diversity in VR-IV can improve interactions with existing VR-VIII mutations and refine transduction, exemplified by AAV.Cap.B22, engineered from AAV-PHP.eB. Notably, the evolution of the AAV6 VR-IV library alone produced Ark313, which shows high transduction efficiency in murine T cells.

To date, few engineering efforts have targeted other VRs, but they contribute to local topological differences among AAVs. Specifically:

  • VR-V, along with VR-IV and VR-VIII, forms the top of the 3-fold protrusion.
  • VR-II constitutes the top of the 5-fold channel.
  • VR-VI and VR-VII form the base of the 3-fold protrusion.
  • VR-I, VR-III, VR-VII, and VR-IX contribute to the 2/5-fold wall.

These VRs also dictate functional differences, influencing receptor attachment, transduction efficiency, and antigenic reactivity among AAVs.

AAV Capsid Library Design

To evolve capsids through multiple rounds of selection, an AAV expression cassette is essential for both production and selection. This cassette typically includes the necessary genetic elements to package the AAV capsid library, produce functional viral particles, and enable the identification of successful variants. Key components of the expression cassette often include:

  • ITRs (Inverted Terminal Repeats): Flanking the expression cassette, ITRs are essential for AAV genome packaging and replication.
  • Promoters: The choice of promoter is critical, as capsid proteins need to be produced in the correct ratio during the packaging step and expressed at high levels during the selection steps. A hybrid promoter is commonly used to achieve this purpose. A strong or tissue-specific promoter is often employed to drive capsid gene expression during selection, while the P40 promoter is used to ensure proper capsid assembly.
  • Capsid Gene with Variable Regions: The AAV capsid gene is modified to incorporate random peptide insertions or mutations in its variable regions (VRs), enabling the generation of a diverse library of capsid variants. These modifications allow for the exploration of new tropisms, transduction efficiencies, and immunogenic profiles.
AAV Capsid Library Design

Premade AAV Capsid Libraries

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.

AAVnerGene offers these to evolve novel capsids with tissue-specific tropism or other applications.
•  high-complexity libraries (>1e9 variants) with various AAV templates (AAV1, AAV2, AAV3B, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.10, AAVrh.74)
•   variable regions (VR-IV, VR-VIII), and random peptides ((NNK)7, (NNK)8, (NNK)9)

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Notes:
1. Complexity. The complexity was calculated according the numbers of clones after electroporation.
2. AAV library production. The AAV library plasmid was transfected as 200 Copies/cells together with our mini-pHelper-Rep.
3. AAV purification: two rounds of CsCl ultracentrifuge.
4. For the premade AAV capsid library, we only provide AAV virus but not the plasmid.

Custom AAV Capsid Libraries

Besides the  high-complexity premade AAV capsid libraries, We also provide custom AAV capsid library construction services, allowing users to select their preferred library templates, randomized positions, inserted variable regions, and desired library complexity. In addition, AAVnerGene specializes in AAV capsid library production and evolution services. For more details, please feel free to send us an inquiry (customer@aavnergene.com).

Steps for AAV Capsid Library Design and Construction:

  • Selection of plasmid AAV backbones: For this purpose, any plasmid containing two functional ITRs is suitable. Examples include our pAAVtri-CMV-EGFP plasmid, which features one ITR119 and one ITR130. These ITRs are essential for proper AAV genome packaging and replication, making such plasmids ideal for constructing AAV expression cassettes.
  • Designing AAV Capsid Expression Strategies: Promoter selection is critical for capsid expression. The right promoter ensures optimal transcription levels, impacting both AAV yield and capsid functionality. Our premade AAV capsid libraries use a CAG/P40 hybrid promoter:
      • CAG promoter drives cap expression during evolution.
      • P40 promoter regulates cap expression during AAV production.
  • Selection of AAV Serotypes: Factors to consider include:

    • Tissue Tropism: Different serotypes exhibit varying affinities for specific tissues or cell types, making it crucial to select one that targets the desired tissue effectively.

    • Transduction Efficiency: The ability of the serotype to efficiently deliver and express the transgene in the target cells is a critical consideration.

    • Availability of Serotype-Specific Antibodies: The presence of antibodies against the serotype, either pre-existing in the host or developed during treatment, can impact the efficacy and safety of the gene therapy.

    • Productivity: The yield of viral particles during production varies among serotypes, influencing scalability and cost-effectiveness.

    • Regulatory Barriers: Regulatory considerations, such as prior clinical use or safety data for specific serotypes, can affect the approval process and feasibility of the therapy.

  • Identification of Variable Regions: The surface-exposed variable regions (VRs) of the AAV capsid play a critical role in determining the vector’s tropism and transduction efficiency. These regions are prime targets for modification to engineer capsids with enhanced or altered properties. Key variable regions include:
    • VR-VIII: A well-characterized region that influences receptor binding and tissue targeting.
    • VR-IV: Another important region that contributes to capsid interactions with host cells and immune responses.
    • Other VRs: Additional variable regions across the capsid surface can also be modified to fine-tune properties such as immunogenicity, stability, and transduction efficiency.
  • Replacement or Insertion of Random Peptides: There are two main strategies for introducing variability in the capsid. Choosing between replacement or insertion depends on research goals. For single amino acid alterations, replacement is ideal, while insertion works better for introducing significant changes or adding new functionalities.
    • Replacement: Swapping specific amino acids or sequences.
    • Insertion: Adding new sequences into the capsid’s variable regions.
  • Determining Random Peptide Insert Length: this is key to library diversity. Pilot studies or varied insert lengths may be necessary to determine the optimal configuration for the specific application. Factors influencing this decision include:

    • Desired Diversity of the Library: Longer inserts can increase sequence diversity, but may also introduce structural instability, while shorter inserts may limit variability but maintain capsid integrity.

    • Structural and Functional Constraints of the Capsid: The insert length must balance the need for diversity with the preservation of capsid stability, assembly, and functionality.

    • Interaction with Target Proteins: The insert length should allow for effective interactions with target proteins or receptors, ensuring the desired tropism or transduction efficiency.

  • Determining Library Complexity: the number of unique variants—determines the diversity of available capsids for selection. Insufficient complexity limits the discovery of optimal capsids for therapeutic or research purposes, so ensuring a diverse pool is crucial. Higher complexity increases the chances of identifying rare, high-performing variants with:
    • Enhanced tissue specificity
    • Immune evasion capabilities
    • Improved transduction efficiency
  • AAV capsid plasmid library construction: This can be done using a variety of methods, including restriction enzyme digestion, ligation-independent cloning, or Gibson assembly. We offer packaging of random peptide libraries from modified AAV capsid plasmid. Our pricing is dependent on quantity and transfection conditions. Pricing increases with lower copies per cell.
  • AAV capsid plasmid library validation: Once the library has been constructed, it must be validated to ensure that it contains a diverse range of sequences and that the library complexity is high enough to allow for effective screening and selection. This can be done by using regular NGS.

  • AAV Capsid Library Production: Once the quality of the library is confirmed, it can proceed to production. However, producing high-quality, highly diverse AAV capsid libraries presents significant technical challenges. A major hurdle is ensuring that each vector in the library contains a unique DNA sequence. Errors or duplications in the transgene region can lead to biased or incomplete library representation, compromising its diversity and utility. To address these challenges, AAVnerGene has developed an optimized production system. By utilizing the mini-pHelper-Rep plasmid and high-efficiency suspension HEK 293T cell lines, AAVnerGene has streamlined the production process, making it more accessible and cost-effective for researchers and biotech companies. This approach ensures the generation of high-quality, diverse AAV capsid libraries, enabling more efficient screening and discovery of novel capsids for gene therapy applications. 

  • AAV Capsid Library Evolution: Once the AAV Capsid library has enough amount and the quality is meet your need,  you can start the capsid evolution.  The evolution process consists of serveral 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. 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.

AAV Capsid Library Construction

Library complexity

Price

Turnaround Time

1.00E+07

$12,8003-4 weeks

1.00E+08

$21,8003-4 weeks
1.00E+09$68,000

4-5 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.