The DNA Shuffling Library is a key tool in AAV capsid engineering, generating diverse capsid variants through recombination and mutation. This enables the selection of optimized capsids with traits like improved tissue tropism, immune evasion, or transduction efficiency.

Key Steps in Creating an AAV DNA Shuffling Library:

1. Selection of Parental Capsid Sequences: The process begins by selecting multiple parental AAV capsid sequences from various AAV serotypes or engineered variants, which act as the genetic material for shuffling. AAVnerGene’s ATHENA-III platforms are built upon the results of NHP selection from ATHENA-I. For each specific tissue or cell type, the highest-performing AAV serotypes are chosen as templates, based on either DNA or RNA levels.
2. Fragmentation of Capsid Genes: The parental capsid genes (Cap) are fragmented randomly using methods like DNase I digestion or restriction enzymes. This creates small DNA fragments that can later recombine to generate hybrid sequences. AAVnerGene’s ATHENA-III platforms are rationally designed based on the structural analysis of AAV capsids, ensuring targeted improvements in performance and functionality.
3. Reassembly Through Homologous Recombination: The fragmented DNA pieces are then subjected to homologous recombination using techniques such as PCR-based overlap extension. During this process, fragments from different parental sequences are combined to create a recombined and shuffled library of capsid gene variants. By carefully selecting and designing these fragments based on capsid structure, the process ensures a higher success rate in generating functional AAV variants, improving overall library quality and performance.
4. Library Construction: The recombined capsid sequences are cloned into an AAV vector backbone to form a plasmid library. Each plasmid contains a unique variant of the shuffled capsid genes. AAVnerGene’s ATHENA-III use a CAG/P40 hybrid promoter to drive cap genes. The CAG promoter is used to drive Cap expression during evolution processes and the AAV2 P40 promoter is used to express Cap protein during AAV production.
5. Library Complexity: The complexity of the AAV DNA shuffling library depends on the number of parental sequences and the extent of recombination. High complexity ensures a large variety of capsid variants, increasing the likelihood of identifying improved capsids. At AAVnerGene, we can generate more than 1e8 AAV variants for each ATHENA-III library and each capsid has an unique barcode.
6. Production of AAV Capsid Variants: The plasmid library is used to produce AAV particles, each displaying a unique shuffled capsid. All the ATHENA-III AAV capsid shuffling libraries are generated at 200 copies/cell. These AAV particles constitute the AAV capsid library, ready for screening and selection.

Screening and Selection:

After generating the AAV DNA shuffling library, the capsid variants are screened for specific properties:

• Tropism: Capsids can be screened for improved targeting of specific tissues or cell types.
• Immune Evasion: Selection can be performed for capsids that evade neutralizing antibodies.
• Enhanced Transduction: Variants with improved transduction efficiency can be identified using high-throughput assays.

Selected capsid variants can be further characterized through next-generation sequencing (NGS) or barcode sequencing to identify the genetic alterations responsible for improved functionality. Overall, an AAV DNA shuffling library is a critical tool for optimizing AAV vectors for gene therapy and research applications, enabling the discovery of novel capsids with enhanced properties tailored to specific therapeutic needs.