ATHENA II AAV Capsid Platform

Designing AAV capsid expression strategies: Selecting the appropriate promoter is crucial for achieving optimal capsid expression levels. The promoter drives the transcription of the capsid gene and determines the strength and specificity of expression. The choice of promoter can also impact AAV production yield. Different promoters may have varying strengths and efficiency in driving transcription, which can affect the overall expression level of the capsid gene and, consequently, the production yield of AAV vectors.
Selection of AAV serotypes: The first step is to choose which AAV serotypes will be included in the library. This may be based on factors such as tissue tropism, efficiency of transduction, and availability of serotype-specific antibodies.

Identification of variable regions: Next, the variable regions of the AAV capsid sequence must be identified. These regions are responsible for determining the tropism and transduction efficiency of the AAV vector, and are typically located on the surface of the capsid.

Replacement or insert random peptides: Both replacement and insertion can be used to introduce variants in the variable regions of AAV capsids. Replacement involves swapping a specific amino acid or sequence of amino acids with another, whereas insertion adds a new sequence into the variable region of the capsid. The choice between replacement or insertion may depend on the specific goals of the AAV capsid library design. If the aim is to explore the effect of single amino acid changes or small sequence alterations, replacement may be a better approach. On the other hand, if the goal is to introduce larger changes or add new functionalities, insertion may be a more suitable strategy.

Determining the length of the random peptide insert: The length of the random peptide insert: in an AAV library depends on several factors, including the desired diversity of the library, the target protein or interaction being studied, and any structural or functional constraints. It’s important to note that the optimal length of the random peptide insert may vary depending on the specific research goals and experimental setup. It may be helpful to conduct pilot studies or explore different insert lengths within the library to determine the most suitable length for your particular application.
Library construction: The PCR products are then cloned into a vector to generate the AAV capsid library. This can be done using a variety of methods, including restriction enzyme digestion, ligation-independent cloning, or Gibson assembly.

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.

Screening and selection: The final step is to screen the library to identify capsids with desired properties, such as tissue tropism or improved transduction efficiency. This can be done using a variety of methods, including in vitro and in vivo selection, high-throughput sequencing, and bioinformatics analysis.