Cross-Packaging and Capsid Mosaic Formation in Multiplexed AAV Libraries.

Area of Science:

• Molecular Biology
• Virology
• Gene Therapy

Background:

• Adeno-associated virus (AAV) variant libraries are crucial for discovering novel capsids for gene therapy.
• Multiplexed production of AAV libraries can lead to cross-packaging and mosaicism, where viral particles contain mixed genomes and capsid proteins.

Purpose of the Study:

• To investigate the prevalence of cross-packaging and mosaicism in AAV libraries.
• To develop and utilize novel assays for assessing capsid composition and packaging fidelity.
• To identify conditions that minimize noise in AAV library production and screening.

Main Methods:

• Generation and screening of simplified AAV libraries.
• Development of novel assays to assess capsid composition and packaging fidelity.
• Experimental manipulation of input DNA levels (dilution) during AAV library production.

Main Results:

• AAV library variants exhibit cross-packaging and mosaicism, particularly at high plasmid levels.
• Dilution of input library DNA significantly enhances capsid monomer homogeneity.
• Dilution increases the correlation between capsid and genome composition, improving library quality for in vivo screens.

Conclusions:

• Cross-packaging and mosaicism are significant challenges in AAV library production.
• Diluting input DNA is an effective strategy to mitigate these issues and improve AAV library quality.
• Optimized AAV library production conditions enhance the reliability of gene therapy screens.