AAV Capsid Screening for Translational Pig Research Using a Mouse Xenograft Liver Model

Area of Science:

• Gene Therapy
• Virology
• Preclinical Research

Background:

• Adeno-associated viruses (AAVs) are crucial gene therapy vectors, particularly for liver applications.
• Traditional rodent models have limitations due to species-specific AAV tropism, necessitating larger, more human-relevant animal models.
• Previous studies utilized human hepatocyte xenografts in mice to evolve AAV variants for liver gene therapy.

Purpose of the Study:

• To select and evaluate efficient AAV capsids for gene delivery in porcine hepatocytes using a chimeric mouse model.
• To compare the predictive accuracy of chimeric mice versus primary porcine hepatocytes for AAV capsid efficiency.
• To identify optimal AAV capsids for potential clinical translation in liver gene therapy.

Main Methods:

• Utilized a DNA-barcoded recombinant AAV library with 47 capsids.
• Administered AAV library to chimeric mice with >90% xenografted pig hepatocytes.
• Employed Illumina sequencing to analyze barcode frequencies in vector genomes and transcripts within porcine hepatocytes.

Main Results:

• AAVLK03 and AAVrh20 capsids demonstrated the highest efficiency for transgene expression in porcine hepatocytes within the chimeric mouse model.
• In vitro validation with primary porcine hepatocytes revealed different capsid efficiencies, with AAV2, AAVAnc80, and AAVDJ outperforming AAVLK03 and AAVrh20.
• Significant discrepancies between in vivo chimeric mouse model and in vitro primary cell results highlight the complexities of AAV tropism.

Conclusions:

• Chimeric mice with xenografted porcine hepatocytes serve as a valuable preclinical model for predicting AAV capsid efficiency in porcine hepatocytes.
• The study underscores the importance of selecting appropriate animal models for AAV vector development in gene therapy.
• Findings suggest that in vitro testing alone may not fully capture in vivo AAV vector performance, necessitating integrated preclinical approaches.