Defining the safety and efficacy of liver-directed AAV gene therapy using a human liver tissue-equivalent platform.

This study reports on the use of human liver tissue equivalents (hLTEs) fabricated using major cell types at ratios that recapitulate native liver structure, physiology, and function, to investigate transduction efficiency, cellular tropism, functional impact, and genotoxicity of 2 adeno-associated virus (AAV) serotypes, AAV5 and AAV3b, encoding eGFP under the strong cytomegalovirus (CMV) promoter to ensure ubiquitous transgene expression in all cells. Additionally, AAV5 encoding eGFP or a bioengineered FVIII transgene (lcoET3) under the phosphoglycerate kinase (PGK) promotor was used to identify unique pathways specific to lcoET3 and/or the effects of different promoters. Overall, AAV5 yielded higher eGFP expression, both AAVs transduced endothelial and stellate cells more efficiently than hepatocytes and Kupffer cells, and both altered liver function biomarkers. Differential gene expression analysis showed that multiple genes involved in hepatotoxicity/inflammation were significantly dysregulated, with each serotype, promoter, and transgene producing a distinct pattern of transcriptional alterations. Integration site analysis identified AAV integrations throughout the human genome, with some in the vicinity of multiple genomic loci associated with oncogenesis. Interestingly, numerous integrations were also found within the human mitochondrial genome. Therefore, hLTEs are a valuable platform for human-relevant safety assessment and to gain critical insights for developing safer and more effective liver-directed AAV gene therapy.