Heterogeneous Cell Infection and Mitochondrial Insertion of AAV Gene Therapy Vectors Validated with a Human Liver Mimic
Background
Adeno-associated virus (AAV)-based gene therapies are being actively developed for the treatment of genetic diseases such as hemophilia. However, animal models commonly used in preclinical studies do not fully reflect human receptor distribution or the immune system, and therefore cannot accurately represent the actual human response. This is the reason why the high safety indicators observed in animal experiments are often not consistently replicated in clinical trials. To address this issue, a technology has emerged that mimics human liver tissue in three dimensions. Human liver tissue equivalents (hLTEs), which contain hepatocytes, endothelial cells, stellate cells, and Kupffer cells in proportions similar to those found in the native liver, precisely represent the physiological activity of the actual liver. This platform is emerging as a new drug screening platform because it can precisely evaluate the safety of therapeutic vectors at a human level.
Key Findings
A research team from the Wake Forest Institute for Regenerative Medicine (WFIRM) precisely compared the transduction efficiency, cellular selectivity, and toxicity of two AAV serotypes, AAV5 and AAV3b, for gene therapy using hLTEs. They designed viruses that express enhanced green fluorescent protein (eGFP) under the control of a cytomegalovirus (CMV) promoter and administered them to hLTEs. The results showed that AAV5 exhibited a higher expression rate than AAV3b.
Analysis of cell type-specific infection revealed an inversion of the expected results. Both serotypes transduced endothelial cells and stellate cells more efficiently than hepatocytes and Kupffer cells, which are the intended targets. This demonstrates the possibility that the therapeutic gene may be delivered to unintended cells, causing adverse effects. In fact, analysis of the hLTE culture medium after AAV administration revealed changes in the levels of liver function biomarkers, as well as dysregulation of gene expression related to liver toxicity and inflammation. The patterns of changes in intracellular transcripts differed depending on the serotype, promoter, and gene.
The researchers also used a bioengineered Factor VIII (FVIII) gene (lcoET3) under the control of a phosphoglycerate kinase (PGK) promoter to precisely track changes in intracellular signaling pathways. The results showed that the types of molecules expressed also differed depending on the type of gene incorporated.
Most importantly, the results of the genotoxicity assessment raised concerns. Analysis of viral genome insertion revealed that viral DNA was inserted into multiple sites near genes associated with cancer development. Furthermore, a significant level of insertion was also found within mitochondrial DNA (mtDNA). This suggests that AAV vectors can penetrate not only the nuclear genome but also the mitochondrial genome, which is responsible for cellular respiration, and potentially threaten cell health.
Significance and Prospects
These results clearly demonstrate the role that the human liver tissue mimic platform presented in this study can play in preclinical screening for gene therapies. It has elucidated cell-specific infection patterns and genotoxic signals such as mitochondrial DNA insertion in a human tissue environment, which could not be demonstrated by conventional animal models. It is expected to serve as a safety net to prevent potential adverse effects before clinical trials.
However, due to the nature of the study, which is based on in vitro tissue mimics, it is difficult to perfectly mimic systemic physiological functions. The fact that the transduction rate of non-target cells such as stellate cells and endothelial cells was higher is one of the challenges that needs to be addressed. It is suggested that further research is needed to modify the viral capsid to reduce off-target reactions and enable selective targeting of hepatocytes.
The analysis that the combination of promoters and exogenous genes can affect not only gene delivery but also the host cell's signaling pathways is also likely to have a significant impact on future drug design guidelines.
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.
The human liver tissue mimic platform presented in this study is suitable for immediate use by pharmaceutical and biotechnology companies that are developing new drugs as a cost- and time-effective screening tool for efficacy and safety. A specific application scenario is the selection of new candidate vectors. By applying dozens of AAV capsid candidates to liver mimics made from patient-derived cells, it is possible to compare which vectors accurately target hepatocytes and minimize the entry of other non-target cells such as stellate cells and endothelial cells.
At the same time, it is also possible to screen in advance whether the therapeutic gene is inserted near oncogenes in the nuclear and mitochondrial genomes, and to narrow down the candidate substances for clinical trials to the safest ones with the lowest risk of genotoxicity. This platform is expected to be established as a key technology that will reduce the risk of clinical trials being terminated due to unexpected adverse effects in the clinical stage, and increase the success rate of commercialization of next-generation, highly safe gene therapies.