Abstract
Introduction Gene therapy offers transformative potential for monogenic disorders, with adeno-associated virus vectors (AAVs) serving as a key platform. While long-term follow-up in hemophilia trials demonstrate sustained therapeutic transgene expression (up to 13 years in hemophilia B and 7 years in hemophilia A), variability in response and durability remain significant challenges. While barriers to vector entry may contribute to poor initial expression, the mechanisms governing long-term persistence and silencing of the transgene appear distinct and are less well understood. Emerging evidence suggests that epigenetic regulation of stably maintained AAV genomes may play a key role in transcriptional silencing, but this has yet to be fully elucidated. Here, we present analyses of liver biopsies from patients with hemophilia A or B treated with AAV-based gene therapy, alongside data from a stably transduced hepatic cell model expressing the same Factor IX (FIX) construct from the UCL/St. Jude's hemophilia B trial (NCT00979238).
Methods Liver biopsies from patients treated with AAV gene therapy (GO-8, AGT4HB) were analyzed by histology and RNAscope for vector DNA/RNA. In parallel, we generated Huh7 clones (hepatocellular carcinoma-derived) stably expressing FIX following repeated AAV2/8 transduction, to facilitate mechanistic studies of AAV genome persistence. Transgene expression, vector genome architecture (Southern blot), and chromatin status (CUT&RUN for H3K4me3, H3K27ac, H3K9me3, H3K27me3) were evaluated. Epigenetic modulation was tested with arsenic trioxide (ATO), known to affect PML bodies and chromatin remodeling. Cells were treated with ATO at four concentrations (1 µM, 500 nM, 250 nM, and 125 nM), harvested after 24 hours and analyzed for FIX protein and mRNA (Western blot, RT-qPCR). We used CUT&RUN to compare histone modification profiles between treated and untreated cells. To assess ATO durability and reversibility, additional experiments included extended 72-hour treatments and washouts.
Results Biopsies from five patients treated with AAV gene therapy (ranging from 2 to 129 months post-treatment) revealed long-term retention of AAV vector DNA within hepatocytes (10–73%, median 24%). However, transgene transcription was limited (<4%), consistent with transcriptional silencing. DNA and RNA patterns in these biopsies closely mirrored findings from Huh7-derived stable clones expressing the same AAV-FIX construct. Despite comparable vector copy numbers, these clones exhibited divergent but stable FIX expression levels (1–6.7 mg/mL), validating their relevance as a model for studying the AAV-host interactions. High-expressing clones exhibited dispersed nuclear vector DNA, while low expressors showed compact foci of transgene DNA in the peri-nucleolar areas. Southern blot analysis was consistent with AAV integration in all clones.
Epigenetic profiling revealed that silencing correlated with repressive histone modifications, especially H3K9me3. To assess whether expression could be modulated, cells were treated with ATO, which induced a dose-dependent reduction in FIX expression at 24 and 72h. This was more pronounced in high expressing cell lines coinciding with a 5-10-fold drop in PML protein levels and increased H3K9me3 enrichment within FIX transgenes. FIX expression partially recovered following 72-hour washout, nearing but not reaching control levels. This demonstrated a dynamic, epigenetically controlled silencing process of AAV transgenes integrated within the host genome.
Conclusions Combined analyses of patient liver biopsies and stable cell models reveal a dynamic process of transgene regulation influenced, at least in part, by the epigenetic modulation of AAV transgene-associated chromatin. This intricate control can either promote transcriptional activity and protein expression in some hepatocytes or induce transcriptional silencing in adjacent cells. Specifically, we show that epigenetic modifications, such as H3K9me3 enrichment, contribute to reduced transgene expression, and that this silencing can be reinforced by agents like arsenic trioxide. These results underscore epigenetic regulation as an important mechanism governing long-term, AAV transgene expression in human liver. Understanding these complex AAV-host interactions is critical and will directly inform the design of future strategies to overcome current challenges and significantly improve the durability of AAV gene therapies.
