AAV GENE THERAPY may face durability challenges due to epigenetic silencing of therapeutic genes, according to new research analysing liver biopsies from patients treated for haemophilia alongside mechanistic studies in a hepatic cell model.
Gene therapy using adeno associated virus vectors has shown promising long-term outcomes in monogenic disorders, including sustained transgene expression lasting up to 13 years in haemophilia B trials and 7 years in haemophilia A studies. However, variability in treatment response and durability remains a major clinical challenge. Investigators sought to explore whether epigenetic regulation of vector genomes contributes to reduced transgene activity over time.
The study examined liver biopsies from five patients with haemophilia A or haemophilia B who had received AAV gene therapy. Samples were collected between 2 and 129 months after treatment and analysed using histology and RNAscope to evaluate vector DNA and RNA within hepatocytes.
Epigenetic Silencing in AAV Gene Therapy
Results showed that AAV vector DNA persisted in hepatocytes long after treatment, detected in 10–73% of cells with a median of 24%. Despite this widespread presence, active transcription of the therapeutic gene occurred in fewer than 4% of hepatocytes, suggesting substantial transcriptional silencing.
To investigate this phenomenon further, researchers developed a stable hepatic cell model derived from hepatocellular carcinoma cells expressing the same Factor IX construct used in a haemophilia B clinical trial. Although these clones contained comparable vector copy numbers, they exhibited markedly different levels of Factor IX protein expression, ranging from 1–6.7 mg/mL.
Cellular Models Reveal Transgene Regulation
Distinct nuclear patterns were observed between cell lines with different expression levels. High expressing clones showed dispersed nuclear vector DNA, whereas low expressing cells displayed compact foci of transgene DNA near peri nucleolar regions. Southern blot analysis indicated integration of the vector genome across all clones.
Epigenetic profiling revealed that reduced transgene expression correlated with enrichment of repressive histone modifications, particularly H3K9me3. These findings suggested that chromatin regulation plays a central role in transcriptional control of integrated AAV genomes.
Epigenetic Modulation Alters Transgene Expression
To assess whether this process could be experimentally modified, the investigators treated cells with arsenic trioxide, a compound known to influence promyelocytic leukaemia protein bodies and chromatin structure. Treatment resulted in a dose dependent reduction in Factor IX expression at both 24 and 72 hours.
This decline was most pronounced in highly expressing cell lines and was associated with a five-to-ten-fold reduction in promyelocytic leukaemia protein levels and increased H3K9me3 enrichment within transgene regions. Partial recovery of Factor IX expression occurred after a 72-hour washout period, indicating that silencing was dynamic rather than permanent.
Taken together, the data suggest that epigenetic regulation represents an important mechanism influencing long term AAV gene therapy expression in human liver cells. Improved understanding of these host vector interactions may inform strategies to enhance the durability and consistency of future gene therapy approaches.
Reference
Simini G et al. Decoding transgene silencing from stably maintained AAV genomes in human liver: epigenetic insights to guide next-generation vector design. Abstract 4310. ASH Congress, 6-9 December 2025.
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