AAV Gene Therapy: Integration Yields Long-Term Expression without Cancer Risk

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AAV Gene Therapy: Integration Yields Long-Term Expression without Cancer Risk

AAV Gene Therapy: Integration Yields Long-Term Expression without Cancer Risk

AAV Virus Integration into Human Genome Not Associated with Cancer but Offers Long-Lasting Expression.

In 1999, a tragic incident occurred when 18-year-old Jesse Gelsinger, suffering from a severe genetic disorder called ornithine transcarbamylase deficiency, participated in a gene therapy clinical trial led by Professor James Wilson at the University of Pennsylvania. After receiving the treatment, Jesse experienced a severe immune reaction and tragically became the first person to die as a result of gene therapy.

Following this tragedy, Professor James Wilson began the quest for safer viral vectors, ultimately leading to the development and widespread use of adeno-associated virus (AAV). AAV is highly regarded for its safety, broad host cell range, and long-lasting expression within the body, making it the most widely used vector for in vivo gene therapy.

In late 2017, Spark Therapeutics developed Luxturna, an AAV gene therapy that gained FDA approval for treating congenital blindness type 2 caused by RPE65 gene mutations. This marked a significant milestone in gene therapy and sparked a new era of gene therapy development.

However, AAV gene therapies targeting the liver face challenges in achieving efficient gene expression and maintaining long-term expression. AAV is typically thought to exist in a free state within cells, but sometimes it can integrate into the host cell’s genome, potentially during DNA repair processes. This raised concerns that AAV vectors might disrupt the host cell’s genome and lead to cancer.

On November 6, 2023, Professor James Wilson’s team published two research papers in the journals Nature Biotechnology and Human Gene Therapy.

These studies represent the most comprehensive exploration to date of AAV integration into the host genome in non-human primates, shedding light on the safety and long-term efficacy of AAV gene therapy.

The research suggests that liver-targeted adeno-associated virus (AAV) integration into the host cell’s genome holds the potential for long-lasting expression without a significant risk of driving cancerous mutations.

AAV Gene Therapy: Integration Yields Long-Term Expression without Cancer Risk

Previous studies in mice, especially neonatal mice, indicated that AAV vector genome integration could lead to liver cancer by disrupting genomic elements that control cancer. However, it remained unclear whether this phenomenon would occur in humans and other primates.

AAV Gene Therapy: Integration Yields Long-Term Expression without Cancer Risk

In the study published in the Human Gene Therapy journal, the research team examined tissue samples, primarily from the liver, of 86 monkeys that had undergone preclinical gene therapy with engineered AAV. They also analyzed tissue samples from 253 monkeys and humans (168 monkeys and 85 humans) who had never been exposed to engineered AAV.

The results showed that the likelihood of random AAV insertions throughout the entire genome was low for the monkeys treated with engineered AAV gene therapy and for those who had only encountered wild-type AAV (naturally occurring AAV) – even 15 years after AAV gene therapy.

In the study published in the Nature Biotechnology journal, the research team conducted over two years of monitoring 12 monkeys who had received liver-targeted AAV gene therapy.

Liver-targeted adeno-associated virus (AAV) gene therapy has been approved for treating hemophilia A and hemophilia B, with several other liver-targeted AAV therapies in clinical development. However, these therapies face two challenges – maintaining AAV persistence in the liver and the difficulty of re-administering the treatment. Previous clinical studies in hemophilia B patients revealed that the therapeutic effect began to decline within the first two months. Additionally, patients with Crigler-Najjar syndrome, a hereditary hyperbilirubinemia, experienced an immediate decrease in serum bilirubin levels after AAV gene therapy, but these levels returned to pre-treatment levels within two months, despite the absence of notable AAV vector immunity or liver inflammation. This suggested that a non-immune mechanism may be responsible for the loss of efficacy.

Surprisingly, after a few months of unstable expression, AAV expression levels remained low but stable in non-human primates (NHP) and human livers. Liver cells continually divide and regenerate, and theoretically, free AAV genomes should be diluted over time. To understand the reasons behind this, Professor James Wilson examined AAV8 and AAVrh10 vector injections in NHP veins for over two years to better define the AAV transduction mechanism influencing gene therapy outcomes.

The results indicated that highly non-immunogenic transgenes were effectively transduced, with expression levels starting to decline within the first 90 days and eventually reaching a lower but stable level. Over 10% of liver cells retained single nucleosome domains of AAV vector DNA, even though the transcribed gene expression might have been lost. Immune transgenes observed a greater reduction in AAV vector DNA and RNA. Moreover, the frequency of AAV vector sequences integrated into the chromosome at different genomic locations was low, and these locations were not near cancer-related genes, with no evidence of significant clonal expansion over the follow-up years.

AAV Gene Therapy: Integration Yields Long-Term Expression without Cancer Risk

These findings suggest that in the liver cells of primates, AAV-mediated transgene expression occurs in two stages: first, there is high but short-lived expression from free AAV genomes, followed by low but persistent stable expression. This indicates that the long-term persistence of AAV gene therapy in primate livers may be attributed to AAV vector integration.