While developing a gene therapy methylmalonic anemia, or MMA, scientists realized that the delivery of a corrected gene could increase the risk of liver cancer—if the gene was delivered to a particular location in the genome. If the gene was sent to a different site, by means of a modified vector, an adeno-associated virus (AAV), cancer did not develop.

The study was conducted in mice, and the portion of the mouse genome that appears unsafe to target has no human analogue. Still, the study’s basic finding—that different gene insertion sites may be associated with greater or lesser cancer risk—may be highly relevant to gene therapy in humans. Moreover, it may be possible to reduce cancer risk by modifying the vectors that deliver genes to specific sites in the cell’s DNA.

These findings appeared January 20 in the Journal of Clinical Investigation (JCI), in an article entitled, “Vector design influences hepatic genotoxicity after adeno-associated virus gene therapy.” The article noted that while gene therapy has demonstrated efficacy in a growing number of clinical trials, the safety of AAV as a vector has been challenged.

AAV-mediated gene delivery was linked to liver cancer by just one study. In contrast, AAV-mediated was not associated with genotoxicity in most other studies. Even a tenuous link between AAV and cancer, however, merited a closer look, reasoned scientists at the National Human Genome Research Institute (NHGRI). These scientists decided to perform a comprehensive study of hepatocellular carcinoma (HCC) in a large number of mice following therapeutic AAV gene delivery.

“Using a sensitive high-throughput integration site-capture technique and global expressional analysis, we found that AAV integration into the RNA imprinted and accumulated in nucleus (Rian) locus, and the resulting overexpression of proximal microRNAs and retrotransposon-like 1 (Rtl1) were associated with HCC,” wrote the authors of the JCI article. “In addition, we demonstrated that the AAV vector dose, enhancer/promoter selection, and the timing of gene delivery are all critical factors for determining HCC incidence after AAV gene delivery.”

Initially, the scientists documented a 50–70% higher occurrence of liver cancer in AAV-treated mice compared with a 10% liver cancer rate in untreated mice. The scientists also determined that the AAV vector triggered the cancer. When the researchers used an alternate AAV vector to deliver the corrected gene in a study of just 10 mice, that vector did not insert where it would elevate the expression of nearby genes, and it did not cause liver cancer. The researchers found that this modification was a safer gene therapy.

“These studies will help us move forward to develop safer gene therapy for methylmalonic acidemia,” said Randy Chandler, Ph.D., lead author and NHGRI staff scientist. “Most of the AAV integrations that caused liver cancer landed in a gene that is not found in the human genome, which suggests that the cancers we observed after AAV gene therapy may have been a mouse-specific phenomenon. However, these studies do convincingly demonstrate that AAV can be a cancer-causing agent, which argues for further studies.”

The researchers also observed that lower doses of AAV resulted in reduced rates of liver cancer compared to a control group of mice. Their data showed that the rate of cancer dropped in proportion to the dose of the AAV.

“Our observations offer the most compelling evidence to date that therapeutic gene delivery mediated by AAV can display marked genotoxicity,” the authors of the JCI article concluded. “The fact that AAV cassette design can markedly alter the incidence of genotoxicity is encouraging and illustrates how a more complete understanding of vector behavior in preclinical models could inform the risks of potential adverse events associated with AAV gene delivery in humans.”

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