July 26, 2007 -- Washington University School of Medicine researchers at the Siteman Cancer Center at Barnes-Jewish Hospital and Washington University School of Medicine in St. Louis have found further evidence linking a method used to deliver gene therapy in humans with the development of liver tumors in mice.

The new research, published in the July 27 journal Science, suggests that ferrying a corrective gene into mice using a disabled virus - an adeno-associated virus (AAV) - inadvertently inserts mutations into the mouse DNA that initiate tumor growth. The same delivery method is also used in some gene therapy clinical trials in humans, but as of yet no studies have found an association between the AAV vector and cancer in patients.

Still, the Washington University scientists, led by Mark Sands, Ph.D., associate professor of medicine and genetics, say the data in the team's latest study raise important safety concerns about the use of AAV vectors in patients receiving experimental gene therapy. "While the findings do not eliminate AAV as a potential therapeutic tool, more research is needed to determine the possible long-term toxicity of the AAV vector in humans," Sands cautions.

The current study is a follow-up to Sands' earlier work, published in 2001 in the journal Gene Therapy, which first suggested an association between AAV and liver cancer in mice. However, that study involved only five mice that had been found to develop liver tumors after the animals lived 18 months, about a year longer than is typical for this type of research. Based on Sands' research in mice, two human AAV gene therapy trials were briefly halted. The AAV vector has been evaluated in gene therapy clinical trials for cystic fibrosis, hemophilia, Parkinson's disease, Duchenne's muscular dystrophy and other diseases.

Sands undertook the new research attempting to confirm his previous findings and to determine how the AAV vector might encourage tumor development. He studies a disease in mice that mimics mucopolysaccharidosis type VII (MPS VII), also know as Sly syndrome in humans. This extremely rare lysosomal storage disease affects only about 1 in 250,000 babies, who are born with two disabled copies of the beta-glucuronidase gene. They are unable to manufacture an enzyme that cells need to properly degrade a certain type of complex carbohydrate.

Babies born with the disease often appear healthy at birth, but within a short time they develop skeletal abnormalities and mental retardation. Most do not survive past early adulthood. Sands has been investigating gene therapy in mice as a stepping stone to a potential treatment for the disease in patients. His research, funded by the National Institutes of Health, uses AAV to insert a corrective human gene for beta-glucuronidase into newborn MPS VII mice. Sands has shown he can essentially cure the disease in mice if they are treated immediately after birth, before symptoms of the disease develop. Waiting even one week means that researchers miss the window of opportunity to effectively intervene against the disease.

The current report in Science details the association between the AAV vector and liver tumors in more than 150 newborn mice that were divided into various treatment groups. The researchers found that 33 percent (6/18) of the MPS VII mice that received the AAV-directed gene therapy developed liver cancer. To eliminate the possibility that the liver cancer was tied to the lysosomal storage disease itself and not the AAV vector, Sands and his team gave bone marrow transplants to another group of MPS VII mice. The procedure extends the lives of the animals, providing even more time for the liver tumors to appear, but after 18 months only 4 percent of the mice (1/25) in this control group developed the cancer. None of the mice (0/12) that received radiation to increase the effectiveness of the bone marrow transplant developed the cancer. "If the hypothesis is that the animals are predisposed to developing liver cancer, then ionizing radiation would almost certainly increase that risk, but it did not," Sands says. "Our results show that the genetic defect that causes the disease is not the source of the tumors."

In normal, healthy mice that received the AAV gene therapy, 54 percent (7/13) developed the liver tumors. This is in contrast to 8 percent (4/52) of normal mice that did not receive gene therapy. The researchers found no tumors in a group of transgenic mice whose DNA was altered with the insertion of the human beta-glucuronidase gene using genetic engineering techniques rather than the viral vector. "The data are quite convincing that there is some correlation between AAV and liver cancer in these mouse studies," Sands notes.

The researchers then took six tumor samples from six different mice to determine where in the mouse genome the vector inserted the functional beta-glucuronidase gene. They found evidence of AAV vector insertion in the tumor samples, but not in normal liver tissue. The scientists also located precise insertion sites in four of the tumor samples. In each case, the vector inserted the gene in the same short stretch of DNA - a 6,000 base pair region at the end of chromosome 12. In two of the samples, the insertion sites were only 12 base pairs apart, Sands notes. "We would have expected the insertion sites to be random among the 3 billion nucleotides that make up the mouse genome," he says. "For the vector to insert the gene within one small area is unusual. We don't know why the vector integrates into this region, but we hope future studies will explain this phenomenon."

The region of the mouse genome where AAV inserts the beta-glucuronidase gene is littered with micro RNAs, tiny snippets of RNA that are too short to be involved in synthesizing proteins, a job reserved for messenger RNA. But micro RNAs can silence genes by interfering with the action of messenger RNA, and they have recently been linked to oncogene expression and tumorigenesis. Employing a tool called microarray analysis that examines the expression of thousands of genes simultaneously, Sands' team also found that genes adjacent to the insertion of the AAV vector were dramatically overexpressed compared to genes in normal liver tissues. In fact, the genes nearby the insertion site were expressed at higher levels than any other genes on chromosome 12.

Although more research is essential to understand the link between AAV and liver tumors, Sands suspects that insertion of the vector triggers the overexpression of genes that initiate tumorigenesis. He also notes that the region of the mouse chromosome 12 where the vector inserts the beta-glucuronidase gene is virtually identical to a region of human chromosome 14, in terms of the genes and micro RNAs that are present. It is important to know whether the AAV vector integrates in that same region in humans receiving gene therapy, Sands says.

His group is now conducting a larger study to determine whether AAV consistently jumps into the same region of the mouse genome. He also plans to establish cell lines from the mouse liver tumors that can be used to decipher the mechanism of tumorigenesis in AAV gene therapy.

Even if future studies point to an increased risk of cancer in patients receiving gene therapy with AAV, Sands says that risk may be acceptable for inherited lethal diseases, such as Sly syndrome, for which no other treatment is available. "In devastating diseases like Sly syndrome, if you can provide patients with a good quality of life for an extended period of time, that's a potentially acceptable use of these vectors," he adds.

Donsante A, Miller DG, Li Y, Vogler C, Brunt E, Russell DW, Sands MS. AAV Vector Integration Sites in Mouse Hepatocellular Carcinoma. Science. July 26, 2007.

A grant from the National Institutes of Health supported this research.