UF Scientists Find Enzyme Protects Cells During Gene Therapy

February 2, 2004

GAINESVILLE, Fla. — An enzyme instrumental to the body’s ability to repair itself appears to also stop a gene therapy virus from damaging a cell once the virus delivers its corrective cargo, University of Florida scientists report today (Feb. 2) in the online edition of the Proceedings of the National Academy of Sciences.

Gene therapy has been heralded as one of medicine’s most promising frontiers, but as with any unchartered territory, dangers lurk. A key concern has been whether the viruses used to ferry therapeutic genes into cells to treat diseases such as Parkinson’s or diabetes also cause a second, unwanted effect: genetic mutations that ultimately trigger cancer. That fear was heightened last year when two boys with an immune system disorder developed leukemia after undergoing gene therapy in France using a retrovirus.

Now UF researchers say their findings add to mounting evidence that the adeno-associated virus, or AAV, is potentially safer than other gene transport viruses that have been associated with harmful side effects.

“One of the major concerns in gene therapy research is that the insertion of a new gene into the genome of the cell might somehow trigger a new tumor or cancer of some kind,” said Dr. Terence R. Flotte, the Nemours eminent scholar and chairman of the department of pediatrics at UF’s College of Medicine. “That is a scenario that has been well-established to cause tumors in mice and birds. One of the observations we’ve had for a long time is that AAV, as compared to other viruses that have been used for gene therapy, doesn’t cause any disease that we know of, including cancer. So the question was, is this risk associated with a retrovirus gene therapy vector also present in the case of AAV?”

Flotte pointed out that UF researchers did not set out to directly assess safety issues associated with using AAV, but the study did yield a better understanding of the mechanism by which it works – insight that led them to presume any risk linked to AAV is lower than that posed by other gene therapy methods.

In the early 1980s, study co-author Dr. Kenneth I. Berns, who now directs the UF Genetics Institute, helped lead the effort to modify and patent AAV for use as a vector for transporting the corrective genes used in gene therapy. UF geneticists, led by Flotte, pioneered the first AAV gene therapy trial in patients with cystic fibrosis. UF also produces the world’s reference standard AAV vector with National Institutes of Health support.

AAV is attractive to scientists in part because of its unique biology. It is apparently harmless to humans and expresses corrective genes for long periods of time in animal models.

UF scientists studied what would happen when AAV was introduced in cell cultures. They also examined its effects on healthy mice whose cells produced the enzyme known as DNA-dependent protein kinase and on mice with an immune deficiency syndrome whose cells lacked the enzyme. Liver cells were taken from the mice and analyzed to see whether they retained AAV’s own genetic material and whether the therapeutic gene had been successfully transferred.

Researchers discovered that once AAV is introduced into a cell, it efficiently transfers the corrective gene it is carrying. But the virus has its own genetic material that could wreak molecular mayhem by seeking to insert itself into a cell’s nucleus, if it weren’t for the cell’s self-repair mechanism, which swings into action. This molecular “fix-it” kit quickly sequesters AAV’s own DNA then handily packs it up into a large new chromosome. In this safe form, the viral DNA rarely invades a cell’s own genetic machinery.

“That mechanism – the fact that when AAV enters a cell it turns on DNA repair – creates a scenario where AAV is probably much safer than a retrovirus from the standpoint of cancer risk,” Flotte said. “This finding is potentially of major significance because there is so much attention on the cancer risk of gene therapy. This is a real direct indication that AAV can get around that.”

Researchers noted that mice with compromised immune systems lacked the enzyme, and in some cases the viral DNA did integrate into their cellular genomes.

That led the scientists to conclude that the enzyme inhibited AAV integration, said Sihong Song, the study’s lead author and an assistant professor of pharmaceutics at UF’s College of Pharmacy.

“So far we think AAV is the safest vector, because this virus itself never causes disease,” Song added. “And so far, several clinical trials using this vector have never shown any vector-related side effects.”

In some cases, UF researchers said, scientists might actually prefer for a viral vector to incorporate itself into a cell’s genetic wiring, and in those instances AAV might not be the best choice, since in many cases it isn’t likely to do so.

“If you’re going to use a bone marrow stem cell that could be important for regenerating some tissue or cell type, then we might want the integrating vector to stay with the cell as the cell repopulates an entire organ or cell type in the body,” Flotte said. “Our findings would suggest that under these conditions this kind of vector (AAV) is not ideal. For something where we want to inject a virus directly into the lung, liver, muscle, retina or brain and just have those cells express the gene, then we might be able to do that more safely with this kind of vector – that is our interpretation.”