Dog study suggests that AAV in gene therapy could induce cancer

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A recent report on the results of a 10-year gene therapy study in dogs, which was presented on December 9, 2019, at the American Society of Hematology (ASH) meeting in Orlando, Florida, has stirred up old fears that by using a virus to insert therapeutic genes into the human genome, scientists may inadvertently be pushing cancer buttons in the treated cells.

AAVImage Credit: Kateryna Kon /

Gene therapy has always been the Holy Grail of medicine when it comes to conditions that are caused by defective genes. Recently, the use of a harmless virus called adeno-associated virus (AAV), which is capable of infecting human cells without any apparent ill-effects, has made it easier to introduce targeted sequences of corrective DNA into a defective cell to repair a gene.

The inserted DNA formed a loop called an episome that floated free within the nucleus rather than becoming a structural part of the host chromosome.

Why AAV is required

For gene therapy, the replacement gene needs to be injected into the target cell so it can become part of its genetic database. Early experiments on gene therapy used a viral vector; it infected the target cell and thus inserted the whole of its genome into the host cell chromosomes. However, when this was found to have induced tumors in some children, this approach was halted.

This is why the AAV appealed to genetic researchers, and this vector has been behind many novel gene therapies introduced recently. For instance, in 2019, the U.S. Food and Drug Administration approved a gene therapy for spinal muscular atrophy, a hereditary neurological condition that eventually kills the individual.

Another such therapy is designed to treat hemophilia B in which the person has a gene defect that prevents the production of an essential clotting factor. This therapy aims to insert a therapeutic gene into the liver by means of doctored-AAV infection of liver cells, making the liver capable of producing the missing protein. It is slotted for approval in 2020.

Currently, target genes are often injected into a modified version of the AAV, which is used as a ferry or a vector to get the gene into the cell in a way that will ensure it remains intact. The use of the AAV was justified by previous observations that it hardly ever incorporates its own genome into the host chromosomes, minimizing the risk that it could introduce cancer-causing mutations. The new study shows that this might indeed happen.

The study

The study involved nine dogs with hemophilia A, caused by the genetic deficiency of the crucial clotting factor called Factor VIII, which was treated by gene therapy. The AAV virus inserted the therapeutic gene successfully. The dogs were sacrificed once the experiment ended after a follow up of up to 10 years, and 6 of the livers were examined.

The findings

Seven of the nine dogs that had received the replacement gene showed a stable increase in the level of Factor VIII in 7 of the animals. In the other two dogs, the increase continued to occur after a period of about three years, and eventually, these dogs had a fourfold rise in Factor VIII by the end of 7-8 years.

The scientists found that in all 6 of the livers examined after the experiment ended, the DNA introduced by the AAV vector had inserted itself into the dog’s genome at multiple loci. In some cases, it was the Factor VIII gene, but in many more, it was a part of a regulatory sequence that was incorporated in spotty fashion across the dog chromosomes. With some of these, the regulatory fragments were perilously located near genes that control cell growth.

In other words, the viral vector was indeed injecting the modified gene into the host cell’s chromosomes. Some insertions occurred at critical sites where the cell’s growth was regulated. As a result, it is possible that it could interfere with normal cell growth regulatory genes to tip the cell into a cancerous phenotype, where non-regulated cell growth and proliferation occurs to form a tumor.

The researchers also found that there were clumps of cells derived from a single infected cell at certain places within the liver, all showing the same insertions of genetic material. This shows that cell division occurred in these cells at a higher rate than in other cells.

They suspect that this could have been the result of growth activation by the inserted regulatory genes, though they will need to do more work to prove it. This would explain why the clones of modified cells occurred, and why Factor VIII levels rose so markedly in 2 dogs.


The current finding was predicted almost two decades ago, ever since it was found that newborn mice injected with high doses of AAV showed its genome becoming part of their own, and causing liver cancer. The fact that newborn mice are not physiologically comparable to adult humans lulled such fears for a while. However, the new study reports findings from older, larger adult dogs treated with AAV-delivered gene therapy.

The scientific community is divided in its response. Some scientists who took part in the first successful gene therapy trial for hemophilia B say the findings weren’t unexpected: the level of integration of the introduced DNA was low, the livers seemed apparently healthy and functional, and factor VIII levels remained constant.

Integration is, in fact, a good thing because it ensures that the inserted DNA will pass down through generations of cells, and the patient will, therefore, continue to show the beneficial effects for many years. On the other hand, an episome doesn’t take part in the normal duplication of DNA that occurs when a cell divides, and will, therefore, pass down to only one of the daughter cells formed. As a result, it could be lost over time, and its effects could be no longer seen.

But other scientists point to the known tendency of cells to acquire mutations. They ask, what if these clones of mutated cells get another mutation that drives growth? They would then give rise to tumors in the tissues treated with such AAV treatments.

The take-home

The current study thus points to the need to search for signs that this is happening in other dog studies carried out over the long term, and in the scheduled biopsies from the hemophilia B patients treated with gene therapy at the St. Jude Children’s Research Hospital. Scientists like Lillicrap are already investigating this possibility in their own experimental animals.

The key recommendation they make is that the current FDA protocol mandating a five-year screening period for liver cancer for all people who have received AAV-vectored genes is insufficient in the light of this new evidence: instead, they need to be followed up much longer.


Kaiser, J. (2020). Virus used in gene therapies may pose cancer risk, dog study hints.


Kaiser, J. (2020). Virus used in gene therapies may pose cancer risk, dog study hints. DOI: doi:10.1126/science.aba7696

Dr. Liji Thomas

Written by

Dr. Liji Thomas

Dr. Liji Thomas is an OB-GYN, who graduated from the Government Medical College, University of Calicut, Kerala, in 2001. Liji practiced as a full-time consultant in obstetrics/gynecology in a private hospital for a few years following her graduation. She has counseled hundreds of patients facing issues from pregnancy-related problems and infertility, and has been in charge of over 2,000 deliveries, striving always to achieve a normal delivery rather than operative.


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