Gene therapies promise hope for patients with hereditary diseases such as hemophilia, in which important clotting factors are absent. The idea: With the help of viruses, intact genetic material should be smuggled into the human cells so that they then produce the necessary coagulation factors. The first gene therapies have already been approved for humans. Further animal studies are running in parallel to assess safety and effectiveness more precisely. Researchers have now presented the results of a ten-year study on dogs. Despite the overall positive results, the researchers warn on the basis of genetic analyzes that gene therapy could in rare cases increase the risk of cancer.
When the first human patient received gene therapy 30 years ago, in November 1990, expectations were high: Intact genetic material, smuggled in with the help of adenoviruses, should permanently cure the then four-year-old Ashanti deSilva of her congenital immune deficiency. Despite initial reports of success, the euphoria quickly passed. Gradually, the inserted genetic material disappeared again from the girl’s cells, the effect wore off. Other children developed leukemia as a result of gene therapies – a sign that the repair genes had been inserted in the wrong places in the genome. Although several gene therapies have now been approved for humans, these challenges still exist: The intact genetic material should remain in the human cells as permanently as possible, but not insert itself in undesired places.
Bleeding tendency significantly reduced
To what extent this works in gene therapy against hemophilia, researchers led by Giang Nguyen from the Children’s Hospital of Philadelphia have been researching dogs for ten years. The nine animals that took part in the study suffered from haemophilia A, a variant of the hemophilia that lacks clotting factor 8. With the help of adeno-associated viruses (AAV), the researchers smuggled intact gene variants for factor 8 into the dogs’ livers.
And indeed: in all nine animals the gene therapy ensured sufficiently high factor 8 levels. Although they only produced between 1.9 and 11.3 percent of the normal amount of factor-8 for dogs, their bleeding tendency was reduced by 97 percent compared to untreated dogs, to less than one spontaneous bleeding per year. This corresponds to the value of people with hemophilia who are regularly injected with the missing coagulation factor. “Our data show that even low factor 8 levels, which are achieved through gene therapy, substantially improve the appearance of the disease,” said the researchers.
Long-term effectiveness
Adeno-associated viruses are known to only seldom integrate their genetic material into human DNA. Instead, the viral DNA with the inserted genes is usually separate from the human genome in the cell nucleus. This minimizes the risk of cancer developing through incorrect insertions, but also ensures that the inserted genetic material can disappear from the human cells again over time.
In the dogs observed by the researchers, the initial factor 8 activity was maintained over the entire ten-year study period. “This is the longest maintenance of a therapeutic factor 8 level that has so far been observed in studies with large animals with haemophilia A, combined with a significant reduction in spontaneous bleeding,” write the Nguyen and colleagues. None of the dogs had any serious side effects. Regular tests of the liver values showed only slight deviations from the norm.
Surprising increase
Instead of falling factor 8 levels, the researchers even observed a slow increase in coagulation factor in two dogs, starting four years after gene therapy. In the course of the study, the level in these animals increased from initially four percent of the normal value to ten and eleven percent, i.e. it almost tripled. The researchers cannot explain this increase with certainty. “The most likely cause is that the inserted DNA has integrated into the genome of the dog where it promotes cell division,” said the researchers. This could have caused the new genes to multiply, causing the higher factor 8 production.
“As far as we know, no other study in large or small animals has shown an increase in factor 8 production after gene therapy,” the researchers write. In the dogs in their study, the increase was harmless to health, because the factor 8 level was still well below the concentrations in healthy animals. “However, factor 8 levels that are too high can lead to thrombosis and would be worrying,” emphasize the researchers. They therefore recommend monitoring human patients after gene therapy over the long term to ensure that too much factor-8 is unexpectedly produced.
Cancer risk may be increased
The researchers also see a reason for warning in other respects: Although none of the dogs developed tumors or precancerous stages during the study, Nguyen and colleagues found indications that the viral and the repaired DNA that was introduced into the dog’s genome was found in some places integrated – and disproportionately often in regions that are associated with the development of cancer. “44 percent of the insertions occurred near genes that are involved in cell growth,” they report. Overall, such insertions into the genome are rare. In these rare cases, however, they can increase the risk of liver cancer.
“Our results raise questions for ongoing gene therapy studies against hemophilia and for other AAV gene therapy studies,” the researchers summarize. “Although clinical studies with a three-year follow-up period for hemophilia A and a ten-year follow-up period for hemophilia B have not reported serious side effects, our data demonstrate the importance of long-term monitoring of patients after AAV gene therapy.”
Source: Giang Nguyen (Children’s Hospital of Philadelphia) et al., Nature Biotechnology, doi: 10.1038 / s41587-020-0741-7