Gene therapy completely corrects hemophilia in laboratory animals

Newborn mice and dogs with hemophilia A were restored to normal health through gene therapy developed by researchers at Washington University School of Medicine in St. Louis. The technique introduced into the animals’ cells a gene that makes clotting factor VIII, a protein missing because of a genetic defect.

“We are really pleased with the results, because the animals produced about 20 times more factor than has been achieved in prior attempts using gene therapy for hemophilia A in dogs,” says senior author Katherine Parker Ponder, M.D., associate professor of medicine and of biochemistry and molecular biophysics.

In addition, the technique using newborn animals had the advantage of not prompting an immune response, which in many other cases eventually blocks the blood clotting activity of introduced factor VIII in hemophilic animals. Since treatment more than a year ago, the blood of the mice and dogs in this study has maintained a normal level of clotting factor activity, and the animals have had no incidents of bleeding. The study will be reported in the April 26 issue of the Proceedings of the National Academy of Sciences.

Hemophilia is an inherited bleeding disorder caused by genetic mutations on chromosome X that prevent normal production of certain blood clotting factors. A defective gene for clotting factor VIII is responsible for hemophilia A, the form occurring in 80 percent of cases. Because females carrying a defective gene can rely on a normal copy of the gene on their second X chromosome, hemophilia almost always occurs in males. One in 5,000 males are born with the disorder.

“Hemophilia greatly restricts patients’ everyday lives,” says Ponder, a hematologist at Barnes-Jewish Hospital. “People with the disease don’t heal well after injuries or surgery. Even running can cause bleeding into the joints.”

For their own safety, hemophiliacs must be near a refrigerated supply of clotting factor at all times. Over the long term, hemophiliacs suffer from joint damage and other complications related to excess bleeding.

Gene therapy for hemophilia A has been especially challenging because the gene for factor VIII is quite large and therefore hard to fit into viral vectors, which serve as the gene delivery vehicle. The researchers eliminated parts of the factor VIII gene and other genetic components to minimize the material needed and used a large viral vector called gamma retroviral vector.

The viral vector carrying factor VIII genes was injected into the blood of 11 newborn hemophilic mice and two newborn hemophilic dogs. The viral vector also contained a short DNA promoter sequence to make the gene active only in liver cells, one of the sites of factor VIII production in non-hemophiliacs.

The normal mechanisms of viral reproduction enabled insertion of the genetic material from the engineered vectors into cells in the animals. After treatment, blood tests demonstrated all of the treated animals were producing factor VIII. The mice achieved an average of 139 percent of normal factor VIII activity and the dogs an average of 115 percent of normal factor VIII activity in a blood clotting assay. This activity level has remained stable for one and a half years. In comparison, untreated animals with hemophilia A have less than one percent normal factor VIII activity.

“This level of expression of factor VIII in dogs is especially interesting, because in other attempts the results in large animals have not been successful,” Ponder says.

The researchers worked with newborn animals for two reasons. First, their livers are still growing. So genes integrated into a liver cell will be reproduced with each new generation of cells, increasing the number of cells containing functional clotting factor genes in the adult animal.

Liver tests done when the animals were about a year old showed that the treated mice had an average of two factor VIII genes per liver cell. In the dogs, an average of one in eight liver cells had the new gene.

Second, newborn mice and dogs have a less mature immune system than do adults, making it less likely they will raise an immune response to the introduced factor VIII. The immune reaction, known as inhibitor formation, diminishes the activity of the clotting factor and has caused failure in previous attempts to correct hemophilia in mice using gene therapy.

The animals in this study have not formed inhibitors against the factor VIII protein after more than a year of follow-up.

“Naturally, the ultimate goal is for gene therapy to work in humans, but humans have a more mature immune system at birth than mice,” Ponder says. “In animals more closely related to humans, there will probably be more risk of inhibitor formation, so the next step needs to be gene therapy trials in primates with hemophilia to see if we can prevent inhibitor formation.”