New Gene Therapy Shows Promise in Hemophilia A Mouse Model, Study Shows

Cells derived from hemophilia A patients — genetically reprogrammed to produce a functional clotting factor VIII (FVIII) that’s missing or not working in people with the blood disease — were successfully grafted into hemophiliac mice, restoring blood levels of FVIII and significantly improving clotting, a study showed. 

If it’s found to be applicable to humans, this new form of gene therapy may be useful not only for treating hemophilia, but as a therapy for any medical condition that requires long-term blood protein replacement.

The study, “Bioengineering hemophilia A — specific microvascular grafts for delivery of full-length factor VIII into the bloodstream,” was published in the journal Blood Advances. 

Hemophilia A is an inherited form of the bleeding disorder, characterized by the lack of blood-clotting FVIII. FVIII normally is produced — with instructions provided by the F8 gene — by specialized cells in the liver, as well as by cells known as endothelial cells, found throughout the body, that line the insides of blood vessels.

Replacement therapy, or the infusion of FVIII concentrates to increase the levels and activity of the missing clotting factor in the body, currently is one of the standard therapeutic strategies used to manage hemophilia A.

“However, patients require repeated IV [intravenous] injections of the factor multiple times per week throughout life, which creates continuous discomfort, augments morbidity, and impairs overall quality of life,” the researchers said.

Alternative approaches such as gene therapy — which treats the underlying cause of the disease and not just its symptoms — have been attempted in animal models. Gene therapy uses harmless viruses as vehicles for replacing the defective F8 gene with a fully functional one.

However, F8 is a large gene that cannot be packaged in existing viral vectors. Small (truncated) versions of the gene also have been used, but so far without success. 

“The gene for factor VIII is enormous,” Juan Melero-Martin, PhD, a researcher in the department of cardiac surgery at Boston Children’s Hospital, and lead author of the study, said in a news story.

“You can try to deliver it with a virus, but either the gene doesn’t fit into the virus, or the virus can carry it but cannot insert the gene permanently into the genome [all genes in a person’s DNA],” Melero-Martin said. “Some people have tried truncating the gene.” 

Given the lack of success, Melero-Martin’s team decided to try a different approach. The team developed a completely new form of gene therapy that uses a patient’s own, engineered cells to create endothelial cells that are able to produce functional FVIII. 

“With our system, we were able to introduce the full factor VIII protein,” Melero-Martin said.

In collaboration with colleagues at the Dana-Farber/Boston Children’s Cancer and Blood Disorders Center in Massachusetts, the researchers collected urine samples from seven patients with severe hemophilia A and isolated epithelial cells lining the surface of the urinary tract. Epithelial cells from urine were chosen over blood cells because drawing blood from patients with hemophilia A can be problematic. 

“For a patient who has a bleeding disorder, it’s not trivial to draw blood,” Melero-Martin said. “We would have to wait until blood being drawn for a clinical purpose, and it was difficult to get a sufficient amount of cells.”

So the tried a different approach.

“If you spin urine, you can capture enough epithelial cells shed off by the urinary tract,” Melero-Martin said. “I was skeptical myself, but it worked beautifully.”

The urine-derived epithelial cells were then converted to induced pluripotent stem cells (iPSCs). iPSCs are a type of stem cell that can be created from most cell types and reprogrammed back to a stem cell state, in which it may give rise to any cell type of choice. 

Next, the team inserted multiple copies of the full-length F8 normal gene into iPSCs and reprogrammed them to create large quantities of modified endothelial cells, dubbed HA-FLF8-iECs. 

“Once you have an iPS line, you theoretically have an unlimited number of cells from a given patient,” Melero-Martin said.

To test the therapy’s efficacy, the investigators implanted the newly derived endothelial cells under the skin of hemophiliac mice. Non-engineered cells that did not contain the modified F8 gene served as controls. 

After seven days, both edited and unedited cells formed networks of blood vessels that connected to the host mice’s bloodstreams. Tests showed that implants of the F8 edited cells produced significantly higher levels of FVIII compared with controls.

A standardized tail-bleeding test was then performed to assess bleeding and coagulation. Compared with controls, the mice implanted with HA-FLF8-iECs had significantly less body weight loss — a measure of blood loss — and shorter bleeding time. 

Analyses of the blood drawn from treated mice showed there was a significant increase in FVIII activity that was, on average, six times higher than controls. 

“After the implant, there was a 600 percent increase in circulating factor VIII,” said Melero-Martin. “We’re super excited about the levels of the protein we achieved. Even by producing just some factor VIII, you can go from severe disease to mild.”

The team already is planning future studies to determine how far the new gene therapy can be pushed. The goals are to produce enough FVIII for human application, as well as to find the best site in the body to place the implant and to determine how long the implants will remain functional. These studies may be performed in larger animal models that will serve as an intermediate step between mice and humans.

If supported by further study, this new gene therapy could be useful not only for treating hemophilia but also any medical condition that requires long-term replacement of a protein, Melero-Martin said. The first goal is to determine if this treatment may be able to eliminate the need for the multiple injections each week that people with hemophilia now require.

“We hope the implant would only need to be replaced once every year, or every two years,” he said. “With our system, there is almost no limit to how big the gene can be.”