Scientists working toward next-generation hemophilia B gene therapy
Longer-lasting clotting protein boosts treatment's effectiveness in mice
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- Hemophilia B is a genetic condition caused by missing or deficient clotting protein known as factor IX, or FIX.
- Scientists in Belgium are working on next-generation gene therapies to extend the protein's half-life and improve treatment effectiveness.
- Now, in a study using a mouse model, the team showed one therapeutic approach boosted clotting factor levels while maintaining good safety.
A gene therapy that delivers instructions to produce a longer-lasting version of the clotting protein missing or deficient in hemophilia B may improve treatment effectiveness while maintaining a favorable safety profile, a study in mice suggests.
Researchers in Belgium, working in a lab, combined the genetic instructions for a highly active form of that clotting protein, known as factor IX or FIX, with those for an engineered version of albumin, a blood protein. The resulting instructions allow liver cells — the body’s main producers of clotting proteins — to make FIX attached to albumin, which helps protect the clotting factor from breakdown.
The end goal was to extend FIX’s half-life, which is the time it takes for half of its circulating protein to be cleared from the bloodstream.
The team then tested a gene therapy delivering this fusion construct, called FIX-R338L-ALB(QMP), in a mouse model of hemophilia B. In the mice, use of the gene therapy significantly increased blood FIX levels and activity, and shortened blood clotting times, compared with a matching therapy delivering genetic instructions encoding the highly active FIX alone.
“These findings support the potential of FIX-R338L-ALB(QMP) fusion constructs encoding [extended half-life] FIX as a promising next-generation gene therapy for hemophilia B,” the researchers wrote, adding that current treatments “show significant interpatient variability in clinical outcomes.”
The study, “Increased gene therapy efficacy through the use of extended half-life clotting factors,” was published in the journal Blood Advances by researchers in the department of gene therapy and regenerative medicine at Vrije Universiteit Brussel.
Hemophilia B is caused by mutations in the F9 gene that result in absent, insufficient, or poorly functioning FIX. Without enough working FIX, blood cannot clot properly, leading to excessive or prolonged bleeding.
Researchers turn to viral vectors for longer-lasting FIX
Gene therapy delivers genetic instructions to liver cells so they can produce FIX over the long term, potentially reducing or eliminating the need for regular infusions of factor replacement therapy. These instructions are commonly carried into cells by a modified adeno-associated virus, or AAV.
Some gene therapies encode FIX-Padua, also called FIX-R338L, a naturally occurring variant with higher clotting activity than the usual protein. Although this approach has improved treatment outcomes, responses vary, and some patients still need preventive FIX infusions, the research team noted.
Increasing gene therapy dosage may improve treatment effectiveness, but can also raise the risk of immune reactions associated with the viral carrier, as well as liver injury.
Previous research had shown that attaching FIX-R338L to albumin allows the clotting protein to benefit from a natural recycling system that rescues the fused protein from breakdown inside cells and returns it to the bloodstream. This approach is used in extended half-life replacement therapies, allowing fewer infusions while maintaining bleeding control.
FIX-R338L’s half-life was further prolonged in subsequent studies by attaching it to human albumin containing three changes, referred to as albumin-QMP.
Building on this work, the team now investigated whether the overall efficacy of gene therapy could be enhanced using viral vectors carrying FIX-R338L-ALB(QMP) — the genetic fusion construct encoding the longer-lasting FIX-R338L.
The team packaged the construct into two viral carriers, AAV8 and AAVDJ/8, that preferentially deliver genetic material to liver cells. AAV8 is a commonly used viral vector, while AAVDJ/8 is an engineered viral vector that has been shown to be less readily recognized by human antibodies than AAV8 and to enter both human and mouse cells.
FIX levels in mice 4 times as high with this gene therapy
In mice without hemophilia, the researchers tested three gene therapy doses using each viral carrier. Across the doses tested, FIX-R338L-ALB(QMP) resulted in significantly higher blood FIX levels than the matching construct encoding FIX-R338L alone. Sustained FIX levels were observed through 33 weeks after treatment, the researchers noted.
The team then tested both constructs in mice with hemophilia B, using AAV8 as a viral vector and the same gene therapy dose. Nine animals received the FIX-R338L-ALB(QMP) construct, and seven received the construct encoding FIX-R338L alone.
Gene therapy delivering FIX-R338L-ALB(QMP) yielded blood FIX levels 4.1 times as high and activity 2.6 times as high as the comparison therapy. Blood samples also clotted significantly faster, the researchers noted.
Neither group had detectable FIX-targeting antibodies at 10 weeks. Such antibodies, which can develop in patients receiving FIX replacement therapy, block the protein’s activity, ultimately making treatment less effective.
The team then tested whether the immune system would continue to tolerate FIX even when deliberately stimulated to react against it. To do so, the scientistis challenged the mice’s immune systems by injecting albumin-linked FIX into the FIX-R338L-ALB(QMP) group and FIX into the FIX-R338L group, each together with an immune-stimulating substance.
After this challenge, one of the six mice in the FIX-R338L-ALB(QMP) group developed FIX-targeting antibodies, compared with three of four in the FIX-R338L group. This suggested that most mice given the FIX-R338L-ALB(QMP) construct had developed immune tolerance to the protein.
This novel [treatment tool] may improve the outcome of future hemophilia B gene therapy clinical trials.
Safety testing found no increase in D-dimer, a blood marker used to assess unwanted clotting, even at the highest gene therapy dose used in mice without hemophilia. Markers of liver toxicity were also not elevated at doses the researchers described as clinically relevant. However, using a higher gene therapy dose in a separate experiment in non-hemophilic mice led to liver injury and immune cell infiltration.
“These findings suggest that the combination of the hyperactive FIX-R338L variant with the albumin-QMP fusion strategy can further enhance the therapeutic potential of AAV-based gene therapy for hemophilia B,” the researchers wrote. “By enabling higher sustained FIX, this approach may improve the overall feasibility of clinical translation, thereby representing a promising advancement for durable and safe hemophilia B treatment.”
The scientists noted that, to their knowledge, “this is the first study that successfully integrates a bioengineered [extended half-life] albumin-based FIX fusion with gene therapy.”
According to the team, “this novel [treatment tool] may improve the outcome of future hemophilia B gene therapy clinical trials.”
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