New gene-editing treatment for hemophilia B able to fix DNA in mice
Scientists developing potentially 'curative intervention' without viruses
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- Scientists in Japan are developing a new gene-editing treatment for hemophilia B that does not use viruses to work.
- The novel strategy corrected hemophilia B mutations in a mouse model, restoring clotting factor IX.
- While further study is needed, the researchers say the treatment is potentially curative for hemophilia B.
A new type of gene-editing treatment being developed by researchers in Japan corrected mutations causing severe hemophilia B in mice, restoring production of the missing clotting factor IX (FIX), which causes the rare bleeding disorder.
The scientists say their novel nonviral treatment — which does not use an inactivated virus to deliver its genetic payload into the body — lays the groundwork for a potentially safer approach to treating hemophilia B that’s less likely to trigger the immune reactions sometimes observed with viral treatments.
In fact, the researchers concluded, based on these lab findings, that this gene-editing system could represent “a truly curative intervention for severe hemophilia B.”
Still, the team noted that “continued preclinical evaluation will be essential to establish the safety and efficacy of this approach and pave the way toward clinical translation,” or the use of the new treatment in humans. The strategy aims for “personalized correction” of the mutations that cause hemophilia B, according to the team.
The research findings were detailed in “Nonviral delivery of a base editor enables personalized correction of hemophilia B nonsense variants in a mouse model,” a study published in the Journal of Thrombosis and Haemostasis.
Hemophilia B is caused by mutations in the F9 gene that produce little to no, or a nonfunctional version of, FIX, a protein needed for blood to clot. As a result, patients experience easy and excessive bleeding. Standard treatment is with factor replacement, in which patients receive regular infusions of FIX to restore clotting and prevent bleeding. However, such treatment is lifelong.
Another option is gene therapy, which uses a virus to deliver a healthy copy of the F9 gene to liver cells so they can produce functional FIX on their own. While gene therapy can restore FIX production for many years, it does not correct the disease-causing mutation, and its use may be limited by immune responses and challenges with repeat dosing.
These limitations led scientists at two Japanese universities to try a different approach.
Scientists aiming to directly repair specific disease-causing mutations
The researchers aimed to develop a more permanent solution to the mutations underlying hemophilia B by using base editing. This form of gene editing changes a single DNA building block without cutting both DNA strands.
Their goal was to directly repair specific disease-causing variants in the F9 gene, while also avoiding the use of a viral vector for delivery. Instead, the scientists used lipid nanoparticles, which are very small, fat-coated, round particles.
The team focused on six nonsense mutations. A nonsense mutation inserts a premature stop signal in the genetic code, causing cells to produce an incomplete and nonfunctional protein. All six mutations were located in exon 8 of the F9 gene, a region that contains instructions for the critical catalytic domain of FIX, which harbors the active site responsible for its action in the clotting cascade.
To test their approach, the researchers first engineered lab-grown human cells carrying each mutation. The editing system combined ABE8e, a molecular editor that converts one DNA building block (A) into another (G), with a modified Cas9 enzyme that recognizes target DNA sequences. Guide RNAs, a type of genetic molecule, directed the editor to the exact target mutation.
This editing system successfully corrected several mutations, particularly c.1067G>A, c.1068G>A, c.1222C>T, and c.1292G>A, according to the researchers, who noted that correction of these mutations increased FIX production. The editing system was less effective for two other mutations, resulting in limited FIX recovery, the data showed.
Unintended edits, known as bystander edits, can sometimes occur when nearby DNA building blocks are changed along with the target mutation. Most bystander edits were harmless because they did not alter the resulting protein. However, some could potentially change amino acids, the building blocks of proteins, and reduce the protein’s function, the team noted.
Lipid nanoparticles carrying the molecular editor, Cas9 enzyme, and guide RNAs were then tested in a mouse model of hemophilia B. To create the model, the researchers used an adeno-associated virus (AAV) vector to deliver and introduce human mutations into the DNA of mice. These mice had little or no detectable FIX, closely resembling severe hemophilia B.
After injection of the lipid nanoparticles into the bloodstream, the editing system entered liver cells. The treatment corrected a large percentage of disease-causing mutations, with editing rates reaching 70.6% for c.1292G>A, 62.8% for c.1068G>A, and 35.9% for c.1222C>T. These corrections restored FIX production, which was sustained for at least 22 weeks, the researchers noted. As a result, clotting times improved.
Few off-target effects seen with new treatment in lab
The researchers also examined off-target effects, which are unintended changes at locations other than the target mutation.
Most potential off-target sites were found in noncoding regions of DNA, where they are less likely to cause harm. Although some possible edits were identified in protein-coding regions, these genes showed very low activity, suggesting limited biological impact, according to the team.
Overall, per the researchers, this study demonstrates that nonviral base editing delivered by lipid nanoparticles can directly repair specific hemophilia B mutations and restore factor IX function.
In this study, we achieved precise correction of pathogenic [disease-causing] variants [of hemophilia B].
“Compared with AAV-based delivery, the transient nature of [lipid nanoparticle]-mediated genome editing tool delivery provides distinct advantages, including the possibility of repeated dosing [and] reduced immunogenicity,” or immune reactions, the researchers wrote.
“In this study, we achieved precise correction of pathogenic [disease-causing] variants” of hemophilia B, the team wrote.
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