General Updates | Emerging non-AAV gene therapy strategies in hemophilia: opportunities and challenges

AAV has established proof of principle in hemophilia, but I do not think it will be the final platform for every patient. Pre-existing capsid antibodies, the difficulty of redosing, variable expression, liver toxicity, dilution of episomal genomes as a child’s liver grows, and several additional challenges inherent in factor 8 gene transfer that I described earlier are all driving the search for alternatives, especially now that the only approved gene therapy for hemophilia A has been withdrawn from the market. The most clinically advanced non-AAV strategy is ex vivo lentiviral gene therapy using a patient’s own hematopoietic stem cells. So CD34 positive cells are collected, modified outside the body with an integrating lentiviral vector and returned after conditioning. In hemophilia A, one active program combines a bioengineered factor VIII called ET3 with a CD68 promoter, which directs expression mainly to the monocyte macrophage lineage rather than to hepatocytes. The initial clinical experience in India is still small but encouraging. The first two participants achieved factor VIII levels of approximately 2% to 5%. After the transduction process was improved, the next three individuals reached roughly 19% to 40%, which is curative functionally, and no spontaneous bleeding was reported during follow-up of up to 27 months. So this approach may provide more stable expression, avoid systemic AAV exposure, and pre-existing capsid immunity. The trade-off is that conditioning can cause transient cytopenias and may carry longer-term risks while an integrating vector raises concerns about insertional mutagenesis. It is also a complex individualized treatment rather than a simple off-the-shelf infusion. Another major direction is targeted gene insertion; the recruiting BEYOND-9 study in hemophilia B uses an AAV8 vector to deliver the factor 9 donor sequence and a lipid nanoparticle to deliver Cas9 messenger RNA and guide RNA, inserting factor 9 into the albumin locus. It is therefore a hybrid rather than a completely AAV-free approach. However, the important difference from conventional AAV therapy is that the therapeutic gene is placed into a defined chromosomal site instead of remaining mainly episomal. In principle, that could improve persistence through cell division and eventually make treatment of younger patients possible. The protocol includes adult, adolescent, and pediatric cohorts. It still requires AAV8 eligibility, however, so it does not solve every limitation of AAV. For hemophilia A, related gene editing work has produced a durable factor VIII expression in non-human primates for more than a year. Even more forward-looking are lipid nanoparticle-only systems designed to deliver the editing machinery and genetic cargo without any viral vector. Those remain preclinical, but they may eventually avoid anti-AAV immunity and permit redosing. The central question now seems to be innate immune toxicity of delivering DNA using those lipid nanoparticle systems because DNA payloads require much higher LNP doses than RNA delivery. A promising clinical program using engineered B cells that are differentiated into long-lived plasma cells producing factor 9 has unfortunately been discontinued very recently. So we have suffered some new setbacks already, but hopefully one or any number of the other approaches will make it and show true benefit. Overall, I do not expect any technology to replace AAV in every setting. AAV offers the simplicity of a single infusion. Now then, stem cell therapy may provide durability despite anti-AAV antibodies, and gene editing may eventually open the door to pediatric treatment. And intuitively, that’s the one approach that has all the makings of the desired curative therapy for hemophilia. But ultimately, only time will tell.

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