Novel Conditioning Strategies Emerge in Gene Therapy, Leaving Busulfan—And Toxicities—Behind

A consensus is beginning to form in hematologic circles that busulfan, an agent used as part of a conditioning process prior to hematopoietic stem cell transplantation that impacts the normal growth of body cells, should be phased out in regimens for patients with conditions such as hemophilia A, Fanconi anemia, and sickle disease due to the significant acute and long-term toxicities associated with the treatment in favor of safer, more targeted agents. These insights, gathered at a session titled “Moving Beyond Busulfan Conditioning” at the American Society of Transplantation and Cellular Therapy’s 2025 Gene Therapy Summit, highlights the evolving trends in gene therapy and shifts in the treatment paradigm poised to occur soon.^1,2

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The session, which was held on September 11, 2025, in Boston, Massachusetts, featured a series of speakers, including chair Sung-Yun Pai, MD, National Institutes of Health; David Wilcox, PhD, professor in the department of pediatrics at Medical College of Wisconsin; Rajni Agarwal, MD, professor of hematology-oncology at Stanford Medicine; and Selami Demirci, PhD, staff scientist in the cellular and molecular therapeutics branch at the National Institutes of Health. Each speaker highlighted a novel approach to conditioning in specific hematologic conditions, showcasing clinical trial data backing up their purported superiority to busulfan.¹

Setting the tone for the conversation, Pai noted the numerous hematologic malignancies that, after decades of research and development, can now be treated with autologous gene therapy. Accordingly, most of them utilize busulfan conditioning, oftentimes in a very high dose. Citing issues of acute and long-term toxicity and its variable completeness of elimination—especially in very young children—Pai acknowledged a consensus in the room that it was time to move on from busulfan when feasible.^1,2

“I think it’s very obvious to the vast majority of people in the room why we would care to move beyond busulfan,” Pai said.¹

Melphalan Conditioning for Hemophilia A

In hemophilia A, an X-linked disorder causing factor VIII deficiency, patients can experience severe, prolonged bleeding episodes and common injuries. Excessive bleeding at the joints is the most common presentation, but bleeding in the digestive tract and intracranial hemorrhage are also severe possibilities. Furthermore, many patients utilizing recombinant factor VIII develop inhibitors, which block clotting factors and make it difficult to control bleeding with traditional treatments. Given the major physical and financial burden, developing novel conditioning regimens—without potential busulfan-associated toxicities—for patients with hemophilia A is a critical focus of many researchers.¹

Wilcox is one such researcher. In a single-center, first-in-human, phase 1 study, Wilcox and his team investigated an ex-vivo, platelet-targeted lentiviral gene therapy, called Pleightlet, that could induce megakaryocytes to synthesize more factor VIII within platelets, shielding the liver and protecting it from the development of inhibitors. Four adult, male patients with severe hemophilia A and inhibitors were enrolled and had infusions manufactured, and 3 were ultimately infused. The study’s primary end point was feasibility of the gene therapy.^1,3

A key aspect of the trial was the conditioning regimen utilized by the investigators. Rather than use traditional autologous stem cell transplant regimens, which can cause myelosuppression and severe complications such as mucositis or stomatitis, Wilcox and his team used a reduced intensity melphalan conditioning regimen in combination with fludarabine (Fludara; Bayer Pharmaceuticals), an agent that suppresses the immune system. By modifying the traditional regimen, concerns about severe bleeding during the gene therapy process could be ameliorated.^1,3

In the trial, the lentiviral gene therapy with fludarabine/melphalan-based conditioning demonstrated efficacy, safety, and robust therapeutic effectiveness, with the average annualized bleeding rate going from 10 to 0, according to Wilcox. There was an expected level of mucositis, and no other acute toxicity reported. Accordingly, there were no serious adverse events, hospital readmissions, exogenous factor VIII, thrombosis, or bleeding. The fludarabine-melphalan conditioning regimen elicited particularly deep and robust responses compared with traditional treatments, and patients experienced improved quality of life following the infusion.^1,3

“[The results] suggest a reduced intensity fludarabine conditioning regimen can be used safely…[with] feasibility and therapeutic effectiveness,” Wilcox said.^1,3

Briquilimab-Based Conditioning for Fanconi Anemia, Severe Combined Immunodeficiency

Next, Agarwal showcased the potential of briquilimab, an anti-human CD117 antibody that is being developed as a non-genotoxic conditioning agent. When undergoing hematopoietic stem cel transplantation, patients require vacating recipient stem cells in the bone marrow so donor engraftment can supplement the missing immune functions in patients with severe combined immunodeficiency (SCID), an inherited primary immunodeficiency disorder. Agarwal and her fellow researchers aimed to investigate a non-toxic approach using a humanized monoclonal antibody, briquilimab, in a phase 1/2 study (NCT02963064).^1,4

Results from the trial, which enrolled patients with SCID who required a second transplant because of engraftment failure and reduced immunity from their previous transplantation, indicated safety and robust myeloid engraftment. Agarwal noted that there were no instances of adverse events or myelosuppression, and patients were suitable for outpatient therapy. Importantly, Agarwal found that the dose of 0.6 mg/kg was optimal for the most uniform, robust engraftment and T- and B-cell production across subgroups.^1,4

“You start seeing a robust myeloid engraftment at the 0.3 mg, but a really more stable, steady response was at 0.6 mg, and the increased dose of 1 mg didn’t give any major benefit,” Agarwal pointed out.¹

Agarwal moved to discussion of another patient population for which poor outcomes necessitate novel approaches: Fanconi anemia (FA). In this condition, caused by mutations in genes involved with DNA repair pathways, the body’s ability to repair DNA damage is altered, putting patients at heightened risk for bone marrow failure and cancer. Using busulfan can worsen the condition of patients, causing widespread tissue damage and systemic toxicities. In a phase 1b trial (NCT04784052), Agarwal and her team developed an alternative approach incorporating briquilimab with anti-thymocyte globulin, cyclophosphamide, fludarabine, and rituximab in patients with FA with bone marrow failure.^1,5

The primary end point of the trial was the safety and engraftment in these patients, with secondary end points including pharmacokinetic measures and immunological recovery. A total of 13 patients were enrolled and 12 were treated, with the results of 3 patients published in the trial. Importantly, there were no treatment-emergent adverse events or acute graft-versus-host disease observed, and patients experienced minimal toxicities. There was a median neutrophil engraftment of 11 days, with robust donor chimerism observed up to 2 years post-procedure, a clear sign of effectiveness.^1,5

‘ESCAPE’ Strategy Preserves Fertility in Sickle Cell Disease

One of possible complications with myeloablative conditioning utilizing busulfan for patients with sickle cell disease is infertility and organ damage, which can turn many patients away from initiating treatment. Demirci’s novel strategy for circumventing busulfan use involves a non-genotoxic, fertility-preserving gene therapy using base gene editing. Demirci noted in his presentation that base editing is often preferred than traditional CRISPR-Cas9 editing because it does not involve DNA cutting, which can create double-strand DNA breaks and unintended genetic changes.^1,6

Demirci and his team utilized multiplex editing of hematopoietic stem cells to modify the CD117 binding site and reactive fetal hemoglobin expression in patients with sickle cell disease. Through this mechanism, the modified CD117 can allow edited cells to “escape” binding by a naked anti-CD117 monoclonal antibody used for conditioning, while unedited host cells are destroyed. Critically, this reduces the likelihood of dangerous toxicities associated with antibody-drug conjugates.^1,6

A series of models in monkeys have demonstrated that dual editing is highly efficient, with the ESCAPE strategy achieving over 90% editing, durable engraftment, and robust fetal hemoglobin activation. Furthermore, the conditioning regimen induced only minor dips in neutrophil and platelet counts that quickly recovered, and the animals involved in the study remained healthy without needing additional supportive care or transfusions. This was the case despite receiving multiple doses of the antibody.^1,6

“I can say that busulfan conditioning remains one of the biggest problems in widely using autologous stem cell transplant-based gene therapies for sickle cell disease,” Demirci concluded. “The ESCAPE strategy can potentially solve this problem because it is non genotoxic.”^1,6

REFERENCES

1. Pai S, Wilcox D, Agarwal R, Demirci S. “Concurrent 3: Moving Beyond Busulfan Conditioning.” Presented: American Society for Transplantation and Gene Therapy 2025 Gene Therapy Summit. September 11, 2025. Joseph B. Martin Conference Center, Boston, Massachusetts.

2. Mayo Clinic Staff. Busulfan (intravenous route). Mayo Clinic. Last Updated September 1, 2025. Accessed September 11, 2025. https://www.mayoclinic.org/drugs-supplements/busulfan-intravenous-route/description/drg-20067303

3. Eapen M, Malec L, Wilcox D, et al. Platelet-Targeted Gene Therapy for Hemophilia A with Inhibitor History. N Engl J Med. 2025;392(4):412-414. doi:10.1056/NEJMc2415164

4. Agarwal R, Weinberg KI, Kwon H, et al. First Report of Non-Genotoxic Conditioning with JSP191 (anti-CD117) and Hematopoietic Stem Cell Transplantation in a Newly Diagnosed Patient with Severe Combined Immune Deficiency. Blood. 2020;10(136):supplement 1. doi:10.1182/blood-2020-137762

5. Agarwal R, Bertaina A, Soco C, et al. Irradiation- and busulfan-free stem cell transplantation in Fanconi anemia using an anti-CD117 antibody: a phase 1b trial. Nat Med. 2025. doi:10.1038/s41591-025-03817-1

6. Demirci S, Mondal N, Austin W, et al. CD117 Antibody Conditioning and Multiplex Base Editing Enable Rapid and Robust Fetal Hemoglobin Reactivation in a Rhesus Autologous Transplantation Model. Blood. 2024;144(516):supplement 1. doi:10.1182/blood-2024-204403