GEN BiotechnologyVol. 2, No. 3 News FeaturesFree AccessBeyond Busulfan: Building a Better Conditioner for Bone Marrow TransplantationJonathan D. Grinstein and Alex PhilippidisJonathan D. GrinsteinE-mail Address: jgrinstein@genengnews.comSenior Editor, GEN Media Group; and GEN.Search for more papers by this author and Alex PhilippidisE-mail Address: aphilippidis@genengnews.comSenior Business Editor, GEN.Search for more papers by this authorPublished Online:19 Jun 2023https://doi.org/10.1089/genbio.2023.29103.jdgAboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail Researchers are looking for less toxic alternatives to busulfan as a bone conditioning regimen, a crucial component of cell and gene therapy protocols.On July 2, 2019, Victoria Gray, an African American mother of four, received an infusion of gene-edited hematopoietic stem cells (HSCs), or her “supercells” as she calls them, at TriStar Centennial in Nashville, Tennessee. She had bravely volunteered for a clinical trial to become the first American patient with sickle cell disease (SCD) to receive CRISPR-edited stem cells in an experimental ex vivo clinical trial.“When [the infusion] went in, my heart rate shot up real high, and it kind of made it hard to breathe, so that was a little scary tough moment for me,” Gray told National Public Radio's Rob Stein. “After that I cried, but they were happy tears. It was kind of overwhelming after all that I had [endured] to finally get what I came for.”1Gray was the first of 31 SCD patients dosed with exa-cel in the Phase II/III CLIMB-121 trial (NCT03745287), an international study clinical trial sponsored by CRISPR Therapeutics and Vertex Pharmaceuticals. The trial has been an unequivocal success, with the U.S. Food and Drug Administration widely expected to grant approval later this year. But although none of the SCD patients reported any severe vaso-occlusive crises after infusion with exa-cel, a third of the cohort did experience severe adverse events related to their conditioning regimen—namely a chemotherapeutic drug called busulfan (Fig. 1).FIG. 1. Chemical structure of busulfan.Gray's physician, bone marrow transplant (BMT) expert Haydar Frangoul, and his colleagues used busulfan because the drug causes sufficient ablation to allow engraftment of the gene-modified cells. A number of successful gene therapy trials have used busulfan.“Busulfan is not a benign drug,” said Frangoul, who is medical director at the Sarah Cannon Pediatric Hematology/Oncology and Cellular Therapy Program at TriStar Centennial. “However, if it is used correctly, and if the patient is well enough, it is well tolerated.”But several biotech companies are exploring alternative nongenotoxic alternatives to the busulfan standard. Frangoul forecasts that “as companies become more apt to realize that their therapies work with a busulfan backbone, I foresee that, in the future, there will be newer, less toxic therapies that will be investigated.”Nothing NewBone marrow transplantation is hardly a new technology. This pioneering form of cell therapy, which is a proxy for transplanting HSCs, has been around for decades and used to treat millions of patients with >100 diseases. In 1968, University of Minnesota immunologist Robert Good performed the world's first successful BMT from a matched relative to treat an 8-year-old boy with severe combined immunodeficiency (SCID) syndrome. Five years later, the first successful transplant using bone marrow from an unrelated donor took place, as a 5-year-old SCID patient in New York was treated with multiple bone marrow infusions from a donor in Denmark.Today, tens of thousands of BMTs are performed by thousands of doctors each year around the world. Bone marrow transplantation has become one of the most highly derisked and validated technologies in medicine.Victoria Gray at the Third International Summit on Human Genome Editing in London 2023.(Credit: The Royal Society)But there are major issues and complications using BMTs in cell and gene therapy. The donor-matching process for BMTs—in which one patient gets matched with one donor to treat one disease—is slow, unpredictable, and inconsistent. The reason being each year some 18,000 people in the United States are diagnosed with life-threatening illnesses where BMTs from a related or unrelated donor or cord blood unit are their best treatment option. However, most patients (∼70%) in need of a transplant do not have a matching donor in their family. They depend on the Be The Match registry to find an compatible donor.2As no two donor-derived BMT products are alike and guaranteed to be safe, there are significant durability and scaling issues for this living medicine. In many instances, donor stem cells within a patient's immune system view that patient's tissues and organs as something foreign and attack them. This graft versus host disease is one of several complications to arise from BMTs. Others include stem cell (graft) failure, organ damage, infections, cataracts, infertility, new cancers, and even death.3Busulfan's Risky HistoryThe process known as conditioning—depleting the resident HSCs and creating space for new HSCs to engraft—is also risky and primed for improvement.Indeed the risks posed by busulfan emerged 4 years before it was approved for clinical use by the FDA. In 1950, the British Medical Journal reported a verdict of “death by misadventure” in the case of a 67-year-old breast cancer patient who was being treated with the drug in an effort to inhibit tumorigenesis. Unfortunately, busulfan also inhibited normal tissues that included the bone marrow. “While the effect on the bone marrow can normally be controlled, in the present case agranulocytosis developed, though no other toxic side effects had been observed during administration,” the BMJ reported.4However, that did not stop the FDA from approving an oral form of busulfan in the palliative treatment of leukemia. Forty-five years later, the FDA expanded busulfan's use by approving an intravenous (IV) form of the conditioning agent for use in chronic myelogenous leukemia patients undergoing stem cell transplantation.Busulfan (C6H14O6S2)—the common name for the methanesulfonate ester that is butane-1,4-diol—is a bifunctional alkylating antineoplastic agent that has shown an immunosuppressive effect on bone marrow. The compound inhibits tumor growth by cross-linking guanine bases in DNA, preventing the double helix strands from uncoiling and separating, thus interfering with cell division.Haydar Frangoul, Medical Director at the Sarah Cannon Pediatric Hematology/Oncology & Cellular Therapy Program at TriStar Centennial.“Busulfan is one of our most potent anti-leukemia drugs. It's also very toxic to the normal bone marrow cells, but it's not very immunosuppressive,” says IV busulfan's lead inventor Borje Andersson, a professor in MD Anderson's Department of Stem Cell Transplantation and Cellular Therapy. As a result, busulfan was originally combined with the immunosuppressive drug cyclophosphamide to get the new stem cells to engraft. As this combination had serious side effects, the cyclophosphamide was later replaced with fludarabine, which is strongly immunosuppressive. This combination not only proved effective, especially in patients with early leukemia who were in remission, but also dramatically increased the safety of transplantation.Busulfan has a long list of side effects. Many are common to other alkylating agents, including intestinal mucosal damage, alopecia, pancytopenia, anemia, impaired spermatogenesis, and increased risk of malignancy. And busulfan has other side effects5 including hepatic veno-occlusive disease, interstitial pulmonary fibrosis (also known as “busulfan lung”), and seizures. But when it is used in patients with adequate organ function, the potential risk is minimized, says Frangoul (see the exclusive interview with Frangoul in GEN Biotechnology, February 2023).6Not a Benign DrugIn the exa-cel trial, busulfan was administered to Victoria Gray intravenously over a grueling 4-day period. Gray experienced almost a dozen severe adverse events (SAEs) before dosing—three instances of sickle cell anemia with crisis, as well as nausea, chest, back, and abdominal pain, all of which were resolved. A single dose of exa-cel was given after the last busulfan dose, with researchers seeing evidence of neutrophil and platelet engraftment on day 30. Postinfusion, Gray experienced sepsis and abdominal pain, but all resolved.“The four days you are receiving busulfan, usually there are no complications,” Frangoul said. “All these SAEs are happening between the time they enroll to the time they infuse. Some patients can experience complications during collection, or have a sickle cell crisis in the meantime. It's a variety of different things. The majority of these things are related to busulfan, if not all.”“Almost everybody experienced SAEs after busulfan was completed, just because it's chemo. You're going to lose your hair and develop mouth sores. These are after the infusion, usually not before,” Frangoul explained. “The fever, neutropenia, losing your hair, hospitalization, this is all attributable to busulfan.” Many of Frangoul's SCD patients continued to have pain crises while waiting for treatment, he added.However, another patient in the exa-cel trial fared much worse than Gray. After the trial's data cutoff in February 2022, an adult SCD patient developed pneumonia and respiratory failure after SARS-CoV-2 infection and died. “The investigator assessed the events as due to SARS-CoV-2 infection, with a potential contribution of busulfan lung injury, and unrelated to exa-cel,” Frangoul said.Another exa-cel trial volunteer, Jimi Olaghere, recalled a rigorous process before, during, and after treatment. Olaghere underwent a lung function test to ensure he was, in his own words, “sick enough to participate in this trial, but at the same time, healthy enough to withstand the pressures of [exa-cel].”7 All told, Olaghere was in the hospital for 2 months from conditioning to the day when he received his new cells. Even then, he did not go straight home to Atlanta because he needed to be seen on a daily basis by the care team.Olaghere lived in a medical hospital apartment for another month—in the middle of the pandemic. “I couldn't really go anywhere—I was stuck in this apartment and feeling crappy most days because I just had chemo,” he said. The entire process from DNA collection to manufacturing the cells through the post-treatment apartment stay took ∼3 months, he added. His post-transplant period took ∼1 month, instead of the usual 60 days, because “I was just so eager to get out, and I was recovering quite well.”Jimi Olaghere, Exa-cel trial volunteer.Olaghere said his health has turned 180° since he was treated with exa-cel: “I can do things now that I couldn't even dream about doing before!” He no longer needs weeks to recover from traveling and can enjoy the rare snow days in Atlanta with his young son.The biggest obstacle in using busulfan in patients with nonmalignant diseases—such as SCD and thalassemia—is the substantial risk of infertility. “Busulfan is a great therapy, and we still use it in patients with cancer who are undergoing bone marrow transplantation or stem cell transplant. However, for a person with SCD and thalassemia, you want to avoid some of those long-term side effects,” Frangoul said.Autologous Ex Vivo Hematopoietic Stem Cell TransplantationGiuseppe Ciaramella, the cofounder and CEO of Orbital Therapeutics, and president of Beam Therapeutics, the company spearheading the commercial development of base editing, says that the conditioning regimen is a major impediment for a transplant treatment of SCD in the current setting, where toxicity and sterility are serious issues with the current standard of care. (Unlike traditional CRISPR genome editing, Beam's base editing approach does not introduce double-stranded breaks in DNA.)A promising alternative to traditional genotoxic conditioning methods such as busulfan or “allogenic” cells derived from a donor is creating nongenotoxic autologous ex vivo HSC therapies. Beam's nongenotoxic approach, called ESCAPE, uses base editing to prevent the gene-edited HSCs from producing an antibody, c-Kit (CD117), which is antagonistic to an essential HSC growth factor called stem cell factor.Blocking this binding is designed to starve the cell, preventing proliferation and growth. In this context, unedited resident HSCs will be inhibited by this antibody, whereas the edited transplanted HSCs will be free to grow. Crucially, the edit only alters a single amino acid in CD117, which allows the receptor to still function by binding to other growth factors critical for the development of hematopoietic lineages.At the recent American Society of Gene & Cell Therapy conference, Beam researchers reported that their anti-CD117 mAb, called mAb7, selectively depleted unedited HSCs as well as progenitor cells, while sparing ESCAPE human stem and progenitor cells both in vitro and in vivo: “We demonstrate proof of concept that mAb7 can potentially be used as a conditioning agent that leads to engraftment of multiplex edited HSPCs.”8Beam opted for ESCAPE after studying that and another conditioning approach based on Magenta Therapeutics' MGTA-117, which Beam had explored for conditioning patients with SCD and beta-thalassemia who receive Beam's base editing therapies, under a collaboration announced in 2020. MGTA-117 was designed to selectively deplete stem cells from patients before transplant or HSC-based gene therapy, to reduce or even abolish the need for high-dose or high-intensity chemotherapeutic agents. Last year, Beam said whichever approaches proved successful could “potentially be paired with BEAM-101 and BEAM-102, as well as other base editing programs in hematology.”9Applying Precision ApproachBeam is applying its ESCAPE approach in its Phase I/II BEACON trial (NCT05456880) assessing BEAM-101 in SCD. The trial's “sentinel” cohort, designed to include three patients treated on a sequential basis, met with a setback earlier this year when the first patient enrolled in BEACON withdrew for what Beam said were personal nonmedical reasons. However, two additional patients have enrolled in the sentinel cohort and are undergoing the screening procedures required to enable mobilization and treatment with BEAM-101. Beam currently has five enrollment sites, a waitlist for volunteers, and expects to fully enroll the sentinel cohort in 2023. This year, Beam also expects to start enrolling patients for an expansion cohort, with interim data expected in 2024 from “multiple” patients.Beam's approach differs from that in the exa-cel trial, which involves disrupting the expression of the BCL11a silencer, restoring expression of the fetal hemoglobin gene to compensate for the mutant beta globin. “It's a very direct effect on those promoters, and, in fact, the change not only prevents the repressor protein but makes the fetal hemoglobin promoter even stronger,” Ciaramella told GEN Biotechnology, adding that the highest regulation of fetal hemoglobin had been reported in the preclinical model. “We believe that has the potential to bring a much stronger clinical resolution to SCD and to avoid some of the issues like hemolysis—continuous inflammation that causes progressive organ damage, which ultimately kills the patient.”Eager to reduce the risk of therapy-associated myelodysplastic syndrome and other busulfan side effects, several research teams are interested in identifying next-generation targeted conditioning approaches, said Geulah Livshits, a senior research analyst at Chardan.“A gentler conditioning regimen that in theory would lead to a better safety profile could open up applications in less severe patient populations, where you don't have to think about potential therapy-associated myelodysplastic syndrome and long-term hospitalization,” Livshits said. She notes that antibody drug conjugates or antibodies targeting CD117 are an area that several companies besides Beam are exploring as a potential alternative nongenotoxic conditioning regimen.Tragic OutcomeBut even that approach might have problems. One company that pursued a CD117-targeting antibody as a busulfan alternative recently met with a tragic outcome. In January 2023, Magenta Therapeutics disclosed the death of a patient in its Phase I/II dose escalation trial of its lead pipeline candidate MGTA-117 in relapsed/refractory acute myeloid leukemia (AML). Magenta acknowledged that the death of the patient (age and gender have not been disclosed) might have been related to the conditioning regimen. The patient experienced what Magenta described as “respiratory failure and cardiac arrest resulting in death”—a Grade 5 SAE, which was “deemed to be possibly related to MGTA-117.”Magenta also halted its collaboration with Beam and a second partnership with AVROBIO that investigated conditioning of patients with MGTA-117. AVROBIO had studied MGTA-117 conditioning in patients to be dosed with that company's investigational lentiviral gene therapies for lysosomal disorders.The patient's death occurred a month after Magenta halted dosing participants at the Cohort 4 dosing level (0.13 mg/kg) of the trial. At that time, Magenta reported observing dose-limiting toxicities in the cohort's second and third participants, with one experiencing a Grade 4 SAE (respiratory) possibly related to MGTA-117. Magenta responded by saying it planned to dose additional trial participants at the lower (Cohort 3) dosing level (0.08 mg/kg).Magenta said it voluntarily paused dosing in the clinical trial after consultation with the trial's safety cohort review committee “and with the highest regard for patient safety.” Days later, Magenta halted development of MGTA-117 and its other treatments designed to facilitate gene therapies and stem cell transplants and began a review of strategic alternatives.That strategic review culminated in May with Magenta agreeing to merge with Dianthus Therapeutics, a developer of next-generation complement inhibitors such as DNTH103, which is in a Phase I trial to treat severe autoimmune diseases.Where did Magenta go wrong? “I do not think they necessarily went wrong,” Frangoul commented. “I think they had a product where their preclinical data looked good from what I see published in the literature. When they took it to the clinic, it caused some side effects. Their study showed excessive toxicities in human, and that is unacceptable.”Frangoul added: “In science, this is what can happen, as you are going to end up with therapies that in the preclinical setting—like in the mouse model or the primate model—looks very encouraging, but when you take it to the clinic you can find unexpected side effects or potential complications.”Too SoonWhether Magenta's experience will damage the prospects of antibody drug conjugates (ADCs) or antibodies such as MGTA-117 as a next-generation conditioning approach is too soon to say, according to Livshits. It is unclear “whether the issue is related to Magenta's specific program or there's some kind of more general class effect,” she said. There could be differences “if the issue is related to the payload falling off the antibody” or “in linker design and different elements of how an ADC could be designed that might trigger a different profile.” Naturally, she says, there will be scrutiny regarding the safety for programs using a CD117 ADC.In the longer term, one area where companies that are operating in the HSC space is in vivo engineering—strategies to directly modify the cells using modalities such as lipid nanoparticles (LNPs) that can deliver gene editing components. However, further research is needed to understand the biodistribution of these approaches, the potential safety profile, and what other considerations need to be addressed, Livshits explained.As for how long it will be until some of these approaches enter clinical trials, Livshits predicts a couple of years, although it varies by company. “I've talked about looking at non-genotoxic conditioning alternatives such as life cycle management, and expansion to less severe disease patients for their lentiviral gene therapy programs as a priority in 2024. But that doesn't necessarily mean that it would be entering the clinic at that point,” she said.(Life-cycle management is an approach that focuses on maintenance, replacement, or repair options throughout the service life of an asset—such as a conditioning alternative—to maximize the asset's benefit at the minimum practicable cost.)Magenta's experience does not necessarily mean that AML is a riskier indication for bone marrow conditioning than other cancers, according to Frangoul. He notes that busulfan was initially developed as a transplant option for patients with malignant diseases. “I do not think the approach is necessarily wrong. It can be reasonable to test some of these therapies in patients with cancer who cannot tolerate busulfan or [similar drugs] and see if it results in engraftment. Then, you might move it along to patients who have thalassemia or SCD to see if it is effective,” Frangoul explained.Frangoul hopes to see advances in the practice of targeting stem cells without causing problems in other organs over the next decade. “Whoever is going to discover this ideal therapy that basically kills the stem cells, and then allows the engraftment of the gene-modified therapies without causing collateral damage, will hit a home run!”Dhavanit Shah, founder and CEO of Garuda Therapeutics, says the solution to reducing the risks associated with conditioning must tag four key bases: technological, clinical, regulatory, and commercial. To Shah, who previously served on the faculty at the Harvard Stem Cell Institute, the answer was simple: figure out how to unlock one of the greatest mysteries in medicine—making HSCs in a dish. This would enable a cheap, scalable, and durable one-size-fits-all approach to BMTs. In doing so, he could create an unlimited supply of healthy young HSCs that are recipient agnostic.Shah is focusing first on nonmalignant disorders, a category that includes hemoglobinopathies such as SCD, because there is less reason to be concerned about conditioning for them compared with malignant disorders. Shah adds that because conditioning also provides some degree of immunosuppression, it will thus render his approach a safer one.“There will be a day where people will not be afraid of [BMTs] because we'll dial down the safety issues not just around cells, but also all the related procedures around the cells,” Shah told GEN Biotechnology. “That will happen in my lifetime, but not in the first patient we are going to dose. Our goal is to do everything the way we do traditionally with conditioning and immunosuppression, and swap the existing product with our product.”Autologous In Vivo Hematopoietic Stem Cell TransplantationA promising and desirable alternative to using ex vivo HSC therapies that require conditioning is to perform gene editing in vivo. One such company dedicated to this approach is Ensoma in Boston.“I became absolutely convinced that the most important thing we needed to do was to be able to in vivo edit safely, because it eliminates this entire argument about allogenic and autologous as well as the costs, time burden, and logistics,” Emile Nuwaysir, CEO of Ensoma, told GEN Biotechnology. “The future of human medicine is about in vivo editing. We enable the ability to durably engineer the hematopoietic system—any component or all components simultaneously with a single administration.”Using single deliveries, Ensoma's technology can in principle fix mutated genes or deliver therapeutic payloads for any component of the blood system to deliver throughout the body. “We'll be able to turn on multiple nodes of the immune system simultaneously for different and combinatorial effects, creating a combination therapy with elements of the immune system,” said Nuwaysir. “That's going to be incredibly important when you think about complex diseases like oncology and autoimmunity.”Ensoma's approach to in vivo gene editing uses a helper-dependent adenovirus with a completely stripped genome, providing ample packaging capacity for the payload. “We can deliver 35 kilobases to the nucleus of the target cell, which is different from an LNP or an RNA-based method where you're dumping the payload into the cytoplasm,” said Nuwaysir. “We have a great toolkit that consists of base editors and a transposase system, and those two things together allow us to do everything from a single base edit or knockout through to a 35-kb insertion.”God and ScienceAlthough it is too soon to tell which if any alternatives will ultimately supplant busulfan-based conditioning, it is a safer bet that patients who benefit from a safer more effective regimen will be just as enthusiastic as Frangoul and Gray were after her treatment, if not more so. “We were able to prove that we can edit human cells and we can infuse them safely into patients and it totally changed their life,” Frangoul concluded.Speaking to a reporter in March, Gray marveled at the CRISPR-based editing approach that so far has shown positive results in the form of restored health and even renewed faith. “It's amazing,” Gray enthused. “It's better than I could have imagined. I feel like I can do what I want now!”Gray was in London to address the Third International Summit on Human Genome Editing about her life journey before and after treatment for SCD. (She received a standing ovation.) While there, Gray visited the British Museum, where she saw a small wooden cross hanging on a wall. “My faith is what brought me this far,” Gray said. “God did his part for what I prayed about for years. And together, hand in hand, God and science worked for me.”
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