Journal Club – Thalassemia

Locatelli, F., Lang, P., Wall, D., Meisel, R., Corbacioglu, S., Li, A. M., de la Fuente, J., Shah, A. J., Carpenter, B., Kwiatkowski, J. L., Mapara, M., Liem, R. I., Cappellini, M. D., Algeri, M., Kattamis, A., Sheth, S., Grupp, S., Handgretinger, R., Kohli, P., Shi, D., … CLIMB THAL-111 Study Group (2024). Exagamglogene Autotemcel for Transfusion-Dependent β-Thalassemia. The New England journal of medicine, 390(18), 1663–1676. https://doi.org/10.1056/NEJMoa2309673

Clinical Question

In transfusion dependent β-thalassemia, does CRISPR–Cas9 editing of BCL11A in autologous stem cells, exagamglogene autotemcel (Exa-cel), decrease or eliminate the need for transfusions?

Background

Adult hemoglobin consists of α- and β-globin chains that form hemoglobin A. In β-thalassemia, mutations in the β-globin gene (HBB) reduce or eliminate β-globin production (β+, β0, or β0-like variants) or produce structurally abnormal chains^1,2. The resulting imbalance between excess α-globin and deficient or abnormal β-globin leads to unstable α-globin aggregates, ineffective erythropoiesis, hemolysis, and chronic anemia.

Without transfusions, ~85% of children with severe transfusion-dependent β-thalassemia die by age 5. ³ Chronic transfusion therapy extends survival but causes iron overload and end-organ damage, limiting life expectancy to 40–50 years despite chelation.^4-6 Newer agents such as luspatercept and mitapivat may reduce transfusion needs, but the only curative option has been allogeneic stem-cell transplantation, which is constrained by donor availability and risks of transplant-related mortality and GVHD.

Increasing fetal hemoglobin (HbF) alleviates α/β imbalance and disease severity. ^7,8 GWAS studies identified BCL11A as a key HbF regulator. CRISPR–Cas9–mediated editing of the BCL11A erythroid enhancer in autologous hematopoietic stem cells can reactivate HbF production. ^9-13 A 2020 phase 1/2 study demonstrated successful engraftment, robust editing, high HbF, and transfusion independence in a patient with β-thalassemia using this approach, with lower risk than allogeneic transplantation.¹² Building on these results, the present phase 3 trial evaluates the efficacy and safety of exagamglogene autotemcel in a larger population of patients with β0/β0, β0/β0-like, and non-β0/β0-like transfusion-dependent β-thalassemia.

Guidelines

The Thalassemia International Federation 2025 (5^th edition) guidelines suggests the following

• Hematopoietic cell transplantation (HCT) be offered to transfusion-dependent β thalassemia (TDT) patients and their parents at an early age, before complications due to iron overload develop, if an HLA identical donor is available (Grade B, Class I)
• Pediatric patients up to the age of 14 years undergoing allogeneic HCT from HLA-identical sibling donors demonstrate excellent clinical outcomes. Allogeneic HCT remains the preferred curative intervention and warrants thorough consideration where no gene therapy is yet commercially accessible for patients below 12 years (Grade B, Class I).
• For patients aged 14 years and older or for those who do not have an HLA-identical family donor, gene therapy instead constitutes an optimal therapeutic option, the ideal candidate is a patient with the following (Grade C, Class IIa)
  • Significant transfusion history (at least 100 mL/kg or at least 10 units/year of packed red blood cells).
  • Adequate control of iron overload (LIC ≤ 7 mg/g dry liver weight and mT2* > 20 ms).
  • Normal ventricular function and respiratory function tests and absence of significant hepatosplenomegaly, gallstone disease, potential for effective fertility preservation, high level of personal motivation
• Patients presenting with at least one of the characteristics listed below should also be considered for treatment (ideally within 24 months) with allogeneic HCT or gene therapy approaches (Grade C, Class IIb):
  • Age between 35 and 45 years.
  • Moderate-to-severe iron overload (LIC > 7 and <15 mg/g dry liver weight and mT2* < 20 and >15 ms).
  • Rare erythrocyte phenotypes or a history of alloimmunization, which could lead to foreseeable difficulties in the identification and long-term availability of suitable red blood cell units
  • Proven intolerance to iron-chelating drugs (due to allergic reactions or excessive side effects) that would likely result in a rapidly progressive worsening of iron overload. ¹⁴

The Italian Society of Thalassemias and Hemoglobinopathies [SITE] suggests the following:

• Patients age <14 years and HLA-identical family donor are the factors associated with the best transplant outcomes in patients with β-TDT
• Gene therapy products can be considered for those between 12-50 years, they recommend caution with patients >50 years old and do not recommend gene therapy in patients aged over 55
• Patients must have no significant iron overload or evidence of organ damage¹⁵

Study Design

• Multicenter, open label (non-blinded), single group, phase 3 study
• Setting: 13 sites in Canada, Germany, Italy, United Kingdom, and the United States
• N=52, single treatment group
• Follow up period: 2 years with option to transition to long term (13 year) follow up study after initial 2 years
• Analysis: (opposite of ITT)
• Outcomes: to be evaluated starting 60 days after last red cell transfusion for post transplantation support or management of transfusion dependent B thalassemia but 16 months or more of total follow up time post exa-cel infusion were required for all end points evaluation
  • Primary end point
    • Transfusion independence for 12 months (defined as weighted avg hemoglobin of at least 9g/dL without transfusion support)
  • Secondary end point
    • Transfusion independence for at least 6 months (defined as weighted Hg of at least 9 g/dL without transfusion)- Key secondary end point
    • Duration of transfusion independence
    • Total hemoglobin and fetal hemoglobin concentrations
    • Reduction in red-cell transfusions
    • Percentage of alleles with intended genetic modifications in peripheral blood and bone marrow cells
    • Change in iron overload measures
    • Change from baseline in patient reported outcomes ((EuroQol Visual Analogue Scale [EQ VAS], Functional Assessment of cancer therapy- general [FACT-G], and Bone Marrow Transplantation Subscale [BMTS])
  • Safety
    • Neutrophil and platelet engraftment
    • Adverse events
    • Mortality
    • Clinical exams including vital signs, physical exam, laboratory assessments, EKG

Populations

• Key Inclusion Criteria
  • Patients >12 years and <35 years with Karnofsky performance of over 80% if >16 years and Lansky performance status of over 80% if <16 years and were eligible for autologous stem cell transplant who:
    • Have a confirmed diagnosis of transfusion dependent B thalassemia defined by
      • Documented and confirmed genotype of homozygous B-thalassemia or compound b-thalassemia including B-thalassemia/Hemoglobin E (HbE) AND
      • A transfusion history of at least 100mL of packed red cells per kilogram of body weight per year OR 10 units of packed red cells per year for 2 years before screening
• Patients of childbearing age agreed to use affective and acceptable methods of contraception from consent through at least six months after treatment infusion
• Willing and able to comply with scheduled visits, treatment plan, laboratory tests, contraceptive guidelines, and other trial procedures

• Key Exclusion Criteria
  • Availability of a healthy, willing 10/10 HLA-matched donor
  • Prior allogeneic HSCT
  • Coexisting α-thalassemia with >1 α-gene deletion or α-gene multiplications
  • Sickle cell–β-thalassemia variants
  • Clinically significant active infections
  • WBC <3×10⁹/L or platelets <50×10⁹/L unrelated to hypersplenism
  • History of major bleeding disorders
  • Medical conditions that could confound results or increase risk (e.g., familial cancer syndromes, significant drug allergies, cardiovascular/CNS disease, uncontrolled seizures, psychiatric illness)
  • Current or prior malignancy, myeloproliferative disease, or major immunodeficiency
  • Advanced liver disease (AST/ALT >3× ULN, direct bilirubin >2.5× ULN, INR >1.5× ULN, cirrhosis/bridging fibrosis, active hepatitis unless biopsy excluded fibrosis, or liver iron >15 mg/g unless biopsy excluded fibrosis)
  • Cardiac T2* <10 ms or LVEF <45%
  • eGFR <60 mL/min/1.73 m²
  • DLCO <50% predicted
  • Prior gene therapy/editing
  • Allergy or intolerance to plerixafor, G-CSF, busulfan, or exa-cel excipients (DMSO, dextran)
  • HIV-1/2, HBV, HCV, or syphilis positivity
  • Participation in another trial within 30 days
  • Pregnancy or breastfeeding

Baseline Patient Characteristics

59 Patients enrolled

1. 52 received exa-cel (this is the full analysis population)
   • 48% were female
   • Mean age 21.5 +/- 6.7 years
   • Racial group: 35% white, 42% asian
   • Mean annualized units of red cells transfused: 35
   • Total Hemoglobin concentration: 10.4 +/- 2.0
   • Total fetal hemoglobin concentration: 0.6 +/- 1.0
   • Spleen intact: 69%
   • Genotype distribution:
     • β0/ β0: 19 (37%)
     • β0/ β0-like:
       • β0/IVS-I-110: 9 (17%)
       • IVS-I-110/ IVS-I-110: 3 (6%)
     • non-β0/ β0-like
       • β+/ β0: 12 (23%)
       • β+/ β+ : 4 (8%)
       • β+/ βE : 5 (10%)
     • Iron Status:
       • Median Liver iron concentration: 3.5
       • Median cardiac iron content by T2 weighted MRI: 34
       • Median serum ferritin concentration: 2891.0
     • Median exa-cel dose: 7.5x10E6 CD34+ cells/kg (median of 1 mobilization cycle)
2. 35 had at least 16 months follow up and were analysis of the primary and secondary end points (full efficacy population)
   • 49% were female
   • Mean age 21.1 +/- 6.1 years
   • Racial group: 43% White, 37% Asian
   • Mean annualized units of red cells transfused: 35
   • Total Hemoglobin concentration: 10.4 +/- 1.9
   • Total fetal hemoglobin concentration: 0.5 +/- 0.6
   • Spleen intact: 74%
   • Genotype distribution
     • β⁰/ β⁰: 10 (29%)
     • β⁰/ β⁰-like:
       • β⁰/IVS-I-110: 7 (20%)
       • IVS-I-110/ IVS-I-110: 3 (9%)
     • non-β⁰/ β⁰-like
       •  β⁺/ β⁰: 8 (23%)
       • β⁺/ β⁺ : 3 (9%)
       • β⁺/ β^E : 4 (11%)
   • Iron Status:
     • Median Liver iron concentration: 4.0
     • Median cardiac iron content by T2 weighted MRI: 34.8
     • Median serum ferritin concentration: 2653.7
   • Median exa-cel dose: 6.4x10E6 CD34+ cells/kg (median of 1 mobilization cycle)

Intervention

• All patients received a combination of granulocyte-colony stimulating factor (G-CSF) and plerixafor for HSPC mobilization followed by apheresis for up to 3 consecutive days to collect CD34+ HSPCs
• Before the exa-cel infusion, patients received a myeloablative, pharmacokinetically adjusted busulfan conditioning regimen for 4 days.
• Exa-cel was manufactured from CD34+ cells with the use of CRISPR-Cas9 and a single guide RNA molecule and infused intravenously through a central venous catheter at least 48 hours but no more than 7 days after completion of the busulfan infusion
• Treatment G-CSF was allowed in the study protocol after day 21 at the discretion of the investigator. 32 (62%) of patients received this prior to engraftment

Outcomes

1. Primary Endpoint
   1. Thirty-two patients (91%; 95% CI, 77–98) achieved transfusion independence at a mean of 35.2 ± 18.5 days after exa-cel infusion (P < 0.001 against a 50% null rate).
2. Key Secondary Endpoint
   1. Thirty-two patients (91%; 95% CI, 77–98) maintained a weighted average hemoglobin ≥9 g/dL for at least six months (P < 0.001 against a 50% null response).
3. Other Secondary Endpoint
   1. Transfusion independence: All patients who achieved transfusion independence remained so throughout follow-up (13.3–45.1 months).
   1. Hemoglobin response: Mean hemoglobin reached 11.4 ± 2.2 g/dL by month 3 and remained ≥12 g/dL thereafter; mean fetal hemoglobin was ≥7.7 ± 2.9 g/dL from month 6 onward with pan-cellular distribution. Patients achieving transfusion independence reached mean hemoglobin 13.1 ± 1.4 g/dL and fetal hemoglobin 11.9 ± 1.9 g/dL. The three non-independent patients showed slower, lower recovery of both.
   1. Transfusion reduction: One non-independent patient had an 84% reduction in annualized transfusion volume. The other two stopped transfusions at 14.5 and 12.2 months post-infusion and remained transfusion-free for 7.3 and 4.0 months, respectively.
   1. BCL11A Editing levels: All patients maintained ≥64% edited BCL11A alleles in blood and marrow from month 2 onward; levels were similar in the three non-independent patients.
   1. Iron overload and erythropoiesis: Serum ferritin fell from 3508 ± 2735.3 to 2295.1 ± 1930.9 pmol/L; liver iron decreased; cardiac T2* remained stable (>20 ms).
   1. Patient-reported outcomes: EQ-VAS, FACT-G, and BMTS scores all improved by month 24, indicating better quality of life.
4. Key Safety Analysis
   1. Assessments of neutrophil and platelet engraftment
      1. All 52 patients in the full analysis population who underwent busulfan conditioning and exa-cel had neutrophil and platelet engraftment with median time 29 days (range, 12 to 56) and 44 days (range 20 to 200) respectively, of note presence of a spleen delayed count recovery
   1. Adverse events
      1. All patients experienced at least one adverse event, 44 patients (88%) experienced adverse events of grade 3 or 4 most of which occurred in the first 6 months after exa-cel infusion
      1. 17 patients (33%) experienced serious adverse events
         1. Serious event attributed to busulfan conditioning including: Veno-occlusive liver disease in 5 patients attributed to busulfan conditioning. All were grade 3 or lower and resolved after defibrotide treatment. One patient had cerebellar and subarachnoid hemorrhage
         1. Two patients had serious events thought to be related to exa-cel including: One patient had headache, acute respiratory distress syndrome, idiopathic pneumonia syndrome all of which occurred in the context of hemophagocytic lymphohistiocytosis (HLH), the second patient had delayed engraftment of platelets (engrafted on day 199) and neutrophils (engrafted on day 56)
   1. Mortality
      1. No deaths, discontinuations, or cancers occurred after exa-cel infusions

Commentary:

In this study, autologous BCL11A-edited hematopoietic stem cells (exa-cel) engrafted after busulfan conditioning produced durable transfusion independence in adolescents and adults (12–35 years) with severe transfusion-dependent β-thalassemia. Treatment led to sustained increases in total and fetal hemoglobin. Of 35 evaluable patients, only three did not achieve transfusion independence during the study window; all three did so with extended follow-up.

Because many patients lack suitable allogeneic donors or face transplant-related risks, this edited autologous approach may substantially broaden access to curative therapy. The safety profile was acceptable, with most serious events attributable to busulfan conditioning. Exa-cel–related events were limited to one case of HLH and one episode of delayed platelet and neutrophil engraftment, both resolving without backup cells. Notably, baseline iron overload—common in β-thalassemia—did not appear to increase the risk of post-transplant hepatic vaso-occlusion, likely reflecting the cohort’s well-controlled iron status (mean ferritin 2653.7 pmol/L, LIC 4.0 mg/g, cardiac T2* 34.8).

This study enabled the first FDA approval of a non-viral CRISPR genome-editing therapy to raise fetal hemoglobin in severe transfusion-dependent β-thalassemia. Unlike lentiviral approaches, ex vivo CRISPR editing does not integrate foreign DNA into the host genome, potentially reducing theoretical risks of insertional oncogenesis. However, genome editing still carries the possibility of off-target mutations. Although the companion study by Yen et al. reported no detectable off-target edits, their detection methods have notable limitations: a small donor sample (n=14); reliance on prediction algorithms to select candidate off-target sites; constraints of GUIDE-seq for unbiased detection; and evaluation of many predicted variant-sensitive sites without the relevant variants represented. ¹⁶ Additional studies are therefore needed to more fully characterize potential off-target effects in this patient population.

This study raises questions about why, despite similar editing levels (≥64%) across all exa-cel–treated patients, some achieved transfusion independence later than others, including the three who reached it only after the study cutoff. Clarifying the specific edit types is important because CRISPR-induced double-strand breaks repaired by nonhomologous end joining can generate diverse outcomes—nonsense, missense, or frameshift mutations—affecting protein expression and function.

Additional considerations involve the consequences of pan-cellular BCL11A loss. BCL11A is essential for hematopoietic stem cell quiescence and lymphoid development; its deletion in mice induces stem-cell aging, raising concerns about potential clonal hematopoiesis or myeloid dysplasia with long-term follow-up. ¹⁷ BCL11A is also highly expressed in lymphoid progenitors and mature B and T cells, where it regulates heavy-chain recombination and B-cell survival. ^18,19  Whether patients in this study achieve full lymphocyte reconstitution—and whether long-term humoral defects might emerge (such as responses to vaccines)—remains unknown.

In summary, this seminal study demonstrates that BCL11A-edited autologous stem-cell therapy (exa-cel) can provide a potentially curative option for patients with transfusion-dependent β-thalassemia. Although important questions remain, the treatment produced durable increases in total and fetal hemoglobin and enabled most patients to achieve sustained transfusion independence.

References

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