Transfusion-dependent β-thalassaemia (TDT) is a severe, rare genetic disease where impaired production of the β-globin chain of adult haemoglobin (Hb) results in a deficiency of functional red blood cells (RBCs), bone marrow expansion and ineffective erythropoiesis, chronic anaemia and haemolysis, leading to serious complications, reduced quality of life and a shortened lifespan.1, 2 Patients require lifelong supportive care with chronic blood transfusions and iron chelation that are burdensome and many patients fail to comply with the standard of care.1-4 Even under optimal monitoring, transfusion and iron chelation treatment, patients with TDT experience a high proportion of disease- and treatment-related complications.5

Allogeneic haematopoietic stem cell (HSC) transplantation is potentially curative, but is limited by donor availability and patient age, and associated with transplant-related complications.1, 2, 6 Gene therapy with betibeglogene autotemcel (beti-cel; LentiGlobin for β-thalassaemia) showed positive results in patients with TDT and β+ genotypes in phase I–II studies7; however, six of nine patients with severe β-thalassaemia genotypes (i.e. β0/β0 or homozygous IVS1-110 mutation) did not achieve transfusion independence (TI). Consequently, beti-cel manufacturing was refined in phase III studies, Northstar-2 (NCT02906202) and Northstar-3 (NCT03207009), leading to increased drug product and peripheral blood vector copy numbers (VCN), and higher gene therapy-derived HbAT87Q levels.

The most recent analysis of the Greek National Registry for Haemoglobinopathies demonstrated that between 2010 and 2015 a total of 2099 patients with TDT were registered, including ~1500 adult patients aged 36–50 years.8 Here, we present the first case report of an adult Greek patient treated with gene therapy in a phase III beti-cel trial (Northstar-3) for TDT of severe genotypes. Signed consent to publish anonymised information was obtained from the patient.

A 33-year-old, Greek Caucasian male teacher, was diagnosed with β-thalassaemia [β+(IVS-I-110)/β0(codon 39) genotype] 6 months after birth. The patient became transfusion-dependent at 9 months, while iron chelation treatment was initiated at 2·5 years. Prior to gene therapy, the patient received iron chelation treatment with deferasirox and deferoxamine and had an intact spleen, with good adherence to standard-of-care treatment.

At baseline, the annualised transfusion number and volume were 39·5/year and 175·5 ml/kg/year, respectively. Satisfactory adherence to chelation treatment led to mild hepatic haemosiderosis (liver iron concentration: 3·3 mg Fe g/dry weight) and normal cardiac T2* values (32.7 ms). Serum ferritin at baseline was 1203 ng/ml. Following HSC collection via granulocyte-colony stimulating factor plus plerixafor mobilisation9 and apheresis, cluster of differentiation (CD)34+ cells were transduced with BB305 lentiviral vector (LVV) encoding a functional βA-T87Q-globin gene (VCN:4.3 copies/diploid genome; transduction efficiency:78%). The patient underwent hypertransfusion before mobilisation and conditioning, maintaining pre-transfusion Hb of ≥110 g/l. After receiving pharmacokinetic-adjusted, single-agent busulfan myeloablation, beti-cel was infused intravenously (7·6 × 106 CD34+ cells/kg). During conditioning and until neutrophil engraftment, the patient received defibrotide as veno-occlusive disease (VOD) prophylaxis. The timeline for the process and outcomes of treatment with beti-cel gene therapy are shown in Fig 1.

An overview of the process and outcomes of treatment with beti-cel gene therapy in an adult patient with a β0/β+ (IVS-I-110) genotype. AE, adverse event; AUC, area under the curve; Hb, haemoglobin; HSC, haematopoietic stem cell; IV, intravenous; LIC, liver iron concentration; LVV, lentiviral vector; RBC, red blood cell; RCL, replication competent lentivirus; VCN, peripheral blood vector copy number. Neutrophil engraftment: unsupported absolute neutrophil count ≥0.5 × 109/l × 3 days; platelet engraftment: platelets ≥20 × 109/l × 3 days without transfusions.

Mobilisation and apheresis were uneventful. During infusion, the patient experienced Grade 2 abdominal pain/discomfort, considered possibly related to treatment. No further beti-cel-related adverse events (AEs) or serious AEs, including VOD, were reported during the study. After infusion, Grade ≥3 AEs were observed, including anaemia, neutropenia, febrile neutropenia, thrombocytopenia and stomatitis, all considered as related to myeloablative conditioning (Fig 1).

The patient received a total of six RBC transfusions after beti-cel infusion (last transfusion 1·3 months after beti-cel). Engraftment was successful; neutrophils and platelets engrafted at days 26 and 58 after infusion respectively. After engraftment, vector-derived HbAT87Q increased between month 1 and 3; subsequently, total unsupported total Hb and HbAT87Q were stable over time (Fig 2A). Total Hb levels, mainly driven by HbAT87Q, reached levels in the normal range and were consistently >120 g/l after month 5. The patient achieved and maintained transfusion independence (TI: weighted average Hb ≥90 g/l without RBC transfusions for ≥12 months). Total Hb was 135 g/l at 24 months after beti-cel, to which HbAT87Q contributed >85% (116 g/l). Peripheral blood VCN was 1·120 copies per diploid genome. The patient started iron removal at month 8, initially with phlebotomies and later deferasirox. Defarasirox was eventually stopped at month 22. Serum ferritin levels declined to near normal ranges (Fig 2C, right panel).

Beti-cel infusion led to increases in total Hb and HbAT87Q, whilst leading to improvements in bone marrow erythropoiesis and markers of ineffective erythropoiesis. (A) After engraftment, total Hb levels, mainly driven by beti-cel–HbAT87Q, allowed discontinuation of RBC transfusions. Total follow-up = 26·6 months. TI (defined as *Weighted average Hb ≥ 90 g/l without RBC transfusions for ≥12 months) was achieved with a total ongoing duration of 22·5 months. Time from infusion to last pRBC transfusion was 1·3 months. †Post-DP infusion, patient received six RBC transfusions on Days 9, 11, 17, 25, 32, and 39. (B) Normocellular bone marrow and reduced erythroid hyperplasia after gene therapy with beti-cel. (C) Markers of ineffective erythropoiesis trended towards normal levels. DP, drug product; Hb, haemoglobin; TI, transfusion independence; pRBC, packed red blood cell transfusion. Scale bar: 50 µm.

Bone marrow was normocellular at baseline, hypocellular at 12 months (reflecting late re-population after myeloablation), and normocellular again at month 24, with an improved myeloid:erythroid ratio (Fig 2B). Markers of ineffective erythropoiesis reverted towards or maintained normal levels after beti-cel. Erythropoietin and soluble transferrin levels (Fig 2C) were 15·3 µ/l and 79 nmol/l at baseline and 17·0 µ/l and 60 nmol/l at month 24. The patient has now enrolled in the long-term follow-up study (LTF-303) for an additional 13-year follow-up.

The patient did not experience any lentiviral vector-related complications, including replication-competent lentivirus or insertional oncogenesis. Integration site analysis at month 24 revealed highly polyclonal integration and no evidence of clonal predominance. Fertility preservation via sperm bank cryopreservation before infusion and subsequent in vitro fertilisation after infusion resulted in the birth of healthy twins.

This Greek patient was one of the oldest patients treated in gene therapy trials for β-thalassaemia. In a trial using the GLOBE vector and intraosseous administration of gene-modified HSCs, in contrast to paediatric patients, adult patients (aged 31–35 years) failed to reach TI.10 The investigators attributed the unfavourable outcome in adults to an ‘aged’ bone marrow and/or a bone marrow (and its stroma) impaired by iron overload that may have inhibited the engraftment of gene-modified HSCs and affected the outcome.10, 11 However, the clinical response observed in our present case argues against the notion of suboptimal engraftment of transduced HSCs due to an impaired ‘aged’ bone marrow niche in adult patients.

One-time gene therapy has been life-changing for the present patient; after more than three decades of frequent RBC transfusions and daily iron chelation, he is overjoyed to be transfusion/chelation-free and enjoys life with his new extended family. He is now full of energy and positivity, working as a schoolteacher.

In summary, after treatment with beti-cel gene therapy in Northstar-3, an adult patient with TDT of a β0/β+(IVS-I-110) genotype has experienced a functional cure, achieving long-term TI with Hb levels within normal ranges. His daily life and its quality have been drastically improved.

Acknowledgements

The patient and his family deserve our greatest gratitude. The authors would also like to acknowledge Ruiting Guo, biostatistician at Bluebird Bio Inc. (Cambridge, MA, USA), for data correctness review against clinical database; Richard A. Colvin, for critical review of the case study and clinical lead of the Northstar-3 study; and Kirstin Stricker (Bluebird Bio Inc.) and Harrie Kerr (LCW Consulting), for editorial assistance. The Northstar-3 study was sponsored by Bluebird Bio Inc. The article is dedicated to the memory of George Stamatoyannopoulos.

Author contributions

Evangelia Yannaki : principal investigator, manuscript writing; Varnavas Constantinou: physician investigator and data manager; Despina Mallouri, Ioannis Batsis, Damianos Sotiropoulos, Ioanna Sakellari, Achilles Anagnostopoulos: physicians-investigators; Vasiliki Papadimitriou, Maria Kammenou: transplantation nurses; Asimina Bouinta, Despoina Papadopoulou: apheresis technicians, Penelope-Georgia Papayanni: study co-ordinator and data manager. All authors reviewed and approved the final manuscript.

Data availability statement

Anonymised data is available on request from Dr Evangelia Yannaki, eyannaki@uw.edu.