Gene Therapy for Beta Thalassemia: Preliminary Results from the PHASE I/II Tiget-Bthal Trial of Autologous Hematopoietic Stem Cells Genetically Modified with GLOBE Lentiviral Vector

Gene therapy for transfusion dependent beta-thalassemia is based on the autologous transplantation of hematopoietic stem cells (HSCs) engineered by lentiviral vectors expressing a transcriptionally regulated human beta-globin gene.Gene therapy (GT) could represent an alternative to HSCT with the following potential advantages: use of autologous stem cells, tailored conditioning with no need for immune suppression post GT, no risk of GVHD or rejection.Our contribution to this field was devoted to the clinical development of a gene therapy protocol based on the high-titer vector GLOBE, a 3^rd generation self-inactivating lentiviral vector encoding for the human beta globin gene. Transfusion dependent beta thalassemia patients (any genotype) undergo peripheral blood stem cell harvest following mobilization with the lenograstim and plerixafor. After transduction of immune-selected autologous CD34+ cells and successful release of the frozen drug substance, patient undergo a conditioning regimen based on myeloablative treosulfan and thiotepa favoring efficient engraftment of corrected cells with reduced extra-medullary toxicity (TIGET-BTHAL; EudraCT number 2014‐004860‐39). The route of administration of gene modified HSCs is intraosseous in the posterior-superior iliac crests with the aim of enhancing engraftment and minimizing first-pass intravenous filter. Three days after gene therapy, previously collected unstimulated autologous peripheral blood leucocytes (1-10 x107 CD3+/kg) are reinfused intravenously to favor immune-reconstitution. After 2 years follow-up, patients will be followed up for a further six years in a long-term follow-up study. On the basis of extensive efficacy and safety preclinical studies the clinical trial TIGET-BTHAL was approved and started in 2015 at Scientific Institute San Raffaele, Milan, Italy. The clinical study foresees treatment of 10 patients: 3 adults (group 1) followed by 3 patients aged 8-17 years (group 2) and 4 patients aged 3-7 years (group 3), with a staggered enrolment strategy based on evaluation of safety and preliminary efficacy in adult patients by an independent data safety monitoring board (DSMB) before inclusion of pediatric subjects. In March 2016 the DSMB approved enrolment of group 2 patients and, in September 2016, of group 3 patients. As of August 2017, seven patients (3 adults aged 31-35 years and 4 pediatric patients aged 6-13 years) with different genotypes (β⁰/β⁰, β⁺/β⁺ and β⁰/β⁺) have been treated with GLOBE-transduced CD34⁺ cells at a dose of 16x10⁶-19.5x10⁶ cells/kg and a vector copy number (VCN)/cell ranging from 0.7 to 1.5. Median follow-up is 13 months (range 8-22). The procedure was well tolerated by all patients, with no product-related adverse events, no evidence of replication competent lentivirus nor of abnormal clonal proliferation on regular peripheral blood and bone marrow analyses. Grade 3-4 adverse events or serious adverse events were principally of infectious origin as expected after a myeloablative autograft. Median time to neutrophil engraftment was 19 days (range 17-25) and to platelet engraftment 15 days (range 10-21). Multilineage engraftment of gene-marked cells was observed in peripheral blood and bone marrow, with a median of 0.58 (range 0.37-1.55) vector copy number/cell in GlyA+ bone marrow erythroid cells at 6 months post GT.Polyclonal vector integrations profiles have been detected in the first 3 patients tested. The three adult patients had a reduction of transfusion requirement but are still transfusion dependent at the last follow-up (22, 18 and 16 months respectively). Among the 4 pediatric patients, 3 have discontinued transfusion shortly after gene therapy and are transfusion independent at the last follow-up (13, 10 and 8 months respectively). One pediatric patient is still receiving regular blood transfusions. A correlation was observed between level of engraftment of gene-marked cells in peripheral blood and bone marrow and transfusion requirement. Preliminary data suggest that the applied clinical protocol for gene therapy with GLOBE LV is well tolerated and leads to significantly reduced transfusion requirement. Follow up analysis are ongoing and updated clinical outcome will be presented.