Lentiviral globin gene therapy with reduced-intensity conditioning in adults with β-thalassemia: a phase 1 trial

Abstract

β-Thalassemias are inherited anemias that are caused by the absent or insufficient production of the β chain of hemoglobin. Here we report 6-8-year follow-up of four adult patients with transfusion-dependent β-thalassemia who were infused with autologous CD34⁺ cells transduced with the TNS9.3.55 lentiviral globin vector after reduced-intensity conditioning (RIC) in a phase 1 clinical trial ( NCT01639690) . Patients were monitored for insertional mutagenesis and the generation of a replication-competent lentivirus (safety and tolerability of the infusion product after RIC-primary endpoint) and engraftment of genetically modified autologous CD34⁺ cells, expression of the transduced β-globin gene and post-transplant transfusion requirements (efficacy-secondary endpoint). No unexpected safety issues occurred during conditioning and cell product infusion. Hematopoietic gene marking was very stable but low, reducing transfusion requirements in two patients, albeit not achieving transfusion independence. Our findings suggest that non-myeloablative conditioning can achieve durable stem cell engraftment but underscore a minimum CD34⁺ cell transduction requirement for effective therapy. Moderate clonal expansions were associated with integrations near cancer-related genes, suggestive of non-erythroid activity of globin vectors in stem/progenitor cells. These correlative findings highlight the necessity of cautiously monitoring patients harboring globin vectors.

Extended Data Fig. 1 |. Cell count recovery after conditioning and infusion of TNS9.3.55 transduced CD34+ HSPCs.

a, Platelet count (×10⁹/l) after treatment. b, Absolute neutrophil count (×10⁹/l) after treatment. Granulocyte colony stimulating factor (G-CSF) was administered for Patient 1 (day 13–16) and Patient 2 (day 16–18) during aplasia. Patient 1 received methylprednisolone intravenously for 3 consecutive days (day 16–18) for treating the engraftment syndrome.

Extended Data Fig. 2 |. β-globin expression in Patient 2 at 12 months post treatment.

a, HPLC chromatograms illustrating globin production of eythroid cells from four individual BFU-Es derived from bone marrow obtained from Patient 2 at 12 months post infusion. Top chromatograms: two representative examples from two individual, non-transduced BFU-E from Patient 2; lower chromatograms: two representative examples from two individual, transduced BFU-E from Patient 2. b, β-globin to α-globin ration in erythroids derived from untransduced and transduced HSCs obtained from Patient 2 at 12 months post infusion. The β/α expression ratio determined by HPLC in single BFU-Es increased from a mean of 0.11 to 0.38 in BFU-Es harboring a single copy of the integrated vector, representing a gain of 0.27.

Extended Data Fig. 3 |. Erythropoietic maturation in bone marrow (Patient 2).

Terminal erythroid differentiation begins with proerythroblasts differentiating into basophilic, then polychromatic, then orthochromatic erythroblasts that enucleate to become reticulocytes. Each distinct stage of terminal human erythroid differentiation can be distinguished using a combination of cell surface markers for glycophorine A (GPA), band 3 and α4 integrin. a, Representative flow cytometry plot of band 3 vs α4-integrin of GPA+ cells in normal erythopoesis: proerythrobalsts (I), early basophilic (II), late basophilic (III), polychromatic (IV), and orthochromatic erythroblasts (V) and reticulocytes (VI). The box plot represents the quantitation of the proportion of cells at each distinct stage of maturation after normalization based on total nucleated erythroid cells (I-V) as 100% as described in ref. The left panel is adapted from ref. b, Bone marrow erythroblasts from Patient 2 were analyzed by flow cytometry at 6, 12 and 24 months post infusion stained with GPA, α4-integrin, and band 3. Plot of band 3 vs α4-integrin of GPA+ cells represents the quantitation of distinct stages of maturation of erythroblasts as described in a. c, Quantitation of the proportion of cells at each distinct stage of maturation after normalization to total nucleated erytroid cells (I-V).

Extended Data Fig. 4 |. Engraftment of transduced cells in bone marrow.

Vector copy number (VCN) in erythroid glycophorine A+ (GPA+) cells and CD45+ cells sorted from bone marrow of patients.

Extended Data Fig. 5 |. Gini and Shannon Index values

Timepoints in months.

Extended Data Fig. 6 |. Annotation of the genes STAT3 and STAT5A on chromosome 17.

Illustration of a cluster of transgene integrations in the first intron of STAT3. Six integration sites were detected at year six, zero were detected pre-transplant. Four out of six integration sites detected were in the same transcriptional orientation as STAT3.

Fig. 1 |. Multilineage engraftment of transduced cells in peripheral blood and bone marrow.

Vector copy number (VCN) per diploid genome in different lymphoid (CD3, CD19) and myeloid (CD14, CD15) lineages sorted from peripheral blood mononuclear cells (PBMC) of patients and whole bone marrow (wBM). Drug product (DP) VCNs were evaluated on infused cells.

Fig. 2 |. Transfusion requirements and VCN in peripheral blood mononuclear cells.

Annual blood transfusion volume (ml/kg/year) and VCN (copy/diploid genome) in peripheral blood mononuclear cells (PBMC) pre- and post-infusion with TNS9.3.55 transduced CD34+ HSPCs.

Fig. 3 |. Long-term clonal abundance and succession in all patients.

a, Longitudinal relative abundance of the 10 most abundant cell clones in whole blood as marked by lentiviral integration sites. The different colors (horizontal bars) indicate the top 10 most abundant cell clones while the remaining sites are binned as low abundance (LowAbund; grey). The x-axis indicates time points after infusion and the y-axis is scaled by proportion of the total cells sampled. The total number of genomic fragments used to identify integration sites are listed atop of each plot. A key to the sites, named for the nearest gene, is shown on the right side of the graphs. The nearest genes possess additional annotations: *site is within a transcription unit; ~ site is within 50kb of human cancer-related genes. b, Cell clones surpassing 1% of relative abundance over time. Longitudinal representation of relative abundance of cell clones in whole blood as marked by lentiviral integration sites.

Fig. 3 |. Long-term clonal abundance and succession in all patients.

a, Longitudinal relative abundance of the 10 most abundant cell clones in whole blood as marked by lentiviral integration sites. The different colors (horizontal bars) indicate the top 10 most abundant cell clones while the remaining sites are binned as low abundance (LowAbund; grey). The x-axis indicates time points after infusion and the y-axis is scaled by proportion of the total cells sampled. The total number of genomic fragments used to identify integration sites are listed atop of each plot. A key to the sites, named for the nearest gene, is shown on the right side of the graphs. The nearest genes possess additional annotations: *site is within a transcription unit; ~ site is within 50kb of human cancer-related genes. b, Cell clones surpassing 1% of relative abundance over time. Longitudinal representation of relative abundance of cell clones in whole blood as marked by lentiviral integration sites.