Correction of beta-thalassemia major by gene transfer in haematopoietic progenitors of pediatric patients

Abstract

Beta-thalassemia is a common monogenic disorder due to mutations in the beta-globin gene and gene therapy, based on autologous transplantation of genetically corrected haematopoietic stem cells (HSCs), holds the promise to treat patients lacking a compatible bone marrow (BM) donor. We recently showed correction of murine beta-thalassemia by gene transfer in HSCs with the GLOBE lentiviral vector (LV), expressing a transcriptionally regulated human beta-globin gene. Here, we report successful correction of thalassemia major in human cells, by studying a large cohort of pediatric patients of diverse ethnic origin, carriers of different mutations and all candidates to BM transplantation. Extensive characterization of BM-derived CD34(+) cells before and following gene transfer shows the achievement of high frequency of transduction, restoration of haemoglobin A synthesis, rescue from apoptosis and correction of ineffective erythropoiesis. The procedure does not significantly affect the differentiating potential and the relative proportion of haematopoietic progenitors. Analysis of vector integrations shows preferential targeting of transcriptionally active regions, without bias for cancer-related genes. Overall, these results provide a solid rationale for a future clinical translation.

Figures

Figure 1. Characterization of thalassemia major BM-CD34+ cells

1. Expression of erythroid lineage markers in BM-CD34+ progenitors: cumulative FACS data of the percentage of CD34+GpA+, CD34+CD36+ and CD34+CD71+ cells plotted for normal (ND, n = 5, 5 and 3, respectively) and thalassemic (THAL, n = 17, 8 and 9, respectively) samples.

2. CFU activity of CD34+ cells analysed as total number of colonies/1000 cells plated and as proportion of the different type of colonies (BFU-E, CFU-GM and CFU-GEMM) in ND (n = 12) and THAL (n = 20) samples. Values are given as mean ± SEM. Asterisks indicate values significantly different between groups (***p < 0.001).

Figure 2. Effect of cytokines treatment on clonogenic activity and progenitors subpopulations of CD34+ cells

1. CFU activity of CD34+ cells and proportion of the different type of colonies (BFU-E, CFU-GM and CFU-GEMM) in thalassemia major samples untreated (THAL, n = 20), activated with cytokines (THAL-act, n = 19) and transduced with the GLOBE vector (THAL-GLOBE, n = 18).

2. Cumulative data from FACS analysis of multipotent (CD34+CD38−CD50+), lymphoid (CD34+CD10+) and myeloid (CD34+CD33+) progenitors subpopulations in thalassemia major untreated (THAL, black bars, n = 3, 6 and 6, respectively) and activated (THAL-act, white bars, n = 3) samples. Values represent the mean ± SEM. Asterisks indicate parameters significantly different between groups (*p < 0.05; **p < 0.01).

Figure 3. Genome expression profiling of thalassemic CD34+ cells and molecular response to cytokine activation

1. Unsupervised cluster analysis of transcripts from thalassemic and normal cells untreated (THAL, n = 9; ND, n = 8) and activated with cytokines (THAL-act, n = 11; ND-act, n = 9).

2. Unsupervised cluster analysis of transcripts from thalassemic and normal cells, untreated and activated with cytokines, including normal donor pediatric cells (ND-P, n = 5). In red overexpressed probe sets, in blue downregulated ones.

Figure 4. Transduction efficiency of thalassemia major CD34+ cells by GLOBE vector and correction of HbA deficiency in transduced erythroblasts

1. Schematic representation of the percentage of transduction as determined by PCR on CFUs derived from transduced CD34+ cells, and of the average VCN/cell, as determined by qPCR on bulk erythroid culture. Single dots and triangles represent individual experiments.

2. Left: Representative FACS analysis for HbA expression at 2 weeks of erythroid culture from normal (ND1), thalassemic (THAL 106, 51) and thalassemic GLOBE-transduced (THAL 106-GLOBE, 51-GLOBE) CD34+ cells after intracellular HbA staining. Right: Cumulative data from FACS analysis for HbA expression in erythroid cells. In the upper panel, the mean proportion ± SEM of HbA+ cells in normal (ND, black bar, n = 5), thalassemia-β0 (THAL-β0, white bar, n = 7) and thalassemia-β0-GLOBE-transduced (THAL-β0-GLOBE, grey bar, n = 7) cultures. In the lower panel, the MFI of HbA-expressing cells in the normal (ND, black bar, n = 5), thalassemic (THAL-β0 and -β+, white bars, n = 7 and 3) and thalassemic GLOBE-transduced (THAL-β0 and -β+, grey bars, n = 7 and 3) cultures. Asterisks indicate parameters significantly different between groups (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 5. Reverse phase HPLC analysis of radiolabelled globin chains synthetized in erythroblastic culture

HPLC profile of globin chains obtained from lysates of 15 × 106 erythroid cells after 14 days of liquide culture started from normal (ND1 and ND2), thalassemic (THAL 35 = THAL-β0 and THAL 51 = THAL-β+) and thalassemic GLOBE-transduced (THAL β0-GLOBE or THAL β+-GLOBE) BM CD34+ cells after metabolic incorporation of radiolabelled leucine. Neo-synthetized single globin chains (α, β, δ, Aγ and Gγ) are indicated on each corresponding peak.

Figure 6. Correction of ineffective erythropoiesis

1. Photographs of May Grünwald–Giemsa-stained cytospin of erythroid cells from normal donor (ND), β-thalassemia (THAL) and GLOBE-transduced β-thalassemia (THAL-GLOBE) samples after 2 weeks of erythroid liquid culture (Microscope: Olympus Provis AX70. Objective: 100×/1.30 oil).

2. Percentage cell number for each erythroblasts population after differential counting. ND n = 4, THAL n = 7, THAL-GLOBE n = 7.

3. Representative FACS analysis of GpA+ erythroid cells for apoptotic/dead cells using Annexin V or propidium iodide labelling. Percentage of double positive cells on the total is reported in the panel.

4. Percentage of apoptotic (GpA+ Annexin V+) or late apoptotic/dead (GpA+ PI+) erythroid cells at day 14 of culture. ND n = 2, THAL n = 3, THAL-GLOBE n = 3 THAL vs. THAL-GLOBE; *p < 0.05.

Figure 7. Distribution of the GLOBE vector integration sites in the human genome and correlation with gene activity in CD34+ cells

1. GLOBE vector integration sites (red bars) in the genome of BM-CD34+ cells from thalassemic patients. LV-CMV-GFP integrations (LV-GFP) and random genomic sequences originated by LM-PCR (RANDOM), are used as control (green and blue bars, respectively). Intergenic, intragenic and TSS-proximal integrations are defined as represented in the scheme. Asterisks indicate frequency significantly different between groups (*p < 0.05; ***p < 0.001).

2. Distribution of target genes in the different expression level categories. The first two bars (CHIP THAL and CHIP CD34+) show the distribution of the genes on the microarray of thalassemic and normal CD34+ cells activated with cytokines, the third bar (RANDOM) represents random control sequences, the fourth and fifth bars (LV-GFP and GLOBE) report the expression values of genes targeted by control LV-CMV-GFP and GLOBE integrations, respectively. The N-value indicates the number of probesets, the n values indicate the number of genes analysed.