Therapeutic Hemoglobin Levels after Gene Transfer in β-Thalassemia Mice and in Hematopoietic Cells of β-Thalassemia and Sickle Cells Disease Patients

Preclinical and clinical studies demonstrate the feasibility of treating β-thalassemia and Sickle Cell Disease (SCD) by lentiviral-mediated transfer of the human β-globin gene. However, previous studies have not addressed whether the ability of lentiviral vectors to increase hemoglobin synthesis might vary in different patients

AnkT9W increases HbA synthesis to therapeutic levels in most of the thalassemic ErPCs.

<p>(A) Net increase of Hb A % (ΔHb A, calculated by subtracting Hb A after treatment from that following treatment) is plotted against VCN (on the X axis) in the three patients groups, β0/0, β0/+ and β+/+. The total Hb A% is also represented as percentage of Hb A before (light grey) and after transduction (dark grey) with AnkT9W in the same groups (B). Figures C and D show the concomitant percentages of Hb F and α-aggregates, both of which are reduced in thalassemic ErPCs after treatment with AnkT9W. The tie bars under the X axes group different specimens from the same individual. Several specimens were harvested and transduced at different times so as to expose the variability in such experiments. “*” Sign indicates specimens treated with AnkT9Wsil2.</p

HPLC profiles of thalassemic specimens analyzed after differentiation from CD34-derived cultures.

<p>The HPLC profiles of the same thalassemic β0/0 (A) and β+/+ (B) specimens were analyzed at steady state (left) and following treatment with AnkT9W (right) after differentiation starting from either CD34⁺ cells (top) or ErPCs (bottom). In the same β0/0 specimen, AnkT9W contributes to increasing Hb A synthesis from from 0% to 62% (VCN = 0.92), in the CD34⁺-derived cells or to 73% (VCN = 0.97), in the ErPCs-derived cells. In the cells from the β+/+ patient, the net Hb increase was 35% (VCN = 0.88), if CD34⁺-derived or 23% (VCN = 0.94), if ErPCs-derived.</p

Mutations in patients.

<p>List of β-thalassemia specimens analyzed in this study.</p

AnkT9W improves Hb synthesis and hematological parameters of thalassemic mice.

<p>Red cell hemolysates were analyzed monthly by HPLC for up to 6 months post BM transplant. Chimeric (A) and total absolute Hb content (B) in T9W-<i>Hbb^th3/+</i> and AnkT9W-<i>Hbb^th3/+</i> chimeras (n = 4 and 6, respectively). Hβ:mα Hb detected by HPLC and relative VCN (C and D) in mice chimeras obtained by transplanting <i>Hbb^th3+</i> BM treated with T9W or AnkT9W vectors into lethally irradiated <i>Hbb^th3+</i> recipient mice. (E, F) Hematologic parameters in WT, <i>Hbb^th3/+</i>, T9W-<i>Hbb^th3/+</i> or AnkT9W-<i>Hbb^th3/+</i> chimeras. On average, the hematocrits in AnkT9W-treated chimeras were 26% and 10% higher, the reticulocyte counts 71% and 49% lower, and the RDWs 35% and 22% lower than in <i>Hbb^th3/+</i> and T9W-<i>Hbb^th3/+</i> mice, respectively. For reticulocyte counts, Hb, RDW, p<0.0001,and for HCT, p = 0.0002.</p

Sequences of exon 1 and 2 in AnkT9W and AnkT9Wsil1/2 silent mutants.

<p>Original and corresponding mutated bases of exons 1 and 2 inside constructs are in bold letters.</p

Hematopoietic parameters, splenomegaly and EMH of AnkT9-treated <i>Hbb^th3/+</i>- are comparable to those of WT-chimeras.

<p>(A) Flow cytometry. CD71/Ter119 antibodies (y/x axes, respectively) were utilized to assess the correction of ineffective erythropoiesis in representative WT and <i>Hbb^th3/+</i> mice and in <i>Hbb^th3/+</i> mice treated with T9W or AnkT9W lentiviral vectors, in spleen (top) and bone marrow (bottom). (B) Mice spleen size. (C) From top to bottom: a WT mouse transplanted with WT bone marrow; an <i>Hbb^th3/+</i> that received <i>Hbb^th3/+</i> bone marrow untreated or transduced with T9W or AnkT9W. Liver and spleen morphology of the same animals are shown in the middle and right panels, respectively. (C, left panel) Blood smears stained with May-Grunwald-Giemsa staining, showing the RBCs morphology of representative mice. Sections of liver and spleen were also analyzed to assess extramedullary hematopoiesis (EMH) and organ morphology. The livers from both T9W- and AnkT9W-treated chimeras were similar to those of normal control mice in which no EMH was detected (C, middle panel). In the spleens of <i>Hbb^th3/+</i> mice, the cross-sectional area of the white pulp (C, right panel) was relatively decreased and the marginal zones were obscured by the large number of nucleated RBCs, reflecting major expansion of the red pulp and erythroid precursors. In mice treated with T9W, and even more so in AnkT9W-treated chimeras, the amount of red pulp was decreased considerably, accounting for only about 40% to 50% of the cross-sectional area, and denoting a considerable reduction of EMH.</p