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

Mutations in patients.

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

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

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

The ankyrin insulator improves translation of the β-globin gene, and its sequence is conserved at the site of integration.

<p>(A) Expression of the chimeric Hb (expressed as a percentage) after HMBA differentiation, in MEL cell clones carrying one VCN of T9W or AnkT9W, and T9Ank2W. One way anova test, p = 0.048. (B) The β-globin mRNA (bottom panel), expressed by T9W (VCN = 0.87,1.4), AnkT9W (VCN = 0.39, 1.07, 1.47) and T9Ank2W (VCN = 1.26, 1.98) was not detected in non-erythroid cells (B-16 melanoma) but only in differentiated MEL cells (AnkT9W, VCN = 2), indicating that the transgene's transcription is tissue specific. (C) Two amplicons were amplified by PCR corresponding to the ankyrin sequence present, respectively, in the 5′ and 3′ viral LTRs. Oligonucleotides were designed to include the ankyrin element and selectively amplify fragments either from the 5′ or 3′ LTR. Only clones bearing a single AnkT9W integrant were used. The amplified fragments exhibited the expected size of 248 bp and 231 bp, indicating that the ankyrin element was faithfully replicated in both LTRs. (D) Southern blot assay of genomic DNA from single integrant clones of MEL cells carrying either T9W or AnkT9W or from MEL control. The genomic DNAs were digested with <i>Xmn</i>I restriction enzyme, which yielded the full β-globin cassette, identified by hybridization using a β-globin <i>BamH</i>I-<i>Nco</i>I probe. Clone #6 of the AnkT9W series was reloaded in higher quantity (left panel) to amplify the signal of the transgenic human β-globin gene, because it was insufficiently loaded and exhibited a weak signal on the first blot (right panel).</p