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

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

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

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

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

AnkT9W increases Hb A synthesis in SCD specimens derived from CD34⁺ cells, maintaining total hemoglobin content unaltered.

<p>(A) HPLC profile of a SCD specimen at steady state (left) or after treatment with AnkT9W (right). (B) Hb A% increase (top) and concomitant Hb F% (middle) and Hb S% (bottom) decrease after treatment with AnkT9W. (C) Increase of Hb A% is plotted against VCN. 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. (D) Total Hb content before and after treatment with AnkT9W, obtained measuring the areas under hemoglobin peaks detected by HPLC.</p

Lentiviral constructs and Hβ:mα Hb synthesis in MEL cells after lentiviral vectors integration.

<p>(A) Black and white rectangles represent exons and introns, respectively, within the β-globin gene. The human ankyrin insulator (in grey, 190 bp) was introduced into the 3′LTR, thereby generating AnkT9W. In the T9Ank2W vector we inserted the ankyrin element between the HS2 and the β-globin promoter. As a control for AnkT9W, the λT9W vector was generated, by replacing the ankyrin element with a fragment from the bacteriophage λ (190 bp, in white). βp: β-globin promoter. E⁺: β-globin enhancer. LCR: locus control region. HS: DNase I hypersensitive sites. LTR: long terminal repeat. SIN: self-inactivating. (B) HPLC analyses of murine Hbs (tetramers of two α-chains associated with two β minor/major chains) in control MEL cells, and chimeric human/mouse Hb (Hβ:mα) in MEL cells infected with approximately one copy of T9W (Hβ:mα = 30.7%), AnkT9W (Hβ:mα = 36.2%) and λT9W (Hβ:mα = 19.16%), respectively. The percentages and absolute amounts of Hβ:mα Hb after HMBA differentiation, in pools of MEL cells carrying three copies of T9W or AnkT9W are shown in C and D, respectively. (E) Expression of human/mouse β-globins expressed over a seven-day differentiation period in MEL cells carrying 3 copies of either T9W or AnkT9W. Values of both globins are normalized by mouse <i>Gapdh</i> expression. (F) Representative polysomal profile obtained. Continuous OD₂₅₄ of 10–50% sucrose gradient with peaks corresponding to the 40 S, 60 S, 80 S and polysomal complexes are indicated. (G) Analyses of ribosomal RNA expression. Total mRNA of MEL cells carrying approximately 3 copies of T9W or AnkT9W was separated into 13 fractions after sucrose gradient ultra-centrifugation. Polysome-bound RNAs were collected between fractions 10 and 13 and represent messenger RNA with higher rate of translation. Values of mouse β-actin or human β-globin expression of each fraction were calculated as proportion of the total RNA.</p

Mutations phenotype.

<p>Description of β0 and β+ mutations.</p