Restoration of hemoglobin A synthesis in erythroid cells from peripheral blood of thalassemic patients

Abstract

Mononuclear cells from peripheral blood of thalassemic patients were treated with morpholino oligonucleotides antisense to aberrant splice sites in mutant beta-globin precursor mRNAs (pre-mRNAs). The oligonucleotides restored correct splicing and translation of beta-globin mRNA, increasing the hemoglobin (Hb) A synthesis in erythroid cells from patients with IVS2-654/beta(E), IVS2-745/IVS2-745, and IVS2-745/IVS2-1 genotypes. The maximal Hb A level for repaired IVS2-745 mutation was approximately 30% of normal; Hb A was still detectable 9 days after a single treatment with oligonucleotide. Thus, expression of defective beta-globin genes was repaired and significant level of Hb A was restored in a cell population that would be targeted in clinical applications of this approach.

Figure 1

Correction of splicing of thalassemic human β-globin pre-mRNA by antisense oligonucleotides. Boxes, exons; solid lines, introns; dashed lines, correct and aberrant splicing pathways; the aberrant 5′-splice sites created by IVS2–654 and IVS2–745 mutations and the cryptic 3′-splice site activated upstream are indicated; heavy bars, oligonucleotide antisense to 3′- or 5′-splice sites; arrows, primers used in the RT-PCR reaction. The length (in nucleotides) of the appropriate RT-PCR products generated on aberrantly and correctly spliced mRNAs (ab and c mRNA, respectively) are indicated below each diagram.

Figure 2

Time course of pre-mRNA and mRNA β-globin expression in cultured mononuclear cells from normal human blood assayed by RT-PCR of total RNA. (Upper) Expression of β-globin pre-mRNA; (Lower) expression of β-globin mRNA (see Materials and Methods). The numbers on the left indicate the size, in nucleotides, of the pre-mRNA and the correctly spliced β-globin mRNA.

Figure 3

(A) Restoration of correct splicing of IVS2–654 β-globin pre-mRNA by antisense morpholino oligomers in cultured mononuclear cells from peripheral blood of IVS2–654/β^E thalassemic patient (YC). RT-PCR analysis of total cellular RNA. On day 14 of culture, the cells were treated for 24 h with morpholino oligomers indicated at the top of the figure. Lanes 1, 2, and 11: control cells, untransfected, transfected with 745–25 mer, and with an α-globin-25mer, respectively. Note lack of production of correct β-globin mRNA. Lanes 3–6: cells transfected with oligonucleotides targeted to the 3′ cryptic splice site (3′−18 mer and -25 mer) at two different concentrations (15 and 45 μM). Lanes 7–10: cells transfected with oligonucleotides targeted to the aberrant 5′-splice site (654–18 mer and −25 mer), at 15 and 45 μM, each. Lane 12, β-globin mRNA from normal human blood. Due to the presence of correctly spliced mRNA derived from the β^E gene (codon 26G→A), a 5′ β^A codon 26 allele-specific forward primer was used (see Materials and Methods). The numbers on the left indicate the size, in nucleotides, of RT-PCR products representing the aberrantly (367) and correctly (294) spliced β-globin mRNAs. Unless otherwise noted similar designations are used in subsequent figures. (B) Linearity of RT-PCR assay. Increasing amounts (indicated on the Left panel) of total RNA from HeLa cells stably expressing correctly spliced β-globin mRNA were subjected to RT-PCR (20 cycles) and the products were analyzed and quantitated as described in Materials and Methods. Aberrantly spliced IVS2–654 RNA and correctly spliced β-globin RNA from HeLa cell lines were mixed in ratios indicated on the Right panel and analyzed as above. The results were averaged from three independent experiments.

Figure 4

Immunoblot of hemolysate of mononuclear cells from patient YC probed with anti-human hemoglobin antibody. Concentrations of 654–18 mer antisense oligonucleotide are indicated above the lanes. Lanes 1–2, hemolysate from cells transfected on day 8 and harvested on day 11; lanes 3–6, hemolysate from cells transfected on day 10 and harvested on day 13 (lanes 3 and 4) and 15 (lanes 5 and 6); lane 7, hemoglobin standards (S); and lane 8, hemolysate of red blood cells from an IVS2–745 heterozygote subject (H).

Figure 5

Correction of splicing of β-globin pre-mRNA in mononuclear cells from IVS2–745 homozygote (DD) and IVS2–745/IVS2–1 heterozygote (ZA) thalassemic patients. On days 7 (A) and 14 (B) of the of culture, the cells from patient DD were treated for 24 h with morpholino oligomers indicated above the lanes. N, RNA from normal blood (lanes 9 and 5 in A and B, respectively). Oligonucleotide targeted to the IVS2–654 aberrant 5′-splice site, uninvolved in splicing of IVS2–745 pre-mRNA served as negative control (lane 2 in A and B). (C and D) Analogous treatments of 7- and 14-day cell cultures, respectively, from patient ZA. α, Oligonucleotide targeted to human α-globin gene as negative control (D, lane 9).

Figure 6

Immunoblot of hemolysate of cultured mononuclear cells from patient DD (lanes 1–4) and ZA (lanes 5–8) probed with anti-human hemoglobin antibody. The concentration of the 745–25 mer antisense oligonucleotide is indicated above the lanes. Lanes: 1–2 and 5–6, hemolysates from cells transfected on day 8 and harvested on day 11; lanes 3–4 and 7–8, hemolysates from cells transfected on day 12 and harvested on day 15; lane 9, hemolysate of mononuclear cells from an IVS2–745 heterozygote subject harvested on day 15 of culture (H); and lane 10, hemoglobin standards (S).

Figure 7

Time course of restoration of correct splicing and Hb A synthesis in mononuclear cells from patient ZA. Mononuclear cells on day 8 of culture were transfected with 45 μM 745–25 mer antisense oligonucleotide and incubated for 1–9 subsequent days with no additional oligonucleotide. Number of days after oligonucleotide treatment is indicated at the top (Upper); other designations as in Fig. 3 and 5. Lanes: 6–10, oligonucleotide-treated cells, lanes 1–5, no oligonucleotide controls. (Upper) Immunoblot of hemolysate of mononuclear cells probed with anti-human hemoglobin antibody. (Lower) Corresponding RT-PCR assay.

Figure 8

Cytoimmunofluorescent staining of Hb A in oligonucleotide-treated cells from patients DD (panels 1–6) and ZA (panels 7–8). Cells were treated with 45 μM 745–25 mer antisense oligonucleotide on day 12 of culture and incubated for additional 3 days. Panels 1, 3, 5, and 7, phase contrast; panels 2, 4, 6, and 8, fluorescence. Panels 1 and 2, no oligonucleotide controls incubated with anti-human β-globin mAb; panels 3 and 4, oligonucleotide-treated cells incubated with nonimmune mouse IgG1; and panels 5–8, oligonucleotide-treated cells incubated with anti-human β-globin mAb.