Correction of the sickle cell mutation in embryonic stem cells

Abstract

Sickle cell anemia is one of the most common genetic diseases worldwide. Patients often suffer from anemia, painful crises, infections, strokes, and cardiopulmonary complications. Although current management has improved the quality of life and survival of patients, cure can be achieved only with bone marrow transplantation when histocompatible donors are available. The ES cell technology suggests that a therapeutic cloning approach may be feasible for treatment of this disease. Using a transgenic/knockout sickle cell anemia mouse model, which harbors 240 kb of human DNA sequences containing the beta(S)-globin gene, we prepared ES cells from blastocysts that had the sickle cells anemia genotype and carried out homologous recombination with DNA constructs that contained the beta(A)-globin gene. We obtained ES cells in which the beta(S) was corrected to the beta(A) sequence. Hematopoietic cells differentiated from these ES cells produced both hemoglobin A and hemoglobin S. This approach can be applied to human ES cells to correct the sickle mutation as well as beta-thalassemia mutations.

Fig. 1.

Genotyping of ES cell clones by Southern blot analysis using mouse (m) α- and β-globin gene probes (A) and human (h) α- and γ-globin gene probes (B). ko, knockout; wt, wild type.

Fig. 2.

Homologous recombination in ES cells prepared from the sickle cell anemia mice. (A) Targeting construct 1 and 2 and the β^S genomic structure. The β-globin gene and flanking sequence of 5.9 kb and 5.0 kb is shown with the three exons indicated by three black boxes for the β^A gene and the first exon indicated by the stippled box for the β^S gene. The positive selection marker, the hygromycin-resistance gene (Hygr), is flanked by the loxP site (hatched boxes). The negative selectable marker, herpes simplex virus thymidine kinase gene (hsvTK), is at the 3′ end of the constructs. The box Pr is a 400-bp probe used for Southern blot analysis. (B) Two types of recombinants resulting from homologous recombination depending on the site of crossing-over: one correcting β^S to β^A, the other not correcting β^S to β^A. (C) Southern blot analysis of DNA of the parental line 96 (P) and the drug-resistant clones (indicated by the numbers). With construct 1, EcoRV digestion produces a 15-kb genomic β^S fragment and a 9-kb recombinant fragment. With construct 2, PvuII digestion produces a 12-kb genomic β^S and a 14-kb recombinant fragment. The 5-kb band A is the result of star activity of the enzyme Pvull, possibly due to low ionic strength, high pH, or high enzyme concentration in the incubation condition (22).

Fig. 3.

Identification of corrected or uncorrected β⁶ sequences in the targeted genes by Southern blot analysis. Genomic DNAs from the parental cell line (P) and the homologous recombinant clones were digested with Bsu36I (B) and hybridized with a ³²P-labeled 1.2-kb Bsu36I fragment 5′ to the β⁶ position as shown under construct 2 (Pr). With construct 1, because of the position of insertion of the hygromycin gene, the corrected genome, β^A, generates a 3.2-kb fragment, whereas the uncorrected β^S generates a 3.4-kb fragment. The 1.4-kb fragment is from the unrecombined allele. With construct 2, the corrected and uncorrected genomes produce a 1.2-kb and 1.4-kb fragment, respectively. (A) Restriction map. (B) Southern blot analysis.

Fig. 4.

Identification of hemoglobin A and S with specific monoclonal antibodies in the hematopoietic cells differentiated from the ES cell clones. Cells on the first slide were stained with anti-hemoglobin S antibody and then incubated with secondary anti-mouse antibody conjugated with Alexa Fluor 488 (green), and on a second slide, the cells were stained with anti-A antibody and then incubated with secondary anti-mouse antibody conjugated with Alex-fluor 555 (red). The numbers indicate hematopoietic cells differentiated from ES cells. 96, parental ES cells; 1-7, uncorrected recombinant clone not producing hemoglobin A; 1-6, 1-34, and 2-41, corrected clones producing both hemoglobin A and hemoglobin S. Blood from the sickle cell anemia mouse serves as control for hemoglobin S, and blood from a human serves as a control for hemoglobin A. Negative indicates no primary anti-hemoglobin A or anti-hemoglobin S antibody was used.