Rh D blood group conversion using transcription activator-like effector nucleases

Abstract

Group O D-negative blood cells are universal donors in transfusion medicine and methods for converting other blood groups into this universal donor group have been researched. However, conversion of D-positive cells into D-negative is yet to be achieved, although conversion of group A or B cells into O cells has been reported. The Rh D blood group is determined by the RHD gene, which encodes a 12-transmembrane domain protein. Here we convert Rh D-positive erythroid progenitor cells into D-negative cells using RHD-targeting transcription activator-like effector nucleases (TALENs). After transfection of TALEN-encoding plasmids, RHD-knockout clones are obtained. Erythroid-lineage cells differentiated from these knockout erythroid progenitor cells do not agglutinate in the presence of anti-D reagents and do not express D antigen, as assessed using flow cytometry. Our programmable nuclease-induced blood group conversion opens new avenues for compatible donor cell generation in transfusion medicine.

Figure 1. TALENs targeting the human RHD gene.

(a) Schematic of the TALEN-targeting sites in the RHD gene. Blue boxes indicate exons. RHD_E1_TALENs and RHD_E4_TALENs represent the TALEN pairs that target sequences (shown in a red colour) in exon 1 and exon 4, respectively. The red, yellow, green and purple rectangular boxes in the TALENs symbolize the TALE repeat units that recognize guanine, thymine, cytosine and adenine, respectively. (b,c) T7E1 assay using 293T cells after transfection with plasmids encoding TALENs targeting RHD exon 1 (b, RHD_E1_TALENs) or exon 4 (c, RHD_E4_TALENs), respectively. The sizes of marker (M) bands are shown on the left (kbp, kilobase pairs). Arrows indicate the expected positions of DNA bands cleaved by T7E1. The numbers at the bottom of the gel indicate mutation percentages measured by band intensities.

Figure 2. Generation of RHD-mutated erythroid progenitor cells.

(a) Schematic representation illustrating the process of RHD-mutated clone generation. Clonal culture of HiDEP-1 erythroid progenitor cells was initiated 3 days after transfection with plasmids encoding TALENs that target RHD. Genomic DNA from each clone was analysed 17 days after the initiation of clonal culture. (b) T7E1-based clonal analysis. The genomic DNA isolated from each clone was subjected to the T7E1 assay. Arrows indicate the expected position of DNA bands cleaved by T7E1. The sizes of marker (M) bands are shown on the left (kbp, kilobase pairs). Clones containing mutations in the target sites were marked with red clone numbers. Untransfected cells and a cell population transfected with the TALEN plasmids were used as the negative control (NC) and positive control (PC), respectively. M: Markers

Figure 3. DNA sequences of RHD-mutated clones.

The RHD gene DNA sequences from the parental cells, clones with biallelic mutations in exon 1 (E1_B; a) or exon 4 (E4_B; b), and a clone with a monoallelic mutation in exon 4 (E4_M; c). TALE-binding sites are in a red font and spacer regions are indicated with green boxes. Deleted bases are indicated by dashes and inserted bases are shown in a blue font. The number of occurrences is shown in parentheses (for example, × 7 and × 5 indicate the number of each sequence). The sequence and sequencing chromatogram for each allele are shown. The locus of each mutation, the PTC generated by the mutation and the distance between the PTC and the exon–intron junction are depicted in a schematic of the RHD gene. Expected protein sequence translated from each allele are displayed, such that mutated protein sequences generated by a nuclease-induced frameshifting mutation are shown in a red font and translation termination is indicated with a dash. nt, nucleotide.

Figure 4. RHD mRNA in the mutated clones.

RT–PCR was performed to detect RHD mRNA in each clone and the amplicons were subjected to electrophoresis (a) and sequencing (b). (a) Representative pictures of electrophoresis. ACTB was used as control. The sizes of marker (M) bands are shown on the left (kbp, kilobase pairs). (b) Schematic representation of RHD mRNA sequences. The number of occurrences is shown on the right of each transcript. Blue and red circles indicate normal and mutated exons, respectively. For some amplicons, the sequence and sequencing chromatogram are shown (spacer regions are indicated with green boxes).

Figure 5. Flow cytometric analysis of D antigen expression in mutated cells.

Parental and RHD-mutated (biallelic, E1_B, E4_B; monoallelic, E4_M) HiDEP-1 cells were induced for differentiation for 4 days and subjected to flow cytometry. D antigen expression was determined in glycophorin A⁺ cells. (a) Representative histograms. (b) The percentage of D antigen-positive cells in the population of glycophorin A-positive cells. ANOVA followed with Bonferroni's multiple comparison was performed (***P<0.001, **P<0.01, ns=not significant; n=3). Error bars represent the s.e.m.

Figure 6. Absence of D antigen-mediated agglutination in RHD-knockout cell lines.

Parental, RHD-knockout (E1_B, E4_B) and RHD-monoallelic mutant (E4_M) HiDEP-1 cells were induced for differentiation for 4 days and subjected to an agglutination test using anti-D blood grouping reagents (a) and a weak D test using anti-D blood grouping reagents and Coombs' reagent (Anti-IgG, -C3d) (b) in 96-well plates and on glass slides. Rh D-positive, D-negative and weak D-positive human peripheral blood cells were used as the controls. A photograph and photomicrographs of each cell line are shown. Scale bar, 500 μm.

Figure 7. The RHD mutation does not affect the differentiation and function of HiDEP-1.

(a,b) At the indicated times after the induction of erythrocyte differentiation, differentiation of each HiDEP-1 cell line was quantified using microscopy after Wright–Giemsa staining. (a) Morphological analysis of differentiation. The sum of two independent experiments is shown. (b) Cell size during differentiation. ANOVA showed that the cell sizes were the same in the parental and RHD-mutant clones. Error bars represent the s.e.m. n=3. (c) Oxygen equilibrium curves of parental and RHD-mutant clones (E1_B, E4_B and E4_M). Human adult peripheral blood (PB) cells were used as the control.