American Society of Hematology

Figure 1.

$Figure 1. Therapeutic gene editing of IVS1-110G>A. (A) Schema of IVS1-110G>A mutation within HBB intron 1 and therapeutic editing strategy. (B) Indicated donors and sgRNAs used for therapeutic editing. Five days after RNP electroporation, amplicon deep sequencing was performed on the SpCas9-treated cells. Following sequence analysis, alleles were classified as edited, unedited IVS1-110G>A or unedited IVS1-110G. (C) Nucleotide quilt showing indels and substitutions at each position around IVS1-110 for indicated donors and SpCas9 RNP treatment groups. β+β0#1 with sgAAVS1 shown as a representative example of an unedited IVS1-110G>A heterozygous donor and β+β+ with sgAAVS1 as a representative example of an unedited IVS1-110G>A homozygous/hemizygous donor. (D) RT-PCR from erythroid progeny with primers spanning the exon 1 to exon 2 junction, demonstrates abrogation of aberrant (A) and increase in normal (N) splicing after therapeutic editing. (E) RT-qPCR of globin genes shows increase in β-globin relative to α-globin expression in erythroid progeny after therapeutic editing. (F) Hemoglobin HPLC shows increase in the HbA fraction after therapeutic editing. (G-H) Flow cytometry shows increase in enucleation fraction and cell size of enucleated erythroid cells after therapeutic editing. (I) RT-PCR from clonal erythroid progeny with primers spanning the exon 1 to exon 2 junction. Indel length of edited IVS1-110G>A allele depicted for individual clones. (J) Fluorescence-activated cell sorting (FACS) of CD34+CD38+ HPC- or CD34+CD38−CD90+CD45RA− HSC-enriched populations 2 hours after therapeutic editing of the β+β+ donor, which was 24 hours after CD34+ HSPC isolation. Indel analysis was performed 5 days after sorting.$

Therapeutic gene editing of IVS1-110G>A. (A) Schema of IVS1-110G>A mutation within HBB intron 1 and therapeutic editing strategy. (B) Indicated donors and sgRNAs used for therapeutic editing. Five days after RNP electroporation, amplicon deep sequencing was performed on the SpCas9-treated cells. Following sequence analysis, alleles were classified as edited, unedited IVS1-110G>A or unedited IVS1-110G. (C) Nucleotide quilt showing indels and substitutions at each position around IVS1-110 for indicated donors and SpCas9 RNP treatment groups. β⁺β⁰_#1 with sgAAVS1 shown as a representative example of an unedited IVS1-110G>A heterozygous donor and β⁺β⁺ with sgAAVS1 as a representative example of an unedited IVS1-110G>A homozygous/hemizygous donor. (D) RT-PCR from erythroid progeny with primers spanning the exon 1 to exon 2 junction, demonstrates abrogation of aberrant (A) and increase in normal (N) splicing after therapeutic editing. (E) RT-qPCR of globin genes shows increase in β-globin relative to α-globin expression in erythroid progeny after therapeutic editing. (F) Hemoglobin HPLC shows increase in the HbA fraction after therapeutic editing. (G-H) Flow cytometry shows increase in enucleation fraction and cell size of enucleated erythroid cells after therapeutic editing. (I) RT-PCR from clonal erythroid progeny with primers spanning the exon 1 to exon 2 junction. Indel length of edited IVS1-110G>A allele depicted for individual clones. (J) Fluorescence-activated cell sorting (FACS) of CD34⁺CD38⁺ HPC- or CD34⁺CD38⁻CD90⁺CD45RA⁻ HSC-enriched populations 2 hours after therapeutic editing of the β⁺β⁺ donor, which was 24 hours after CD34⁺ HSPC isolation. Indel analysis was performed 5 days after sorting.

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