preparation-gene-modification-nonhuman-primate-hematopoietic-stem

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, ...

Fred Hutchinson Cancer Research Center

Sorting Out the Best: Enriching Hematopoietic Stem Cells for Gene Therapy and Editing.

Engineering resistance to CD33-targeted immunotherapy in normal hematopoiesis by CRISPR/Cas9-deletion of CD33 exon 2.

Elements within the γ-hemoglobin promoters ( and ) function to bind transcription complexes that mediate repression of fetal hemoglobin expression. Sickle cell disease (SCD) subjects with a 13-bp deletion in the  promoter exhibit a clinically favorable hereditary persistence of fetal hemoglobin (HPFH) phenotype. We developed TALENs targeting the homologous  promoters to de-repress fetal hemoglobin. Transfection of human CD34 cells with TALEN mRNA resulted in indel generation in  (43%) and  (74%) including the 13-bp HPFH deletion (∼6%). Erythroid differentiation of edited cells revealed a 4.6-fold increase in γ-hemoglobin expression as detected by HPLC. Assessment of TALEN-edited CD34 cells  in a humanized mouse model demonstrated sustained presence of indels in hematopoietic cells up to 24 weeks. Indel rates remained unchanged following secondary transplantation consistent with editing of long-term repopulating stem cells (LT-HSCs). Human γ-hemoglobin expressing F cells were detected by flow cytometry approximately 50% more frequently in edited animals compared to mock. Together, these findings demonstrate that TALEN-mediated indel generation in the γ-hemoglobin promoter leads to high levels of fetal hemoglobin expression  and , suggesting that this approach can provide therapeutic benefit in patients with SCD or β-thalassemia.

Olivier Humbert

Stem Cell and Gene Therapy Program

Fred Hutchinson Cancer Research Center

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells

Olivier Humbert

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Fred Hutchinson Cancer Research Center

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells

Stem Cell and Gene Therapy Program