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H - Clinical Translation of Gene and Cell Therapies -> Vector Product Engineering, Development or Manufacturing

Genome Editing of Human Hematopoietic Stem Cells to Induce Fetal Hemoglobin for Autologous Cellular Therapy of Sickle Cell Disease

Type: Oral Abstract Session

Presentation Details
Session Title: Late-Breaking Abstracts I
Location: Salon H
Start Time: 5/17/2022 9:00
End Time: 5/17/2022 9:15

Sickle cell disease (SCD) is a severe genetic blood disorder that affects millions of individuals worldwide. Individuals that co-inherit homozygous sickle mutations in HBB and natural genetic variants that cause hereditary persistence of fetal hemoglobin are typically asymptomatic. Thus, genome editing strategies to re-activate developmentally silenced fetal hemoglobin (HbF, α2γ2) represent promising clinical approaches to replace defective sickle hemoglobin (HbS, α2βS2). Recent reports from an early-stage clinical trial targeting BCL11A erythroid specific enhancer to induce fetal hemoglobin have been encouraging.
To identify which target when edited in the γ-globin gene promoters would elicit the most robust HbF induction, we compared editing of BCL11A (-115) and ZBTB7A (-198) binding motifs. We electroporated Cas9-3xNLS ribonucleoprotein complexes (RNPs) into human primary CD34+ hematopoietic stem and progenitor cells (HSPCs) and attained high editing rates ranging from 76.2% to 85.7%. 17 weeks after transplanting edited CD34+ cells into immunodeficient NBSGW mice, we observed consistent and high indel mutations frequencies of 63.5% to 92.7% in all hematopoietic lineages with no apparent impairment of multilineage differentiation. We observed associated higher levels of HbF induction in erythroid progeny of CD34+ cells edited at the -115 γ-globin promoter target (31.8%) than those edited at the -198 ZBTB7A binding site (13%-18%).
To further test our -115 γ-globin promoter editing approach in SCD patient cells, we edited plerixafor-mobilized CD34+ HSPCs from one healthy donor and three individuals with SCD. 17 weeks after transplanting edited cells into NBSGW mice, we observed consistently high rates of editing (75.1%) in all hematopoietic lineages, and substantial levels of HbF (27.6%) in erythroid progeny compared to controls (<2.9%). Single cell western showed expression of gamma globin in a majority of edited erythroblasts (58%) compared to unedited controls (<6%). Moreover, RBCs derived from edited CD34+ cells from SCD patients exhibited significant three-fold reduction in hypoxia-induced sickling.
We characterized potential genotoxicities associated with our -115 γ-globin promoter editing approach. With CHANGE-seq discovery and rhAMP-seq validation, we did not detect off-target mutations above background at 194 identified sites. PacBio-Hifi long-range sequencing revealed that the only high-frequency large deletion observed (4.9 kb) results from simultaneous double strand beaks in HBG1 and HBG2 promoters. By digital droplet PCR, we detected an average of 23.9% of this 4.9 kb deletion in engrafted cells at 17 weeks that has no apparent impact on erythropoiesis and HbF induction.
To support plans for a future clinical trial, we have advanced our efforts to manufacture of GMP-grade Cas9 and optimize editing at clinical-scale using the Maxcyte electroporation system. In summary, our preclinical results suggest that disruption of the -115 BCL11A repressor binding site in the γ-globin gene promoters can induce therapeutically effective levels of HbF and is a promising approach for genomic SCD therapy.

Varun Katta1, Kiera O’Keefe1, Racheal Wood1, Cicera R. Lazzarotto1, Thiyagaraj Mayuranathan1, Jonathan Yen1, GaHyun Lee1, Yichao Li1, Naoya Uchida2, Shondra M. Pruett-Miller3, John Tisdale2, Akshay Sharma4, Mitchell J. Weiss1, Shengdar Q. Tsai1

1Hematology, St Jude Children's Research Hospital, Memphis, TN,2Molecular and Clinical Hematology Branch, National Heart, Lung and Blood Institute, Bethesda, MD,3Cell and Molecular Biology, St Jude Children's Research Hospital, Memphis, TN,4Bone Marrow Transplantation and Cellular Therapy, St Jude Children's Research Hospital, Memphis, TN
 V. Katta: None.

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