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91 Editing the LRF Repressor Binding Site in the γ-Globin Promoters Induces Therapeutically Relevant Fetal Hemoglobin Levels for the Treatment of β-Hemoglobinopathies

Program: Oral and Poster Abstracts
Type: Oral
Session: 113. Hemoglobinopathies, Excluding Thalassemia—New Genetic Approaches to Sickle Cell Disease: Fetal Hemoglobin Regulation And Reticulocyte Maturation In Sickle Cell Disease
Hematology Disease Topics & Pathways:
Biological, Therapies, gene therapy
Saturday, December 5, 2020: 10:30 AM

Giacomo Frati, PhD1,2*, Panagiotis Antoniou, MSc2,3*, Giulia Hardouin2,3*, Bochra Mlaya, MSc2,3*, Leslie Weber4*, Tristan Felix2,5*, Antonio Casini6*, Marion Rosello7*, Anne Chalumeau2,5*, Mario Amendola8*, Adrian J. Thrasher, MBBS, PhD, FMedSci9*, Giandomenico Turchiano, PhD10*, Filippo Del Bene7*, Wassim El Nemer, PhD2,11,12*, Anna Cereseto6*, Jean-Paul Concordet13*, Carine Giovannangeli14*, Marina Cavazzana, MD, PhD15,16 and Annarita Miccio, PhD1,2

1Imagine Institute, Laboratory of chromatin and gene regulation during development, INSERM UMR1163, Paris, France
2University of Paris, Paris, France
3Imagine Institute, INSERM UMR1163, Paris, France, Paris, France
4Imagine Institute, Paris, FRA
5Imagine Institute, Paris, France
6CIBIO, University of Trento, Trento, Italy
7Institut de la Vision, Paris, France
8Genethon, Evry, France
9UCL Great Ormond Street Institute of Child Health, London, United Kingdom
10UCL GOS Institute of Child Health Molecular and Cellular Immunology, LOndon, United Kingdom
11Integrated Biology of the Red Cell, INSERM Unit 1134, Paris, France
12Laboratory of Excellence GR-Ex, Paris, France
13INSERM U565, CNRS UMR7196, Museum National d'Histoire Naturelle, Paris, France
14INSERM U565, CNRS UMR7196, Museum National d'Histoire Naturelle, Paris, France, Paris, France
15Biotherapy Department, Hopital Necker - Enfants Malades, Paris, France
16IMAGINE Institute, Université Paris Descartes, Sorbonne Paris Cité, Paris, France

β-hemoglobinopathies are genetic anemias caused by a reduced or abnormal synthesis of the adult β-globin chain. In β-thalassemia, the reduced (β+) or absent (β0) production of adult β-chains causes α-globin precipitation and death of red blood cell (RBC) precursors. In sickle cell disease (SCD), a single amino acid change (β6Glu→Val) in the adult hemoglobin (Hb) βS-chain causes Hb polymerization with consequent red blood cell (RBC) sickling, vaso-occlusive crises, organ damage and reduced life expectancy. The co-inheritance of genetic mutations causing a sustained fetal γ-globin chain production in adult life (hereditary persistence of fetal hemoglobin, HPFH) reduces the clinical severity of β-hemoglobinopathies. HPFH mutations in the promoter of the two γ-globin genes, HBG1 and HBG2 disrupt the binding sites (BS) for transcriptional repressors (e.g., BCL11A and LRF).

Recently, we demonstrated that CRISPR/Cas9-mediated disruption of the LRF BS in the HBG promoters via non-homologous end joining and microhomology-mediated end joining (MMEJ) repair mechanisms mimics the effect of HPFH mutations by impairing the LRF binding and re-activating the γ-globin expression (Weber, Frati et al., Science Advances, 2020). Efficient editing of the LRF BS (≥ 3 γ-globin promoters in >70% of SCD hematopoietic stem/progenitor cells (HSPCs)) resulted in a robust HbF reactivation and a concomitant reduction in βS-globin levels recapitulating the phenotype of asymptomatic SCD-HPFH patients. RBCs derived from edited HSPCs displayed HbF levels sufficient to correct the SCD cell phenotype. Similarly, LRF BS targeting in β0-thalassemic cells results in HbF reactivation potentially correcting the α/β-like globin imbalance.

Xenotransplantation of human HSPCs edited using several gRNAs targeting the LRF BS showed a robust engraftment of edited cells that were capable to differentiate into multiple lineages. HBG editing in engrafted cells ranged from 26% to 76% with a decrease (-33%) of editing events compared to the input HSPCs, partially due to a reduced occurrence of MMEJ-mediated events in hematopoietic stem cells (HSCs). Erythroid progenitors (BFU-E) obtained ex vivo from engrafted human cells, showed a relevant γ-globin expression (~40% of the total β-like chain) despite of the reduction in the number of edited promoters per BFU-E after transplantation. Moreover, mature RBCs ex vivo differentiated from edited human cells ensure therapeutically relevant HbF levels.

Sequencing of top-scoring off-targets identified by GUIDE-seq showed a relatively high off-target activity within an intergenic site devoid of known regulatory elements both in vitro and in vivo in primary human cells treated with one of the gRNAs targeting the LRF BS. Although the occurrence of this off-target event in repopulating cells suggests that it has no detrimental effect on HSC engraftment and multilineage differentiation, we tested high fidelity Cas9 variants to reduce off-target activity in primary HSPCs. Finally, we used CAST-Seq assay to evaluate the potential chromosomal rearrangements in edited primary human cells in vitro and in vivo.

To minimize the potential genotoxicity associated to Cas9 nuclease-mediated double-strand breaks (DSBs) in the genome, we explored the base editing system to introduce C>T point mutations in the LRF BS without generating DSBs. The absence of the canonical SpyCas9 NGG PAM close to the LRF BS, prompted us to use known and novel base editors containing non-NGG Cas9 variants that allowed the editing of up to 6 out of 8 cytosines of the LRF BS in erythroid cell lines and in primary HSPCs from SCD patients. These C>T conversions include not only known HPFH mutations but also mutations that can further impair LRF binding. In the majority of cases, we detected no insertions or deletions in base-edited samples, as compared to nuclease-edited samples. The 4.9-kb deletion that can be generated upon cleavage of the two identical HBG promoters by the Cas9 nuclease was barely detected in base-edited samples. Importantly, disruption of the LRF BS by non-NGG enzymes led to HbF de-repression, without affecting erythroid differentiation.

This work identifies the LRF BS as an effective and safe therapeutic target for the treatment of β-hemoglobinopathies.

Disclosures: Casini: Alia Therapeutics: Current Employment, Current equity holder in publicly-traded company. Thrasher: Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Generation bio: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership; 4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership.

*signifies non-member of ASH