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673 Base Editing Eliminates the Sickle Cell Mutation and Pathology in Hematopoietic Stem Cells Derived Erythroid Cells

Program: Oral and Poster Abstracts
Type: Oral
Session: 801. Gene Editing, Therapy and Transfer II
Hematology Disease Topics & Pathways:
sickle cell disease, Biological, HSCs, Diseases, bioengineering, Therapies, red blood cells, Genetic Disorders, Hemoglobinopathies, Biological Processes, Technology and Procedures, gene therapy, DNA repair, Cell Lineage, erythropoiesis, gene editing, hematopoiesis, transplantation
Monday, December 7, 2020: 12:00 PM

Jonathan S Yen, PhD1, Gregory A. Newby, PhD2*, Thiyagaraj Mayuranathan1*, Shaina N. Porter, PhD3*, Yu Yao, MD1*, Kaitly J. Woodard, BS1, Kalin Mayberry, BS1*, Kelcee Everette2*, Jingjing Zhang, MS1*, Jordana M Henderson, MS4*, Anton P Mccaffrey4*, Shondra M. preutt-Miller, PhD3*, John F. Tisdale, MD5, Shengdar Q. Tsai, PhD1, David R. Liu, PhD6,7,8* and Mitchell J. Weiss, MD, PhD1

1Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN
2Broad Institute of MIT and Harvard, Cambridge, MA
3Center for Advanced Genome Engineering (CAGE), St. Jude Children's Research Hospital, Memphis, TN
4Trilink Biotech, San Diego, CA
5Cellular and Molecular Therapeutics Branch, NHLBI, National Heart, Lung, and Blood Institute, Bethesda, MD
6Howard Hughes Medical Institute, Harvard University, Cambridge, MA
7Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA
8Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA

Sickle cell disease (SCD) is a chronic, life-altering multisystem disorder that affects millions of individuals worldwide. Strategies for genetic therapy of autologous SCD hematopoietic stem cells (HSCs) include lentiviral vector (LV) delivery of an anti-sickling β-like globin gene and genome editing, either to revert the SCD mutation by homology-directed repair (HDR) or to induce the expression of fetal hemoglobin (HbF, α2γ2) in red blood cell (RBC) by non-homologous end-joining repair (NHEJ). Base editing is a newer technology that offers the potential for facile generation of more precise genetic alterations with improved safety features. Adenosine base editors (ABEs) convert A-T base pairs to G-C pairs at loci targeted by a single guide (sg) RNA and Cas9 nickase. In contrast to LV vectors and standard Cas9 genome editing, ABEs function through mechanisms that are independent of double-stranded DNA breaks (DSBs), which can cause large deletions, structural DNA rearrangements and TP53-mediated DNA damage responses leading to cell death or malignant transformation.

Base editors cannot generate the T-to-A transversion required to revert the mutant SCD codon (Val, GTG) to wild-type (Glu, GAG). However, ABEs can convert the Val codon to Ala (GCG) to generate the naturally occurring, non-sickling variant hemoglobin “Makassar” (HbG). Hemoglobin Makassar heterozygotes and 1 reported homozygote exhibit normal RBC indices, indicating that the variant is benign. We used protein directed evolution to generate a new ABE (ABE8e-NRCH) that accesses a nearby CACC PAM to convert HbS alleles to HbG efficiently in heterologous cells. To demonstrate therapeutic proof of concept, we electroporated ABE8e-NRCH mRNA and targeting sgRNA or ABE8e-NRCH/sgRNA ribonucleoprotein (RNP) complex into 3 different SCD donor CD34+ hematopoietic stem and progenitor cells (HSPCs). After 48 hours, conversion of the HbS allele to HbG was 58±5% with ABE8e-NRCH mRNA/sgRNA and 34±5% with RNP (n=3). On target editing was maximal at 144 hours: 80±2% with ABE8e-NRCH mRNA/sgRNA and 44±7% with ABE8e-NRCH protein (n=3). The indel rate resulting from inadvertent DSBs was <1%, and missense bystander mutations were not detected. In vitro differentiation of base-edited CD34+ cells generated late stage erythroid cells with 76% HbG/14% HbS protein after editing with ABE8e-NRCH mRNA/sgRNA, and 52% HbG/37% HbS after editing with RNP. The sickling rate of in vitro-generated reticulocytes exposed to hypoxia (2% O2) was 52% in controls derived from unedited CD34+ cells, 16% after editing with ABE8e-NRCH mRNA/sgRNA and 27% after editing with RNP.

To assess base editing in repopulating HSCs, we transplanted ABE8e-NRCH–edited SCD CD34+ cells into NBSGW mice. The editing frequency before transplantation was ~64% for ABE8e-NRCH mRNA/sgRNA and 40% for RNP at 48 hours after electroporation. Mice were euthanized after 16 weeks and base editing was analyzed in human donor-derived cells. Overall editing rates were preserved in repopulating cells: 68%±3% for ABE8e-NRCH mRNA/sgRNA and 36%±3% for RNP (n=4). The editing frequencies were similar in donor cell–derived myeloid, erythroid, and B cell-lineages, indicating that base editing did not alter hematopoietic development. Bone marrow erythroblasts derived from base-edited and control CD34+ HSPCs exhibited similar maturation profiles and enucleation. Erythroblasts generated in vivo from HSPCs from SCD patient exhibited potentially therapeutic levels of HbG protein: 58±3% with ABE8e-NRCH mRNA/sgRNA and 27%±3% with RNP (n=4). Adenine base editor conversion of the HbS allele to the Makassar variant in autologous HSCs represents a new therapeutic approach for SCD.

Disclosures: Yen: Beam Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Henderson: Trilink Biotech: Current Employment. Mccaffrey: Trilink Biotech: Current Employment. Liu: Pairwise Plants: Consultancy, Patents & Royalties; Prime Medicine: Consultancy, Patents & Royalties; Editas Medicine: Consultancy, Patents & Royalties; Beam Therapeutics: Consultancy, Patents & Royalties. Weiss: Rubius Inc.: Consultancy, Current equity holder in private company; Cellarity Inc.: Consultancy, Current equity holder in private company; Novartis: Consultancy, Current equity holder in private company; Esperion Therapeutics: Consultancy, Current equity holder in private company; Beam Therapeuticcs: Consultancy, Current equity holder in private company.

*signifies non-member of ASH