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951 Conversion of HbS to Hb G-Makassar By Adenine Base Editing Is Compatible with Normal Hemoglobin Function

Program: Oral and Poster Abstracts
Session: 113. Hemoglobinopathies, Excluding Thalassemia: Basic and Translational: Poster I
Hematology Disease Topics & Pathways:
Fundamental Science, Sickle Cell Disease, Biological, Translational Research, Sickle Cell Trait, Clinically Relevant, Hemoglobinopathies, Diseases, Gene Therapy, Therapies, Emerging Technologies, Gene Editing, Technology and Procedures, Transplantation
Saturday, December 11, 2021, 5:30 PM-7:30 PM

S. Haihua Chu, PhD*, Manuel Ortega, PhD*, Paty Feliciano, PhD*, Valerie Winton, PhD*, Chavonna Xu*, Daniel Haupt*, Teresa McDonald*, Salette Martinez, PhD*, Alexander Liquori*, Jeffrey Marshall, PhD*, Daisy Lam*, Jenny Olins, MS*, Conrad Rinaldi, M.S.*, Kyle Rehberger, BS*, Colin Lazarra*, Jeremy Decker*, Bob Gantzer, M.S.*, Tanggis Bohnuud, PhD*, David Born, PhD*, Luis Barrera, PhD*, Bo Yan, PhD*, Ian Slaymaker, PhD*, Michael S. Packer, Ph.D.*, Sarah Smith*, Carlo Zambonelli, PhD*, Matt Lee, PhD*, Nicole M. Gaudelli, Ph.D.*, Adam J. Hartigan, PhD* and Giuseppe Ciaramella, Ph.D.*

Beam Therapeutics, Cambridge, MA

Conversion of the pathogenic sickle allele to a naturally occurring, non-pathogenic hemoglobin variant, Hb G-Makassar, represents a long-term and durable treatment strategy for sickle cell disease (SCD). Using our engineered adenine base editor, we achieved highly efficient base editing in mobilized sickle trait (HbAS) and non-mobilized homozygous sickle (HbSS) CD34+ cells that led to >70% conversion of sickle allele to Makassar allele in in vitro erythroid differentiated (IVED) cells derived from ex vivo edited CD34+ cells. At this level of editing, >70% bi-allelic Makassar editing could be achieved in HbSS IVED cells, with ~20% of cells being mono-allelically Makassar edited. These mono-allelically edited cells behaved similarly to sickle trait (HbAS) cells, when exposed to hypoxic conditions in vitro. In vivo proof of concept xenotransplantation studies demonstrated that Makassar edited HbAS CD34+ cells achieved long-term, multi-lineage hematopoietic engraftment as well as Makassar globin protein expression in human erythroid glycophorin A+ cells in the bone marrow of immunocompromised mice.

Although the Makassar variant is naturally occurring in human genetics and present in individuals in Southeast Asia with normal hematologic parameters in both heterozygous and homozygous states, we sought to further characterize Makassar hemoglobin and assess its biophysical and biochemical properties. Recombinant Makassar globin was co-expressed with alpha globin in E. coli and tetramers were purified to homogeneity. Recombinant tetramers were assessed for identity, purity, globin content, and heme content demonstrating comparability to hemoglobin tetramers isolated from primary sources (whole blood). Several characterization methods were employed, to assess size, molecular weight, oligomerization state, tetramer composition, and oxygen binding properties. These studies indicated Makassar globin could properly assemble into hemoglobin tetramers, displaying biochemical properties characteristic of hemoglobins. Furthermore, we assessed polymerization potential using a temperature jump method previously employed for kinetic measurements of sickle-fiber formation and found Makassar hemoglobin did not polymerize in vitro under conditions where sickle hemoglobin (HbS) readily polymerizes, consistent with behavior observed previously by others. Finally, a crystal structure of Hb G-Makassar has been determined at the 2.2 Å resolution and showed high similarity to the HbA (wildtype hemoglobin) structure with a RMSD of 0.385 Å for all the Cα atoms, which indicates that the glutamic acid to alanine (E6A) substitution in beta-hemoglobin does not seem to induce any significant conformational change in hemoglobin structures.

Altogether, our biophysical and biochemical characterization shows that the Makassar variant behaves as a functional hemoglobin. By replacing the pathogenic sickle globin with a benign hemoglobin variant with normal function, our base editing approach provides a promising autologous investigational cell therapy for the treatment of SCD.

Disclosures: Chu: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Ortega: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Feliciano: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Winton: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Xu: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Haupt: Beam Therapeutics: Current Employment, Current holder of stock options in a privately-held company. McDonald: Beam Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Martinez: Beam Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Liquori: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Marshall: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Lam: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Olins: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Rinaldi: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Rehberger: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Lazarra: Beam Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Decker: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Gantzer: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Bohnuud: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Born: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Barrera: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Yan: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Slaymaker: Beam Therapeutics: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Packer: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Smith: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Zambonelli: Beam Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Lee: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Gaudelli: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Hartigan: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company. Ciaramella: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

*signifies non-member of ASH