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Base editors are fusions of a deaminase and CRISPR-Cas ribonucleoprotein that allow programmable installment of transition mutations without double-strand DNA break intermediates. The breadth of potential base editing targets is frequently limited by the requirement of a suitably positioned Cas9 protospacer adjacent motif. To address this, we used structures of Cas9 and TadA to design a set of inlaid base editors (IBEs), in which deaminase domains are internal to Cas9. Several of these IBEs exhibit shifted editing windows and greater editing efficiency, enabling editing of targets outside the canonical editing window with reduced DNA and RNA off-target editing frequency. Finally, we show that IBEs enable conversion of the pathogenic sickle cell hemoglobin allele to the naturally occurring HbG-Makassar variant in patient-derived hematopoietic stem cells.

Von Willebrand factor (VWF) is a multimeric hemostatic protein that is synthesized in endothelial cells, where it is stored for secretion in elongated secretory organelles called Weibel-Palade bodies (WPB). The hemostatic activity of VWF is strongly related to the length of these bodies, but how endothelial cells control the dimensions of their WPB is unclear. In this study, using a targeted short hairpin RNA screen, we identified longin-SNARE Sec22b as a novel determinant of WPB size and VWF trafficking. We found that Sec22b depletion resulted in loss of the typically elongated WPB morphology together with disintegration of the Golgi and dilation of rough endoplasmic reticulum cisternae. This was accompanied by reduced proteolytic processing of VWF, accumulation of VWF in the dilated rough endoplasmic reticulum and reduced basal and stimulated VWF secretion. Our data demonstrate that the elongation of WPB, and thus adhesive activity of their cargo VWF, is determined by the rate of anterograde transport between endoplasmic reticulum and Golgi, which depends on Sec22b-containing SNARE complexes.

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 thebone 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.