Installation of HbG-Makassar by base editing restores hemoglobin function: a transformative therapy for sickle cell disease

doi:10.21203/rs.3.rs-3995314/v1

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Installation of HbG-Makassar by base editing restores hemoglobin function: a transformative therapy for sickle cell disease

https://doi.org/10.21203/rs.3.rs-3995314/v1

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Adenine base editing offers a viable gene-based therapy for sickle cell disease (SCD), converting sickle hemoglobin (HbS, βΕ6V) to G-Makassar hemoglobin (HbG, βE6A), a naturally occurring, non-pathogenic variant. However, HbG functionality alone and with HbS has been largely uncharacterized. We present a mouse model used to characterize purified HbG-Makassar as well as HbGG and HbGS red blood cell function. Purified HbG-Makassar behaves as a functional hemoglobin, including no polymerization under hypoxia. Structural characterization of oxy and deoxy states of HbG-Makassar showed no change in the topology of the hemoglobin fold with the βΕ6Α mutation. Red blood cell function assays, sickling propensity under hypoxia, blood counts, and mitochondrial retention measures place HbGS RBCs as intermediate in severity between HbAS and HbSS, organ function was comparable to HbAS. HbGG resembled HbAA for most metrics. Taken together our results suggest direct correction of HbS to HbG-Makassar could provide a transformative therapy for SCD.

Health sciences/Molecular medicine

Health sciences/Medical research/Translational research

Health sciences/Medical research/Experimental models of disease

gene therapy

CD34

hemoglobin

Townes mice

X-ray crystallography

oxygen binding

HbG-Makassar

polymerization

HbS

sickle cell disease

Sickle cell disease (SCD) is a monogenic disorder caused by a single point mutation in the beta-globin gene, producing an abnormal hemoglobin (HbS) that polymerizes under hypoxia, resulting in rigid, poorly deformable red blood cells (RBCs).^1,2 Clinically, this causes vaso-occlusion, severe pain crises, organ damage, and early mortality.^3–5 All pharmacologic therapies, including hydroxyurea and newer second line therapies, are palliative, as is chronic transfusion therapy. Allogenic hematopoietic cell transplantation may be curative for individuals with SCD; but matched sibling donors, which produce the best outcomes, are available to only ~ 10% of individuals with SCD.⁶ Autologous transplant with gene modified cells eliminates the donor barrier, avoids graft versus host disease, and can directly address the underlying pathology of SCD by increasing the amount of functional hemoglobin relative to HbS.^7,8 In December of 2023, the FDA approved genetic medicines exa-cel and lovo-cel for individuals with SCD 12 years of age and over. Most study participants achieved the clinical endpoint, 80% reduction of vaso-occlusive crisis (VOC), validating the utility of genetic medicines that induce fetal hemoglobin or express a non-sickling hemoglobin β subunit^11–13 for the treatment of SCD.^9,10 Recently, preclinical data demonstrated that gene editing therapies utilizing adenine base editing can achieve more potent, uniform, and durable fetal hemoglobin induction ^14,15 without requiring double strand breaks in the genome. While the sickle point mutation in the hemoglobin β subunit (βE6V) cannot be directly corrected to wild-type (βE6) by an adenine base editor (ABE), it can be converted to a naturally occurring hemoglobin variant, known as HbG-Makassar (HbG, βE6A). This presents a promising alternative base editing approach to treat SCD, as it has the potential to convert the pathogenic HbS to a non-pathogenic form of hemoglobin.

The HbG-Makassar variant was initially discovered in 1969 with heterozygous and homozygous individuals identified in Southeast Asia.¹⁶ HbG-Makassar and its potential interaction with HbS have not been well characterized due to low population frequency of HbG-Makassar and the challenges associated with separation of HbG-Makassar and HbS using gel electrophoresis or high-performance liquid chromatography.¹⁷ Characterization of heterozygous HbG-Makassar individuals across Southeast Asia did not reveal any hematological phenotype that correlated with HbG-Makassar expression.^18,19 Recently, a larger study involving individuals with heterozygous HbG-Makassar/beta thalassemia in Malaysia indicated that the HbG-Makassar behaves as a benign hemoglobin variant.²⁰ An HbGG individual was reported to have normal red cell indices and was also pregnant at the time of the report.²¹ In a more recent study, the conversion of HbS to HbG-Makassar through base editing in a SCD mouse model showed the promising potential of this gene editing approach to correct the pathophysiology associated with sickle cell disease.²² Several reports investigating the biochemical and biophysical properties of recombinant HbG-Makassar hemoglobin have demonstrated non-sickling, nonpathogenic properties of the HbG-Makassar variant.^23–26 However, the biophysical properties, including deformability and sickling, of mature RBCs containing HbGS and HbGG have not been described. Furthermore, the lack of structural studies on HbG-Makassar prevents assessing the impact the βE6A mutation has on the hemoglobin fold and consequently, on its function. Despite the nonpathogenic nature of HbG-Makassar, compound heterozygotes of Hb variants can portray disease states. For example, HbC (βΕ6Κ) is recognized as a non-sickling variant.^27,28 However, when combined with HbS, HbC produces a milder disease state with an abnormal red blood cell due to the impact of HbC on hydration.^29,30 Thus, to further biologically de-risk a base editing strategy predicated on the direct conversion of HbS to HbG-Makassar, it is crucial to understand the impact of both HbGG and HbGS on red blood cell function. In this work, we generated transgenic human mouse models expressing human HbGG and HbGS from a well-established Townes mouse model and characterized the purified HbG-Makassar protein as well as red blood cell function. Functional and structural characterization of HbG, coupled with red blood cell functional assays and organ characterization, revealed that HbG-Makassar is indeed a functional hemoglobin variant with normal characteristics when compared to HbS. Our work provides compelling evidence that the direct correction of HbS to HbG-Makassar using base editing is a viable and promising approach to treat individuals suffering with sickle cell disease.

Generation of animal models. A humanized beta globin locus mouse (JAX stock 013071 B6;129-Hbbtm2^{(HBG1,HBB*)Tow}/Hbbtm3^{(HBG1,HBB)TowHbatm1}(Hba)^Tow/J) with the Makassar allele was created at the Jackson Laboratory by knocking-in the Makassar point mutation using CRISPR/Cas9 and donor oligos (Supplemental Information) to generate WT/Makassar heterozygotes (HbAG) which were backcrossed to generated HbGG homozygotes. HbGS genotypes were subsequently generated by crossing HbAG to HbSS and HbAS Townes mice. Genotypes were determined by Sanger sequencing. We analyzed the red cell function of HbAA, HbAS, HbSS, HbGG, and HbGS mice. All mice were maintained and studied according to the National Institute of Health Guide for the Care and Use of Laboratory Animals following an approved protocol by Emory University Institutional Care and Use of Animals Committees.

Purification and characterization of hemoglobin variants. Whole blood carrying the desired hemoglobin variant was collected from appropriate sources (Table S1) and resuspended in 100 mL of IEX binding buffer (10 mM sodium phosphate dibasic pH 6.5). Cells were homogenized (5600 psi, 1 passage) and lysate clarified via centrifugation (4°C, 36000 x g, 45 min). Proteins were purified using a method described previously to separate hemoglobin variants using a MonoS HR16/10 column.³¹ Recombinant hemoglobin HbG for structural studies was purified as described previously.^32,33 Purified proteins were stored at -80°C. Success in isolating individual hemoglobin variants was assessed via mass spectrometry (Figure S1). Methods used for characterizing O₂ binding, and polymerization kinetics of purified hemoglobins can be found in the supplemental information.

Structural characterization of HbG. All crystallization conditions were prepared and optimized using a Mosquito robot (SPT Labtech) at 20°C. For the HbG R2-state structure, drops were prepared by mixing 0.5 µL of HbG isolated from Townes mice (13 mg/ml in 20 mM Tris-HCl pH 7.5, 150 mM NaCl, and 1 mM TCEP) and 0.5 µL of reservoir solution (0.1 M TRIS pH 8.0; 26–36% (v/v) PEG 6,000) and equilibrated against 70 µl of reservoir solution. The crystals were transferred to a cryoprotectant solution (0.1 M Tris pH 8, 36% PEG 6,000, 18% glycerol) and flash-cooled in liquid nitrogen. For HbG in its T-state, the crystallization condition was identified and optimized in an MBraun anaerobic glovebox. Drops were prepared by mixing 0.5 µL of recombinant HbG isolated from E. coli (25 mg/ml in 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM TCEP, and 20 mM sodium dithionite) and 0.5 µL of reservoir solution (0.055 M Citric acid, 0.045 M Bis-Tris propane, pH 4.5, 22% PEG 3,350), and equilibrated against 70 µl of reservoir solution. The crystals were transferred to a cryoprotectant solution (0.055 M Citric acid, 0.045 M Bis-Tris propane, pH 4.5, 24% PEG 3,350, 20% glycerol), and flash-cooled in liquid nitrogen.

Data collections were performed at the Highly Automated Macromolecular Crystallography (AMX) beamline of the National Synchrotron Light Source II. Diffraction data were processed using XDS³⁴ and scaled using AIMLESS. ³⁵ The crystal structures were determined by molecular replacement techniques implemented in Phaser ³⁶ using coordinates of the human hemoglobin structure (PDB ID 2DN2 or PDB ID 2DN1).³⁷Following molecular replacement, simulated annealing was performed to remove model bias using PHENIX.refine. ³⁸ All models were refined by iterative rounds of model building and the addition of water molecules was performed using Coot.³⁹ Non-crystallographic symmetry restraints, TLS (translation, libration, and screw), and positional and B-factor refinement were used on all structures. The data collection and refinement statistics are summarized in Table S2. The residues visualized in the structures from 141 and 146 residues for the α and β subunit respectively, are listed in Table S3.

Hematology measurements. Whole blood, collected in EDTA, was used for all rheological devices and measurements. Complete blood counts were obtained using hematology analyzers Element HT5 (HESKA, Loveland, CO, USA) and ADVIA 2120i (Siemens, Malvern, PA, USA).

Sickling assay. RBCs were stained with Hoechst 33342 and subjected to 2% sodium metabisulfite (MBS) by volume as previously described.⁴⁰ Images were collected by light microscopy at baseline and every minute for thirty minutes post-MBS addition. Number of sickled RBCs, reported as a percentage of the total were quantified by two individuals blinded to the RBC genotype.

Rheology measurements. Red cell deformability (elongation index, EI) was measured using oxygen gradient ektacytometry using the laser-assisted optical rotational red cell analyzer (LORRCA, RR Mechatronics, Zwagg, Netherlands) with the oxygenscan test under normoxic (EImax) and hypoxic (EImin) conditions as previously described.^41,42

Viscosity was measured using a Beckman cone and plate viscometer. 500 µL of whole blood was run through a multipoint viscosity test starting at 6 rpm with a shear rate of 45 (1/s) then raised to 30 rpm at a shear rate of 225 (1/s).^43,44 The two average values of viscosity across those conditions were then recorded, and the hematocrit to viscosity ratio calculated.⁴³

Assessment of mitochondrial retention. RBC mitochondrial retention was measured by flow cytometry using MitoTracker Deep Red (Invitrogen, cat# M46753) on washed RBCs as previously described.⁴⁵ The flow panels were run on a FACSymphony A5 and A3 flow cytometers. Analysis was then performed through FlowJo v10 software.

Measure of erythroid maturation. In a sterile environment, femurs, tibias, and humeri were extracted from mice of each genotype. The compact bone was cut at the caps to expose the bone marrow and spun into a solution of bone marrow harvest media (RPMI, 10% heat inactivated FBS, 20 U/mL DNase, 4 U/mL heparin) at 10,000 rpm for 30 seconds. The erythroid maturation panel was made up of Ter119 (BD Biosciences cat# 563827), CD44+ (Biolegend cat# 103012), Annexin V (Biotium cat# 29004), and zombie dye. Ter119 and CD44 + were used to characterize the erythroid population with Annexin V measured apoptosis, as previously described.⁴⁶ The flow panels were run on a FACSymphony A5 and A3 flow cytometers. Analysis was then performed through FlowJo v10 software.

Pathology. Mice were weighed at the time of sacrifice. Kidneys, spleens, and livers were harvested from three males and three females of each of the five genotypes and washed in PBS prior to being weighed. Organs were preserved in formalin before paraffin embedding. The paraffin blocks were sliced using a microtome to obtain 5 µm thick sections. Sections were stained with hemolysin and eosin and imaged under a Keyence microscope. Images were scored for glomerular sclerosis by two physicians blinded to genotype as previously described.⁴⁷

Statistical analysis: Comparisons across genotypes were performed using Dunn Pairwise test with statistically significant values being selected at adjusted p < 0.05; all performed using STATA 18.0 (College Station). Statistical analysis for data obtained on FlowJo was completed on Prism.

mRNA production for ABE editors used in CD34 + cells. All adenine base editor mRNA was generated as previously described.^15,48 Editors were cloned into a plasmid encoding a dT7 promoter. PCR amplification of the mRNA template was used in subsequent in vitro transcription. The NEB HiScribe High-Yield Kit was used as per the instruction manual but with full substitution of N1-methyl-pseudouridine for uridine and co-transcriptional capping with CleanCap AG (Trilink). Reaction cleanup was performed by lithium chloride precipitation.

CD34 + cell culture and electroporation. Mobilized peripheral blood from HbAS patients was obtained and enriched for Human CD34 + HSPCs and frozen in single-use aliquots (HemaCare, M001F-GCSF/MOZ-2). The CD34 + cells were cultured un X-VIVO 10 (Lonza) containing 1% v Glutamax (Gibco), 100 ng/mL of TPO (Peprotech), SCF (Peprotech) and Flt-3 (Peprotech) and cultured for 48 hours prior to electroporation. Electroporation of hCD34s was conducted with MaxCyte Atx with OC25x3 cassettes and HSC-3 program as previously described. ⁴⁸

CD34 + isolation from HbSS donors. Mobilized peripheral blood CD34s from SCD patient were generously provided by Dr. John Manis (BCH). Non mobilized HbSS CD34s were obtained from red cell exchange bags collected under an Emory approved IRB protocol. Peripheral blood mononuclear cells (PBMCs) were isolated using density centrifugation by Ficoll-Paque (GE healthcare) per manufacturer’s protocols of apheresis product. RBCs were removed using GlyA (StemCell Technologies RBC depletion kit); CD34 + cells were isolated by magnetic separation with UltraPure human CD34 + positive enrichment kit with LS columns (Miltenyi Biotech).⁴⁹

Erythrocyte differentiation post ABE electroporation. CD34 + cells underwent three phase vitro erythroid differentiation (IVED) 48 h post electroporation as previously described.⁴⁸ Single cell IVED clones were obtained by limiting dilution of CD34 + cells 48 h post-electroporation into 96 well U-bottom plates that were confirmed by NGS to be the genotypes of interest.

Ultra-high-performance liquid chromatography (UHPLC) Analysis

UHPLC analysis was previously described.¹⁵ The separation conditions were further optimized for the separation of HbG from HbS. A reverse-phase column at a temperature of 75°C was used. Mobile phases were 0.1% v trifluoroacetic acid (TFA) in water (A) and 0.08% v TFA in acetonitrile (B) with a flow rate of 0.25 mL/min. A gradient of 38–48%B 0–23 min; 48–99%B 10-23-26 min; and 99 − 38%B to 26–28 min was applied. Sample injection volume was 10 µL. UV spectra at a wavelength of 220 nm with a data rate of 5 Hz were collected throughout the analysis.

Genomic DNA extraction and NGS

Genomic DNA from cells was isolated using the Quick Extract (Lucigen) per manufacturer’s recommended protocol. Genomic DNA samples were amplified and prepared for high throughput sequencing as previously reported.⁴⁸

Generation of Townes HbG-Makassar animal models. To evaluate the function of HbG containing RBCs, we generated a knock-in mouse model utilizing the Townes mice that carry several human hemoglobin genes, replacing the endogenous mouse hemoglobin genes.⁵⁰ We targeted the Glu6 of HBB to install HbG-Makassar mutation as well as a silent mutation in the Pro5 amino acid residue, replicating our base editing strategy.⁴⁸ Townes mice expressing HbG-Makassar hemoglobin were then crossed to HbAS Townes mice to generate HbGS animals.

Functional characterization of purified Hb G-Makassar hemoglobin. To determine the impact of βE6A mutation in the function of HbG-Makassar, we performed comparative oxygen binding studies with purified HbA, HbS, and HbG-Makassar. To eliminate confounding results due to the source of isolation we first compared functional parameters between HbA and HbS isolated from human blood and from Townes mice. Purified HbA and HbS have similar p50 values and hill coefficients irrespective of the purification source (Fig. 1A and 1B). Oxygen equilibrium curves show HbG-Makassar has similar functional parameters as HbA, indicating that the βE6A mutation has a negligible impact on the ability of HbG-Makassar to bind and release oxygen (Fig. 1C). To assess how the βE6A mutation affects hemoglobin polymerization,⁵¹ we performed comparative polymerization assays with purified HbS, HbG-Makassar, and HbA. HbS polymerized with delay times that varied as a function of HbS concentration (Fig. 1D). HbG-Makassar did not exhibit polymerization within the designated assay time frame, (compare Figs. 1E and 1F). This was observed even when the HbG-Makassar concentration in the assay was increased to a level that induced a 40 s delay in polymerization for HbS. Similarly, no polymerization behavior was observed for HbA in the same assay condition (Fig. 1F). The polymerization potential of hemoglobin mixtures was also assessed to characterize the editing outcomes resulting in HbGS. Notably, both HbAS and HbGS mixtures showed a slower polymerization rate compared to HbS alone. Under similar in vitro polymerization conditions, HbGS mixtures behave similarly to HbAS mixtures.

To assess the impact of βE6A on the hemoglobin structure, we determined the 1.94-Å and 2.24-Å resolution X-ray crystal structures of HbG-Makassar in the R- or liganded state and T- or unliganded state (table S2). The R-state structure was determined at 1.94-Å resolution and contained one copy of the functional heterotetramer (α1β1–α2β2) in the asymmetric unit (Fig. 2A and 2B). A heme cofactor is seen in all four subunits and its Fe center is coordinated with a water molecule as an axial ligand (Fig. 2A). This structure shows a high degree of similarity to the R2-state structure of HbA (PDB ID 1BBB) ⁵² as indicated by the RMSD of 0.390-Å and 0.218-Å for all Cα atoms in the α and β subunits, respectively (Fig. 2B). The T-state structure was solved at 2.24-Å resolution with two copies of the functional heterotetramer in the asymmetric unit. Analysis of the electron density map revealed there are no non-proteinogenic axial ligands bound to the heme iron in both α- and β-subunits, indicating we captured deoxygenated or unliganded state (Fig. 3C). The T-state hemoglobin is characterized by the formation of a large cavity in the center of the heterotetramer, which is formed due to the rearrangement of two αβ dimers when the hemoglobin is unliganded or deoxygenated (compare Figs. 2B and 2D). The T-state structures of HbG-Makassar and HbA (PDB ID 2DN2)⁵² also revealed high conformational similarity with an RMSD of 0.352-Å for all of Cα atoms (Fig. 2D). Taken together, our structural and biochemical characterization suggests HbG-Makassar would function analogously to HbA.

Characterization of red blood cells expressing HbG. Following the biochemical confirmation of normal hemoglobin function of purified HbG-Makassar, we next wanted to assess the functional parameters of HbG in whole blood. Conventional hematology lab values showed that the blood indices of HbGG mice resembled those of HbAA mice, while HbGS indices more closely resembled the indices of HbSS than HbAS mice. Mice with HbGG had hemoglobin levels of 2.8 to 12.5 g/dL, compared to HbAA mice with hemoglobin levels of 5.5 to 12.6 g/dL and HbAS with hemoglobin levels of 10.9 to 15.7 g/dL. The Hb values of mice with HbGS were not significantly different from mice with HbSS. HbAS mice had higher Hb levels than HbGS mice (p < 0.001) (Fig. 3A). The white blood cell counts (WBC) of HbAA, HbGG, and HbAS mice were not significantly different; WBC of HbGS mice were comparable to the WBC of HbSS mice (p < 0.27), suggesting similar amounts of inflammation. HbGS mice had a significantly higher WBC count than HbAS mice. (p < 0.0006) (Fig. 3B).

HbAA and HbGG mice had similar absolute reticulocyte counts (ARC), suggesting that the RBC of HbGG mice had similar lifespans and rates of hemolysis as of HbAA mice. The ARC from HbGS and HbSS mice were not significantly different (Fig. 3C). Mean corpuscular volume (MCV) was significantly lower in RBC from HbGG mice compared to the RBC of HbAA mice (p < 0.006). The MCV of HbGS RBC was significantly lower than that of HbSS mice (p < 0.001) (Fig. 3D), suggesting that HbG contributes to red cell dehydration.

The deformability and point of sickling of RBCs from animals of each genotype was assessed using oxygen gradient ektacytometry. The elongation index minimum (EImin) measures deformability under hypoxia; RBCs from HbAA and HbAS mice had better deformability under low oxygen tension than HbGG (p = 0.002) and HbGS (p < 0.001) mice. However, HbGS RBC deformability under hypoxia was better than that of HbSS RBC (Fig. 4A). The elongation index maximum, (EImax), which measures the RBC deformability under normoxia, demonstrates that HbAA and HbAS RBC have better RBC deformability when oxygenated than HbGG (p < 0.001) and HbGS (p < 0.01) (Fig. 4B). HbAA, HbAS, and HbGG do not sickle and therefore do not have point of sickling (PoS) values. HbGS mice had very low point of sickling compared to HbSS, typically below a physiologic threshold of pO₂ > 15% in the bone marrow reference (Fig. 4). Visualized sickling under chemical hypoxia was similar to the PoS using oxygen gradient ektacytometry, with no sickling in HbGG RBCs, and minimal sickling in HbGS RBCs. Whole blood from animals with HbGG, HbGS, HbAS and HbSS show comparable HVR, indicating similar oxygen carrying capacity. HbAA had a significantly higher HVR than all other genotypes (Fig. 4D).

Given the low MCV and concern for RBC dehydration of RBCs containing HbG, we assessed dense red blood cell percentages using an ADVIA hematology analyzer.⁵³ Increased red blood cell density can contribute to HbS polymerization⁵⁴ and clinical complications.⁵⁵ Due to their short lifespan, low hemoglobin levels and high percent reticulocytes, sickle mouse RBCs are typically not dense.⁵⁶ However, HbG-containing RBCs exhibited 5 to 14.8% dense red blood cells (Fig. 4E).

RBCs from mice with the HbGG genotype had significantly more mitochondrial retention compared to the RBCs of HbAA mice; however, this difference was eliminated when corrected for the higher level of reticulocytes found in HbGG compared to HbAA. (Fig. 5A-B). In assessing the functionality of the erythropoiesis of Makassar mouse, we investigated erythroid maturation as a measure of ineffective erythropoiesis. Here, we saw the same distribution of erythroid maturation in the bone marrow of mice with HbAA and HbGG genotypes, suggesting that HbGG mice do not have ineffective erythropoiesis. HbGS erythroid maturation was delayed at the basophyllic stage compared to HbAS (Fig. 5C-D).

Organ pathology. We next assessed organ pathology in mice of all genotypes, at 24–28 weeks of age. Liver weight per total mass showed comparable size between HbAA and HbGG, with HbGS intermediate to that of HbAS and HbSS (Fig. 6A). Spleen weight per total mass was comparable between HbAA and HbGG mice; the total mass of HbGS spleens was significantly larger than HbAS (p = 0.01), and comparable to HbSS (Fig. 6B). Mice with HbGG had no significant difference in glomerular sclerosis compared to HbAA mice. There was no significant difference in glomerular sclerosis between HbGS and HbAS mice. The total sclerotic score of HbGS mice is intermediate between that of HbAS and HbSS mice (Fig. 6C-D).

Ex vivo HbG-Makassar gene editing in CD34s. To characterize the ex vivo base editing outcomes that install the Makassar hemoglobin variant in HbSS hCD34s, we used an adenine base editor (ABE) engineered to convert HbS to HbG (Fig. 7A). In both mobilized and non-mobilized homozygous HbSS CD34s we achieved > 58% Makassar editing at 48 hours post-EP with mRNA encoding our base editor and guide RNA targeting HbS. Makassar editing continued to increase to > 80% as cells differentiated (Fig. 7B). Ultra-high-performance liquid chromatography (UHPLC) was used to quantify HbG relative to HbS in erythroid cells. HbG-Makassar protein levels correlated closely with those of DNA editing rates; HbS was reduced to < 16% of total beta globins (Fig. 7C). To understand the distribution of editing outcomes that gave us > 80% bulk HbG-Makassar editing, we single cell cloned CD34s 2 days post-EP to obtain allelic editing on a clonal level of 183 clones. ~75% ± 7% of cells were bi-allelically Makassar edited, ~ 14.2% ± 6% were mono-allelically edited, 6.7% ± 1.7% were Makassar bi-allelically edited with one allele harboring Makassar and rare bystander edits,⁴⁸ and 2.5% + 0.98% were completely unedited (Fig. 7D). Similar allelic editing frequencies were also observed from single BFU-E colonies obtained through a colony forming unit assay that were sequenced (Supplemental Figure S2). This distribution of allelic editing is similar to those obtained using a different NRCH-PAM ABE and guide RNA pair to install the Hb-G Makassar variant.²²

To further characterize the impact of the HbGS heterozygous state derived from primary hCD34s, we isolated single erythroid colonies derived in vitro from HbSS CD34s that were base edited and assessed colonies that were confirmed by NGS at the genomic level to be heterozygous HbGS for expression of beta globins. The percent globin expression detected by UHPLC in these HbGS cells displayed a similar 60:40 ratio of HbG-Makassar globin to HbS globin seen in HbGS Townes mice (Fig. 4G), and to individuals with sickle trait (HbAS) (Fig. 7E). To functionally assess sickling in these erythroid precursors, we subjected cells that were identified via sequencing to be unedited, mono-allelically edited and bi-allelically edited for HbG and exposed them in vitro to hypoxic (2% O₂) conditions for > 48 hours and noted that indeed, mono-allelic edited HbSS cells, now HbGS, displayed reduced evidence of sickling in vitro (Fig. 7F, Supplemental Fig S3). Altogether, our ex vivo editing data suggests that installation of the HbG-Makassar variant can be achieved in CD34s with high efficiency and can lead to > 90% of red blood cells that no longer express HbS protein or achieve therapeutically relevant reduced levels of HbS.

Converting HbS to HbG-Makassar through adenine base editing is a promising therapeutic strategy.^7,15,22,48 This approach has the additional benefit of eliminating both sickle allele and the associated pathogenic protein, leading to minimal residual HbS of < 15%. We and others have shown exceptionally high editing efficiency, particularly in the bi-allelic conversion of HbS to HbG-Makassar using ABEs.^22,48

In this study, we demonstrated that human HbG-Makassar purified from the Townes mouse has functional and structural properties similar to human HbA. Purified HbG-Makassar is a non-sickling variant that does not polymerize like HbS, even under hypoxia conditions, and results are in alignment with previous studies.^24–26 This observation could be attributed to the reduced ability of HbG-Makassar to form polymer nucleation due to the substitution of valine to alanine present in the β chain of HbG-Makassar. Considering the functional similarities between HbG-Makassar and HbA, it was anticipated that HbGS mixtures would polymerize to the same extent as HbAS in vitro, and our functional characterization indeed demonstrated HbG-Makassar behaves like a wild-type hemoglobin in heterozygous conditions in vitro.

Sickle RBCs are known to be rigid and poorly deformable under normoxia; this worsens with hypoxia-induced polymerization of HbS.^64–66 We noted abnormal deformability in HbGG and HbGS RBC; nonetheless, sickling was absent in HbGG. Sickling in HbGS occurred only at levels of hypoxia that are unlikely to occur in living animals. Poor deformability in the absence of sickling has been observed previously in non-pathogenic hemoglobin variants even in the homozygous state, such as HbCC.^67,68 In this case, poor deformability is attributed to HbC activation of the Gardos channel, causing RBC dehydration. Individuals with HbC have high levels of dense red blood cells, similar to the mice with HbG. The rheological properties of HbGG RBCs resembled that of HbAA RBCs, except for metrics affected by dehydration, such as %DRBC, EImin and EImax. We therefore conclude that while HbG affects red cell hydration, it does not create a disease state in HbGG. HbGG erythroid maturation was not significantly different from HbAA; further evidence for normal erythropoiesis in HbGG mice is supported by spleen size, which was not significantly different from HbAA mice. In cases of ineffective erythropoiesis, the spleen becomes a site of red cell production, resulting in enlargement.⁶⁹ Our assessment of organ function continued with liver and kidney; again, HbSS mice exhibited typical pathology of liver enlargement and glomerular sclerosis; there was no significant difference between HbAA, HbAS, HbGS and HbGG mice.

HbGS hematologic indices and measures of red cell function were often intermediate between that of HbAS and HbSS. However, the HbGS state represents a less frequent (< 15%) editing outcome with an ex vivo base editing approach, with bi-allelic editing by far predominating (> 70%). A minor population of RBCs are likely to have a 60:40 ratio of HbG:HbS, and even these RBCs will exhibit significant functional improvement over the original HbSS RBC. Overall, we expect to significantly improve the sickle cell phenotype in > 90% of cells with a base editing approach.

By directly replacing, and thereby eliminating, in edited cells the pathogenic HbS from red blood cells with a normally functioning, naturally occurring and benign hemoglobin variant, adenine base editing strategies that install HbG-Makassar have the potential to provide life-long transformative therapy for individuals with SCD.

Acknowledgements The authors would like to thank Dr. Nico Tjandra from the National Heart, Lung, and Blood Institute at the NIH for providing the expression plasmids for recombinant hemoglobin. In addition, the authors thank the Drennan Lab at MIT for providing access to equipment needed to perform the anaerobic crystallographic studies. This research used resources of the National Synchrotron Light Source II, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under Contract No. DE-SC0012704. The Center for BioMolecular Structure (CBMS) is primarily supported by the National Institutes of Health, National Institute of General Medical Sciences (NIGMS) through a Center Core P30 Grant (P30GM133893), and by the DOE Office of Biological and Environmental Research (KP1605010).

Author contributions M.A.O designed, executed, analyzed, and supervised all biochemical experiments. P.F. designed, executed, and analyzed all structural studies. C.X. performed biochemical and structural studies. S.H.C., D.L and E.B. performed ex vivo primary human CD34 experiments V.W. performed all mass spectrometry analyses. S.J.L. supervised biochemical and structural work. Z.K. oversaw mouse breeding, molecular biology with B.H., M.A.P., and J.Z., pathology with Y.Z., B.H. and M.A.P., and rheology assays with J.D., E.N.E., J.Z., C.K.K., and B.H. assisting. A.P., B.H., and K.G. performed statistical analysis. V.S. designed the red cell function and mouse pathology experiments. M.A.O, Z.K., S.J.L., S.H.C., K.G. and V.S. wrote and edited the manuscript.

Competing interests All authors from Beam Therapeutics disclose a conflict of interest and are shareholders of Beam Therapeutics.

Materials and correspondence For materials and correspondence pertaining to the ex vivo editing of CD34 cells, biochemical and structural characterization of HbG-Makassar and generation and characterization of HbG-Makassar Townes mice model please contact [email protected], [email protected], and [email protected] respectively.

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Installation of HbG-Makassar by base editing restores hemoglobin function: a transformative therapy for sickle cell disease

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