Evaluation of the Impact of a Naturally Occurring Beta Hemoglobin Variant, Hb G-Makassar, on Mature Red Blood Cell Function and Pathology in a Sickle Cell Disease Mouse Model

Background:

Gene based therapy may provide a cure for individuals with sickle cell disease (SCD); base editing is a novel strategy with the ability to directly edit the mutation responsible for disease. The sickle hemoglobin (HbS) point mutation cannot be converted to wild-type HBB by an adenine base editor, however it can be converted to a naturally occurring hemoglobin variant, HbG-Makassar (HbG). HbG is a non-pathogenic, non-sickling hemoglobin variant. In vitro crystal structure analysis of HbG shows that it is highly similar to that of wildtype hemoglobin (HbA). However, few HbGG individuals (with no overt red cell disease) and no HbSG individuals have been identified; thus, the red blood cells (RBCs) of HbGG and HbSG individuals have not been evaluated functionally. The Makassar base editing strategy produces greater than 80% homozygous Hb-G product and has been evaluated in vitro, however erythroid culture of edited hematopoietic stem cells do not produce fully mature, enucleated red cells. Therefore, mouse models of HbGG and HbSG could be used to evaluate the function of mature, enucleated HbGG or HbSG RBCs.

Methods:

A humanized beta globin locus mouse (Townes) with the Makassar allele was created at by knocking-in the Makassar point mutation to generated WT/Makassar heterozygotes (HbAG) which were backcrossed to generated HbGG homozygotes. HbSG genotypes were subsequently generated by crossing to HbSS Townes mice. We analyzed the red cell function of HbAA, HbAS, HbSS, HbGG, and HbSG mice.

Whole blood was collected through terminal enucleation. Complete blood counts were measured on a hematology analyzer (Heska and, ADVIA). Red cell deformability (elongation index, EI) was measured using oxygen gradient ektacytometry (LoRRCA) under normoxic (EImax) and hypoxic (EImin) conditions. Viscosity measurements were obtained on a Beckman cone and plate viscometer. Percent sickle cells were quantified through chemically induced sickling with sodium metabisulfite and counted by an individual blinded to genotype using light microscopy.

Kidney, liver, and spleen were fixed, preserved in paraffin, stained using hematoxylin and eosin stains and imaged using a Keyence microscope.

Flow cytometry was performed on bone marrow and whole blood to determine the erythroid maturation and mitochondrial retention of RBCs.

Analysis was performed with Stata 18.0 (College Station) across genotypes using Dunn Pairwise test with statistically significant values being selected at adjusted p < 0.05.

Results

Homozygous HbG mice (HbGG) had comparable white blood cell count (WBC) and absolute reticulocyte count (ARC) to that of mice with homozygous HbA (HbAA). HbGG mice had lower hemoglobin and mean corpuscular volume (MCV) in comparison to mice with HbAA. HbGG mice had a higher percentage of dense RBCs than HbAA mice. HbSG mice had comparable values to HbAS mice for MCV but had significantly lower hemoglobin and ARC. HbSG mice had significantly higher values for WBC, and % dense RBCs against HbAS mice. HbSS and HbSG mice had comparable values for hemoglobin, and ARC. Hematocrit to viscosity ratio (HVR) shows comparable oxygen transport effectiveness of HbGG, HbSG, HbAS and HbSS RBC. Further red cell function testing is shown in Figure 1 and Figure 2.

Mitochondrial retention was highest in HbSS mice, followed by HbSG, while HbAS, HbAA, and HbGG had similar values. Microscopy analysis of renal sections of HbGG and HbSG mice sacrificed at 24-28 weeks did not show the characteristic morphologic changes seen in HbSS mice at 24-28 weeks.

Conclusions-

HbGG appears functionally similar to HbAA, with minimal sickling under hypoxia, no anemia, normal spleen size, and no organ damage at 24 weeks of age. HbGG does exhibit signs of dehydration, as evidenced by elevated %DRBC and reduced RBC deformability. RBC function, sickling under hypoxia, Hb, WBC, ARC, and mitochondrial retention measures places HbSG as intermediate in severity between HbAS and HbSS. However, organ pathology, spleen and liver weights for HbSG were more comparable to HbAS than HbSS. Base editing strategies to install Makassar therefore can potentially lead to >90% of RBCs that have therapeutic benefit due to rheological properties comparable to HbAA and can be seen as a promising treatment strategy for patients with sickle cell disease.