Single-Cell RNA Sequencing of Sickle Cell Reticulocytes to Identify Beta-Globin Genotypes and Associated Gene Expression Differences

Abstract

Background: Sickle cell disease (SCD) is a genetic condition typically caused by a single point mutation in both copies of the beta-globin (HBB) gene, resulting in sickle (S) hemoglobin (HbS) production instead of adult (A) hemoglobin (HbA). GPH101 (nulabeglogene autogedtemcel) is an investigational gene-edited autologous hematopoietic stem cell-based therapy in clinical development for SCD that is designed to directly correct the underlying mutation, thereby decreasing HbS production and restoring HbA expression. Previous allogeneic hematopoietic stem cell transplant data, mostly in the context of using HLA-matched related donors, has demonstrated a competitive advantage for homozygous (AA) or heterozygous (AS) hemoglobin due to ineffective erythropoiesis of SS erythroid progenitors. At early timepoints following infusion of nulabeglogene autogedtemcel, red blood cells (RBCs) expressing corrected HbA cannot be distinguished from HbA in transfused blood. Because RBCs are enucleated, tracking of gene editing at the genomic level is not possible. However, reticulocytes are immature RBCs that still contain some RNA and could be used to evaluate allelic correction. We sought to develop a single-cell RNA sequencing (scRNAseq) method to enable enumeration of potential gene-editing outcomes in peripheral reticulocytes. Methods: Reticulocytes were sorted from peripheral blood from healthy donors (AA), donors with sickle cell trait (AS), and donors with sickle cell disease (SS) by fluorescence-activated cell sorting (FACS) based on a Live/CD235a+/CD45-/CD71+/TO+ phenotype and mixed at given ratios before application to the 10x genomics workflow for scRNAseq. Individual mRNA molecules were tagged with a unique molecular identifier (UMI) to enable transcript quantification. A custom bioinformatics analysis pipeline was developed to identify HBB variants within the scRNAseq data to identify individual reticulocytes expressing either normal HBB, sickle HBB, or both. Results: Genotypes of individual cells could be clearly distinguished from scRNAseq data using a custom bioinformatics pipeline. Additionally, differential mRNA expression profiling between AA and SS samples was performed and previously known results from bulk RNAseq studies were produced, including upregulation of HbA2 subunit delta and alpha hemoglobin stabilizing protein. An examination of cell populations underlying these expression differences will be presented. Conclusion: Our data demonstrate the utility of scRNAseq for evaluating differential HBB editing outcomes in erythroid progenitors from patients treated with nulabeglogene autogedtemcel to support clinical development. Further evaluation of the clinical utility of the scRNAseq pipeline is planned.