Base editor-mediated correction of a BTK Mutation in XLA Patient Hematopoietic Stem Cells restores Human B cell development

Abstract

IntroductionX-linked agammaglobulinemia (XLA) due to Bruton's tyrosine kinase (BTK) mutations causes lack of circulating mature B lymphocytes (<1%), lack of serum immunoglobulins (IgG/A/M) and increased susceptibility to infections. Correction of BTK mutation in hematopoietic stem/progenitor cells (HSPC) should restore B cell development and humoral immunity. Here, we develop a novel approach that leverages adenine base editors (ABEs) to correct HSPCs from a XLA patient. Materials and methodsPeripheral blood HSPCs were collected from an XLA BTK:c.46C>T (p.Gln16Ter) patient and healthy donors (HD) after G-CSF/plerixafor mobilization. HSPCs were cultured ex vivo (StemSpan™ SFEM II with SCF, FLT3L, TPO) for 2 days before base editing (BE) with mRNA-encoded ABEs (CELLSCRIPT LLC) and gRNAs (Synthego) including ABE8e-SpRY with a gRNA that positions the target adenine in nucleotide 6 of the target site (A6), and ABE8e-SpG or ABE8e-SpCas9-NG with gRNA A7. BE-HSPCs were transplanted two days later into NSG mice conditioned with busulfan (20 mg/kg). Analyses were performed at 12, 16 weeks and animals sacrificed at 20 weeks to assess engraftment and B cell immune reconstitution. ResultsHSPCs BE with ABE8e-SpRY/gRNA A6(SpRY/A6) achieved highest correction at 86.6% BTKc.46C >T and restored BTK protein expression in ex vivo differentiated (14 days, DMEM+G-CSF) CD33+ myeloid cells (Fig.1A) which restored peripheral blood (PB) CD19+CD20+ B cells to HD level (Fig.1C). Phenotype analysis of PB confirmed predominantly mature naïve B cells in BE comparable with HD instead and mostly transitional B cells in mice with naïve-XLA cells (Fig.1F) with CD179a+ that corresponded to Pre-B cells (Fig.1G). Analysis of bone marrow confirmed restored immature (BE: 16.3 vs naïve:1.0, p = 0.001) and mature B cells (BE: 1.2 vs naïve:0.11 p = 0.002) in mice with BE-HSPCs to HD levels while naïve mice comprised of mostly Pre-B-II cells (mean BE: 64.7% vs naïve:81.3%, p = 0.0006). ConclusionsOur studies demonstrate a novel base editing strategy for a XLA missense mutation that overcame the bone marrow arrest of B cells to restore mature B cells in humanized NSG peripheral blood. These findings demonstrate preclinical efficacy of an ABE-based treatment for BTK. The highly efficient-specific correction with base editing holds great promise for gene therapy for XLA disease. [Display omitted]