In vivo HSPC gene therapy with base editors allows for efficient reactivation of fetal g-globin in b-YAC mice

Abstract

Base editors are capable of installing precise genomic alterations without creating doublestrand DNA breaks. In this study, we targeted critical motifs regulating g-globin reactivation with base editors delivered via HDAd5/3511 vectors. Through optimized design, we successfully produced a panel of cytidine and adenine base editor (ABE) vectors targeting the erythroid BCL11A enhancer or recreating naturally occurring hereditary persistence of fetal hemoglobin (HPFH) mutations in the HBG1/2 promoter. All 5 tested vectors efficiently installed target base conversion and led to g-globin reactivation in human erythroid progenitor cells. We observed 23% g-globin protein production over b-globin, when using an ABE vector (HDAd-ABE-sgHBG-2) specific to the-113A.G HPFH mutation. In a b-YAC mouse model, in vivo hematopoietic progenitor/stem cell (HSPC) transduction with HDAd-ABE-sgHBG-2 followed by in vivo selection resulted in .40% g-globin1 erythrocytes in the peripheral blood. This result corresponded to 21% g-globin production over human b-globin. The average-113A.G conversion in total bone marrow cells was 20%. No alterations in hematological parameters, erythropoiesis, and bone marrow cellular composition were observed after treatment. No detectable editing was found at top-scoring, off-target genomic sites. Bone marrow lineage-negative cells from primary mice were capable of reconstituting secondary transplant-recipient mice with stable g-globin expression. Importantly, the advantage of base editing over CRISPR/Cas9 was reflected by the markedly lower rates of intergenic HBG1/2 deletion and the absence of detectable toxicity in human CD341 cells. Our observations suggest that HDAd-vectorized base editors represent a promising strategy for precise in vivo genome engineering for the treatment of b-hemoglobinopathies.