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

Base editors are capable of installing precise genomic alterations without creating double-strand DNA breaks. In this study, we targeted critical motifs regulating gamma-globin reactivation with base editors delivered via HDAd5/35(++) 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 gamma-globin reactivation in human erythroid progenitor cells. We observed similar to 23% gamma-globin protein production over beta-globin, when using an ABE vector (HDAd-ABE-sgHBG-2) specific to the -113A>G HPFH mutation. In a beta-YAC mouse model, in vivo hematopoietic progenitor/stem cell (HSPC) transduction with HDAd-ABE-sgHBG-2 followed by in vivo selection resulted in >40% gamma-globin(+) erythrocytes in the peripheral blood. This result corresponded to 21% gamma-globin production over human gamma-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 gamma-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 CD34(+) cells. Our observations suggest that HDAd-vectorized base editors represent a promising strategy for precise in vivo genome engineering for the treatment of beta-hemoglobinopathies.

Automated summary

Base editors delivered via HDAd vectors efficiently induced gamma-globin reactivation in human erythroid progenitor cells, offering a promising strategy for the treatment of beta-hemoglobinopathies. Important advantages of base editing over CRISPR/Cas9 include lower rates of off-target deletions and absence of detectable toxicity in human CD34(+) cells.