In Vivo Efficacy of Bcl11a Erythroid-Enhancer Deletion in Humanized Mouse Models of Anemia

Autologous cell therapy holds great promise for the treatment of beta thalassemia major and hemoglobinopathies like sickle cell anemia. Gene editing of a patient's own stem cells to reactivate the silenced gamma globin gene is one approach under active development. Prior to directly testing these new therapies in patients, we can answer some basic questions about their in vivo efficiency and efficacy in humanized mouse models of anemia. These models have their endogenous adult alpha and beta globin genes replaced with human alpha, gamma, and beta globin genes. These mice synthesize high level of human fetal hemoglobin during fetal life and complete their fetal-to-adult hemoglobin switch after birth. Experimental strategies designed to reactivate the silenced fetal gamma globin genes in adult erythroid cells are easily tested in vivo in these humanized hemoglobin switching mouse models. The silenced human fetal gamma globin genes can be activated by mutating the erythroid-specific enhancer of Bcl11a by gene editing. CRISPR sgRNAs, designed to target the +62 kb DNase I hypersensitive site in the second intron of Bcl11a, were microinjected along with Cas9 mRNA, into fertilized mouse embryos collected from humanized hemoglobin (Hb A) mice. The indel mutations that were generated in the founder animals were characterized and bred to homozygosity. The data demonstrates that the sgRNAs tested were successful in creating multiple unique mutations at the erythroid enhancer target sites. These mutations were transmitted through the germline allowing the effect of individual edited alleles to be analyzed. The majority of the mutations showed marginal increases in the number of F-cells over control animals. Significantly, despite having homozygous mutation of the erythroid-enhancer in all cells, fetal hemoglobin expression remains heterocellular. Importantly, the therapeutic efficacy of reactivating fetal hemoglobin with specific Bcl11a erythroid-enhancer mutations for the treatment of beta thalassemia major and sickle cell anemia was directly measured in vivo in these humanized models of disease. The reactivation of gamma globin in these humanized mouse models provides us with an opportunity to further interrogate the Bcl11a enhancer element, identify additional factors involved in hemoglobin switching and elucidate the mechanism driving pancellular vs heterocellular fetal hemoglobin expression.