Reactivation of γ-globin in adult β-YAC mice after ex vivo and in vivo hematopoietic stem cell genome editing

Abstract

Disorders involving β-globin gene mutations, primarily β-thalassemia and sickle cell disease, represent a major target for hematopoietic stem/progenitor cell (HSPC) gene therapy. This includes CRISPR/Cas9-mediated genome editing approaches in adult CD34 cells aimed toward the reactivation of fetal γ-globin expression in red blood cells. Because models involving erythroid differentiation of CD34 cells have limitations in assessing γ-globin reactivation, we focused on human β-globin locus-transgenic (β-YAC) mice. We used a helper-dependent human CD46-targeting adenovirus vector expressing CRISPR/Cas9 (HDAd-HBG-CRISPR) to disrupt a repressor binding region within the γ-globin promoter. We transduced HSPCs from β-YAC/human CD46-transgenic mice ex vivo and subsequently transplanted them into irradiated recipients. Furthermore, we used an in vivo HSPC transduction approach that involves HSPC mobilization and the intravenous injection of HDAd-HBG-CRISPR into β-YAC/CD46-transgenic mice. In both models, we demonstrated efficient target site disruption, resulting in a pronounced switch from human β- to γ-globin expression in red blood cells of adult mice that was maintained after secondary transplantation of HSPCs. In long-term follow-up studies, we did not detect hematological abnormalities, indicating that HBG promoter editing does not negatively affect hematopoiesis. This is the first study that shows successful in vivo HSPC genome editing by CRISPR/Cas9.

Adenoviral Vectors Meet Gene Editing: A Rising Partnership for the Genomic Engineering of Human Stem Cells and Their Progeny

“…At 10 weeks post-transplantation, there was a~5-fold reduction of HBB mRNA and a~30-fold increase in HBG mRNA levels in red blood cells when compared to controls. These results indicate that a switch in the balance of adult to fetal globin expression was achieved [113]. In another study, Li and co-workers using fiber-modified HC-AdVs encoding RGNs targeting BCL11A gene enhancer or BCL11A protein binding sequences obtained over 20% indel formation at these motifs in CD34 + cells [114].…”

“…Disruption of binding motifs for the HBG repressor protein BCL11A is a promising strategy to reactivate HBG expression and fetal γ-globin synthesis to complement the absence of functional adult β-globin in β-thalassemic and sickle cell disease (SCD) patients. In this regard, transduction of mobilized peripheral blood CD34 + cells from healthy donors with fiber-modified HC-AdVs encoding HBG-specific RGNs led to around 20% of target motif disruption in these cells [113]. Moreover, no indels were observed in the top 10 candidate off-target sites, as assessed by mismatch-sensing nuclease assays and, importantly, the erythroid differentiation capability of the gene-edited hematopoietic progenitors was maintained [113].…”

“…In this regard, transduction of mobilized peripheral blood CD34 + cells from healthy donors with fiber-modified HC-AdVs encoding HBG-specific RGNs led to around 20% of target motif disruption in these cells [113]. Moreover, no indels were observed in the top 10 candidate off-target sites, as assessed by mismatch-sensing nuclease assays and, importantly, the erythroid differentiation capability of the gene-edited hematopoietic progenitors was maintained [113]. Cell transplantation assays in lethally irradiated immunodeficient mice revealed indel frequencies ranging from 19% to 25% at HBG alleles in human CD45 + cells isolated from bone marrow at 10 weeks post-transplantation.…”

“…Cell transplantation assays in lethally irradiated immunodeficient mice revealed indel frequencies ranging from 19% to 25% at HBG alleles in human CD45 + cells isolated from bone marrow at 10 weeks post-transplantation. Upon in vitro differentiation of these bone marrow-derived CD45 + cells, the frequencies of γ-globin + cells were~50% and~27% in the transduced and non-transduced groups, respectively, as determined by flow cytometry [113]. In addition, β-YAC/CD46 mice were also used in this study to overcome the known block on human erythrocytic lineage differentiation in NSG mice.…”

Therapeutic gene editing in haematological disorders with CRISPR/Cas9

“…In contrast, in vivo gene correction of HSCs would constitute a much more affordable approach that most hospitals could implement with off‐the‐shelf reagents available from commercial vendors, which could be injected directly into patients and would enable broad applicability. In vivo HSC gene therapy with retroviral vectors has been performed in pre‐clinical murine models with some success (Wang et al , , ; Richter et al , ), and a single study has also shown in vivo CRISPR/Cas9 gene editing in a mouse model of β‐thalassaemia (Li et al , ). This study delivered the Cas9 and sgRNA by means of an adenoviral vector that was injected intravenously into mice after having mobilized HSPCs from the bone marrow into the blood.…”