Potent and uniform fetal hemoglobin induction via base editing.

Inducing fetal hemoglobin (HbF) in red blood cells can alleviate ß-thalassemia and sickle cell disease. We compared five strategies in CD34+ hematopoietic stem and progenitor cells, using either Cas9 nuclease or adenine base editors. The most potent modification was adenine base editor generation of γ-globin -175A>G. Homozygous -175A>G edited erythroid colonies expressed 81 ± 7% HbF versus 17 ± 11% in unedited controls, whereas HbF levels were lower and more variable for two Cas9 strategies targeting a BCL11A binding motif in the γ-globin promoter or a BCL11A erythroid enhancer. The -175A>G base edit also induced HbF more potently than a Cas9 approach in red blood cells generated after transplantation of CD34+ hematopoietic stem and progenitor cells into mice. Our data suggest a strategy for potent, uniform induction of HbF and provide insights into γ-globin gene regulation. More generally, we demonstrate that diverse indels generated by Cas9 can cause unexpected phenotypic variation that can be circumvented by base editing.

Ex vivo prime editing of patient haematopoietic stem cells rescues sickle-cell disease phenotypes after engraftment in mice.

Sickle-cell disease (SCD) is caused by an A·T-to-T·A transversion mutation in the ß-globin gene (HBB). Here we show that prime editing can correct the SCD allele (HBBS) to wild type (HBBA) at frequencies of 15%-41% in haematopoietic stem and progenitor cells (HSPCs) from patients with SCD. Seventeen weeks after transplantation into immunodeficient mice, prime-edited SCD HSPCs maintained HBBA levels and displayed engraftment frequencies, haematopoietic differentiation and lineage maturation similar to those of unedited HSPCs from healthy donors. An average of 42% of human erythroblasts and reticulocytes isolated 17 weeks after transplantation of prime-edited HSPCs from four SCD patient donors expressed HBBA, exceeding the levels predicted for therapeutic benefit. HSPC-derived erythrocytes carried less sickle haemoglobin, contained HBBA-derived adult haemoglobin at 28%-43% of normal levels and resisted hypoxia-induced sickling. Minimal off-target editing was detected at over 100 sites nominated experimentally via unbiased genome-wide analysis. Our findings support the feasibility of a one-time prime editing SCD treatment that corrects HBBS to HBBA, does not require any viral or non-viral DNA template and minimizes undesired consequences of DNA double-strand breaks.

Multiplex Base Editing to Protect from CD33-Directed Therapy: Implications for Immune and Gene Therapy.

On-target toxicity to normal cells is a major safety concern with targeted immune and gene therapies. Here, we developed a base editing (BE) approach exploiting a naturally occurring CD33 single nucleotide polymorphism leading to removal of full-length CD33 surface expression on edited cells. CD33 editing in human and nonhuman primate (NHP) hematopoietic stem and progenitor cells (HSPCs) protects from CD33-targeted therapeutics without affecting normal hematopoiesis in vivo , thus demonstrating potential for novel immunotherapies with reduced off-leukemia toxicity. For broader applications to gene therapies, we demonstrated highly efficient (>70%) multiplexed adenine base editing of the CD33 and gamma globin genes, resulting in long-term persistence of dual gene-edited cells with HbF reactivation in NHPs. In vitro , dual gene-edited cells could be enriched via treatment with the CD33 antibody-drug conjugate, gemtuzumab ozogamicin (GO). Together, our results highlight the potential of adenine base editors for improved immune and gene therapies.

An RPS19-edited model for Diamond-Blackfan anemia reveals TP53-dependent impairment of hematopoietic stem cell activity.

Diamond-Blackfan anemia (DBA) is a genetic blood disease caused by heterozygous loss-of-function mutations in ribosomal protein (RP) genes, most commonly RPS19. The signature feature of DBA is hypoplastic anemia occurring in infants, although some older patients develop multilineage cytopenias with bone marrow hypocellularity. The mechanism of anemia in DBA is not fully understood and even less is known about the pancytopenia that occurs later in life, in part because patient hematopoietic stem and progenitor cells (HSPCs) are difficult to obtain, and the current experimental models are suboptimal. We modeled DBA by editing healthy human donor CD34+ HSPCs with CRISPR/Cas9 to create RPS19 haploinsufficiency. In vitro differentiation revealed normal myelopoiesis and impaired erythropoiesis, as observed in DBA. After transplantation into immunodeficient mice, bone marrow repopulation by RPS19+/- HSPCs was profoundly reduced, indicating hematopoietic stem cell (HSC) impairment. The erythroid and HSC defects resulting from RPS19 haploinsufficiency were partially corrected by transduction with an RPS19-expressing lentiviral vector or by Cas9 disruption of TP53. Our results define a tractable, biologically relevant experimental model of DBA based on genome editing of primary human HSPCs and they identify an associated HSC defect that emulates the pan-hematopoietic defect of DBA.

Activation of γ-globin expression by hypoxia-inducible factor 1α.

Around birth, globin expression in human red blood cells (RBCs) shifts from γ-globin to ß-globin, which results in fetal haemoglobin (HbF, α2γ2) being gradually replaced by adult haemoglobin (HbA, α2ß2)1. This process has motivated the development of innovative approaches to treat sickle cell disease and ß-thalassaemia by increasing HbF levels in postnatal RBCs2. Here we provide therapeutically relevant insights into globin gene switching obtained through a CRISPR-Cas9 screen for ubiquitin-proteasome components that regulate HbF expression. In RBC precursors, depletion of the von Hippel-Lindau (VHL) E3 ubiquitin ligase stabilized its ubiquitination target, hypoxia-inducible factor 1α (HIF1α)3,4, to induce γ-globin gene transcription. Mechanistically, HIF1α-HIF1ß heterodimers bound cognate DNA elements in BGLT3, a long noncoding RNA gene located 2.7 kb downstream of the tandem γ-globin genes HBG1 and HBG2. This was followed by the recruitment of transcriptional activators, chromatin opening and increased long-range interactions between the γ-globin genes and their upstream enhancer. Similar induction of HbF occurred with hypoxia or with inhibition of prolyl hydroxylase domain enzymes that target HIF1α for ubiquitination by the VHL E3 ubiquitin ligase. Our findings link globin gene regulation with canonical hypoxia adaptation, provide a mechanism for HbF induction during stress erythropoiesis and suggest a new therapeutic approach for ß-haemoglobinopathies.

Dual function NFI factors control fetal hemoglobin silencing in adult erythroid cells.

The mechanisms by which the fetal-type ß-globin-like genes HBG1 and HBG2 are silenced in adult erythroid precursor cells remain a fundamental question in human biology and have therapeutic relevance to sickle cell disease and ß-thalassemia. Here, we identify via a CRISPR-Cas9 genetic screen two members of the NFI transcription factor family-NFIA and NFIX-as HBG1/2 repressors. NFIA and NFIX are expressed at elevated levels in adult erythroid cells compared with fetal cells, and function cooperatively to repress HBG1/2 in cultured cells and in human-to-mouse xenotransplants. Genomic profiling, genome editing and DNA binding assays demonstrate that the potent concerted activity of NFIA and NFIX is explained in part by their ability to stimulate the expression of BCL11A, a known silencer of the HBG1/2 genes, and in part by directly repressing the HBG1/2 genes. Thus, NFI factors emerge as versatile regulators of the fetal-to-adult switch in ß-globin production.

Base editing of haematopoietic stem cells rescues sickle cell disease in mice.

Sickle cell disease (SCD) is caused by a mutation in the ß-globin gene HBB1. We used a custom adenine base editor (ABE8e-NRCH)2,3 to convert the SCD allele (HBBS) into Makassar ß-globin (HBBG), a non-pathogenic variant4,5. Ex vivo delivery of mRNA encoding the base editor with a targeting guide RNA into haematopoietic stem and progenitor cells (HSPCs) from patients with SCD resulted in 80% conversion of HBBS to HBBG. Sixteen weeks after transplantation of edited human HSPCs into immunodeficient mice, the frequency of HBBG was 68% and hypoxia-induced sickling of bone marrow reticulocytes had decreased fivefold, indicating durable gene editing. To assess the physiological effects of HBBS base editing, we delivered ABE8e-NRCH and guide RNA into HSPCs from a humanized SCD mouse6 and then transplanted these cells into irradiated mice. After sixteen weeks, Makassar ß-globin represented 79% of ß-globin protein in blood, and hypoxia-induced sickling was reduced threefold. Mice that received base-edited HSPCs showed near-normal haematological parameters and reduced splenic pathology compared to mice that received unedited cells. Secondary transplantation of edited bone marrow confirmed that the gene editing was durable in long-term haematopoietic stem cells and showed that HBBS-to-HBBG editing of 20% or more is sufficient for phenotypic rescue. Base editing of human HSPCs avoided the p53 activation and larger deletions that have been observed following Cas9 nuclease treatment. These findings point towards a one-time autologous treatment for SCD that eliminates pathogenic HBBS, generates benign HBBG, and minimizes the undesired consequences of double-strand DNA breaks.

ZNF410 Uniquely Activates the NuRD Component CHD4 to Silence Fetal Hemoglobin Expression.

Metazoan transcription factors typically regulate large numbers of genes. Here we identify via a CRISPR-Cas9 genetic screen ZNF410, a pentadactyl DNA-binding protein that in human erythroid cells directly activates only a single gene, the NuRD component CHD4. Specificity is conveyed by two highly evolutionarily conserved clusters of ZNF410 binding sites near the CHD4 gene with no counterparts elsewhere in the genome. Loss of ZNF410 in adult-type human erythroid cell culture systems and xenotransplantation settings diminishes CHD4 levels and derepresses the fetal hemoglobin genes. While previously known to be silenced by CHD4, the fetal globin genes are exposed here as among the most sensitive to reduced CHD4 levels.. In vitro DNA binding assays and crystallographic studies reveal the ZNF410-DNA binding mode. ZNF410 is a remarkably selective transcriptional activator in erythroid cells, and its perturbation might offer new opportunities for treatment of hemoglobinopathies.

Genome editing of HBG1 and HBG2 to induce fetal hemoglobin.

Induction of fetal hemoglobin (HbF) via clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of DNA regulatory elements that repress γ-globin gene (HBG1 and HBG2) expression is a promising therapeutic strategy for sickle cell disease (SCD) and ß-thalassemia, although the optimal technical approaches and limiting toxicities are not yet fully defined. We disrupted an HBG1/HBG2 gene promoter motif that is bound by the transcriptional repressor BCL11A. Electroporation of Cas9 single guide RNA ribonucleoprotein complex into normal and SCD donor CD34+ hematopoietic stem and progenitor cells resulted in high frequencies of on-target mutations and the induction of HbF to potentially therapeutic levels in erythroid progeny generated in vitro and in vivo after transplantation of hematopoietic stem and progenitor cells into nonobese diabetic/severe combined immunodeficiency/Il2rγ-/-/KitW41/W41 immunodeficient mice. On-target editing did not impair CD34+ cell regeneration or differentiation into erythroid, T, B, or myeloid cell lineages at 16 to 17 weeks after xenotransplantation. No off-target mutations were detected by targeted sequencing of candidate sites identified by circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), an in vitro genome-scale method for detecting Cas9 activity. Engineered Cas9 containing 3 nuclear localization sequences edited human hematopoietic stem and progenitor cells more efficiently and consistently than conventional Cas9 with 2 nuclear localization sequences. Our studies provide novel and essential preclinical evidence supporting the safety, feasibility, and efficacy of a mechanism-based approach to induce HbF for treating hemoglobinopathies.

Identification of rare and novel deletions that cause (뫧)0-thalassaemia and hereditary persistence of foetal haemoglobin in Indian population.

OBJECTIVES: Hereditary persistence of foetal haemoglobin (HPFH) and (δß)(0) -thalassaemia are conditions caused by large deletions that involve δ- and ß-globin genes in the ß-globin cluster, and they are characterized by increased haemoglobin (HbF) levels in adults. Significant phenotypic diversity is observed between the different mutations that cause these conditions. Molecular characterization of these deletions is important for accurate molecular diagnosis, and they will also provide the information on the cis-acting genetic regulatory elements present in the ß-globin cluster. METHODS: We performed gap-PCR, multiplex ligation-dependent probe amplification (MLPA), quantitative fluorescent multiplex PCR (QF-MPCR) and DNA sequencing to detect and characterize the deletions in the ß-globin cluster. RESULTS: We characterized six different deletions resulting in (δß)(0) -thalassaemia or HPFH in 51 unrelated families. CONCLUSION: With the help of multiple genetic tools, we performed comprehensive genetic analysis of HPFH and (δß)(0) -thalassaemia in Indian population and could define the molecular basis of these conditions in this population. We also identified two novel HPFH mutations, 49.98 kb (HPFH-9) and 86.7 kb (HPFH-10) deletions, in this population.