Despite nearly complete understanding of the genetics of the β-hemoglobinopathies for several decades, definitive treatment options have lagged behind. Recent developments in technologies for facile manipulation of the genome (zinc finger nucleases, transcription activator-like effector nucleases, or clustered regularly interspaced short palindromic repeats–based nucleases) raise prospects for their clinical application. The use of genome-editing technologies in autologous CD34+ hematopoietic stem and progenitor cells represents a promising therapeutic avenue for the β-globin disorders. Genetic correction strategies relying on the homology-directed repair pathway may repair genetic defects, whereas genetic disruption strategies relying on the nonhomologous end joining pathway may induce compensatory fetal hemoglobin expression. Harnessing the power of genome editing may usher in a second-generation form of gene therapy for the β-globin disorders.

The β-hemoglobinopathies, namely sickle cell disease (SCD) and β-thalassemia, result from genetic mutations in the β-globin gene and are among the most common monogenic diseases in the world.1  SCD results from a nonsynonymous A to T mutation in codon 6 of the β-globin gene leading to a Glu-Val replacement,2,3  whereas β-thalassemias are caused by diverse point mutations or deletions.4-9  Treatment options are largely supportive. Transfusion and iron chelation are mainstays in the thalassemias whereas pain management, hydration, and hydroxyurea are used in SCD.10-16 

The hemoglobin tetramer is composed of 2 α-like globin chains encoded by any of the 3 genes in the α-globin cluster on chromosome 16 and 2 β-like globin chains encoded from any of the 5 genes in the β-globin locus on chromosome 11. The expression of the 3 genes at the α-globin locus (ζ, α1, α2) and the 5 genes at the β-globin locus (ε, Gγ, Aγ, δ, β) are developmentally regulated. It has been appreciated for many years that levels of fetal hemoglobin (HbF; α2γ2), subject to developmental silencing in the months after birth, is a modifier of disease severity in patients with β-globin disorders.16-23  This protective effect of HbF has motivated the therapeutic strategy to reinduce its expression in adult life. Hydroxyurea, a cytotoxic agent that inhibits ribonucleotide reductase, induces HbF modestly through an unknown mechanism. However, it has dose-limiting myelosuppressive effects and some patients are nonresponders to therapy.10-13  Although bone marrow transplant (BMT) represents the sole established curative option for patients, its use is limited by donor availability and graft-versus-host disease (GVHD). A clinical trial has demonstrated successful gene addition of an antisickling form of β-globin to a transfusion-dependent βEβ0 thalassemia patient who gained transfusion independence as a result of gene transfer.24  Several additional somatic gene therapy trials for β-thalassemias and SCD are ongoing.25  Despite a deep understanding of molecular defects and gene control mechanisms, treatment options for the majority of patients remain limited.3 

The emergence of designer nucleases for eukaryotic genome editing has ushered in an era of unprecedented control over the genome. The development of zinc finger nucleases (ZFNs),26-35  transcription activator-like (TAL) effector nucleases (TALENs),36-40  and meganucleases41-44  established genome editing as a valuable laboratory technique. The emergence of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) nuclease system,45-49  which utilizes a single guide RNA (sgRNA) to direct the Cas9 nuclease for site-specific cleavage, has engendered tremendous excitement about potential clinical applications. The breakneck speed at which new variations on the general theme are developed is truly remarkable. Other Cas9-like systems include the CRISPR/Cpf1 nuclease platform,50  dimeric RNA-guided FokI nucleases,51,52  and use of Cas9s derived from a variety of prokaryotic species.53,54  It is unlikely that the discovery of novel CRISPR-based systems and Cas9-like nucleases capable of eukaryotic genome editing will end soon.55  The relative benefits of the newly developed CRISPR-based systems, ZFNs, and TALENs are still subject to debate. Although CRISPR-based systems are often cited as the most efficient,56  ZFNs are the only editing technology that has been brought thus far to a clinical trial. The CCR5 gene has been targeted by ZFNs in autologous CD4+ T cells from patients with HIV. The gene-modified cells were subsequently reinfused, which led to a decrease in the blood level of HIV in most patients.57  Notably, this study demonstrated that reinfusion of autologous genome-edited primary human cells could be achieved, well tolerated, and possibly lead to clinical benefit.

Genome-editing–based therapies rely on gene correction or disruption. Double-strand break (DSB) induction by an engineered nuclease is repaired by the endogenous repair pathways of homology-directed repair (HDR) or nonhomologous end joining (NHEJ).58  Genetic correction strategies exploit the HDR pathway to insert custom sequences into the genome through codelivery of an extrachromosomal repair template in conjunction with an engineered nuclease. The creation of a DSB improves HDR frequency.59  As such, wild-type (or customized) sequences can be provided as an extrachromosomal donor for repair following site-specific cleavage by the nuclease. In contrast, genetic disruption strategies rely on the NHEJ pathway following nuclease-induced DSB to produce local insertions/deletions (indels).47,48,58  Introduction of 2 engineered nucleases can result in targeted deletion or inversion, duplication, local indels at nuclease cleavage sites, or translocations/chromosomal rearrangements.60-73  Here, we review genome-editing approaches for genetic correction and disruption strategies for the β-hemoglobinopathies as summarized in Figure 1 and Table 1.

Figure 1

Network of potential targets for genome-editing–based therapy of the β-globin disorders. Therapeutic genome-editing strategies rely on genetic correction through the HDR pathway or genetic disruption through the NHEJ pathway. Genetic correction/repair strategies involve direct modification of the β-globin gene cluster through (1) correction of the sickle mutation in the β-globin gene or (2) insertion of the HPFH-associated single-nucleotide polymorphisms (SNPs) into the Gγ or Aγ promoters. Genetic disruption strategies involve targeted disruption of (1) BCL11A coding sequence, (2) the minimal critical sequences in the +58 DHS within the erythroid-specific BCL11A enhancer, (3) the HbF-associated sequences within the Aγ-δ intergenic region, or (4) other genes with a known role in γ-globin regulation such as MYB, KLF1, LRF/ZBTB7A, or EHMT1/EHMT2. HbA, adult hemoglobin.

Figure 1

Network of potential targets for genome-editing–based therapy of the β-globin disorders. Therapeutic genome-editing strategies rely on genetic correction through the HDR pathway or genetic disruption through the NHEJ pathway. Genetic correction/repair strategies involve direct modification of the β-globin gene cluster through (1) correction of the sickle mutation in the β-globin gene or (2) insertion of the HPFH-associated single-nucleotide polymorphisms (SNPs) into the Gγ or Aγ promoters. Genetic disruption strategies involve targeted disruption of (1) BCL11A coding sequence, (2) the minimal critical sequences in the +58 DHS within the erythroid-specific BCL11A enhancer, (3) the HbF-associated sequences within the Aγ-δ intergenic region, or (4) other genes with a known role in γ-globin regulation such as MYB, KLF1, LRF/ZBTB7A, or EHMT1/EHMT2. HbA, adult hemoglobin.

Close modal
Table 1

Potential targets for therapeutic genome editing for the β-globin disorders

TargetRepair strategyEfficiencyAdvantages/Disadvantages
Repair of SCD allele HDR Low-moderate Single target allele; inadvertent generation of β-thalassemia alleles 
Repair of β-thalassemia allele HDR Low-moderate Heterogeneous target alleles 
Recreation of nondeletional HPFH HDR Low-moderate Inadvertent generation of γ-null alleles; identified HPFH patients support mutation tolerance/clinical benefit 
Recreation of deletional HPFH NHEJ Low-moderate Insufficient efficiency of targeted deletion; identified HPFH patients support mutation tolerance/clinical benefit 
Other targets in β-globin cluster NHEJ — Targets unknown 
BCL11A NHEJ High HSC/B-cell dysfunction due to BCL11A requirement; haploinsufficient patients have significant HbF induction 
BCL11A enhancer NHEJ High Erythroid-specific BCL11A loss; haploinsufficient patients have significant HbF induction 
α-Globin NHEJ Moderate-high Balance α-β chains; inadvertent generation of α-thalassemia cells 
KLF1 NHEJ High Broad role in cell proliferation and cellular development 
MYB NHEJ High Broad role in cell proliferation and cellular development 
LRF/ZBTB7A NHEJ High Broad role in cell proliferation and cellular development 
EHMT1/EHMT2 NHEJ High Role in hematopoiesis unknown 
LIN28B pathway NHEJ High Role in hematopoiesis unknown 
TargetRepair strategyEfficiencyAdvantages/Disadvantages
Repair of SCD allele HDR Low-moderate Single target allele; inadvertent generation of β-thalassemia alleles 
Repair of β-thalassemia allele HDR Low-moderate Heterogeneous target alleles 
Recreation of nondeletional HPFH HDR Low-moderate Inadvertent generation of γ-null alleles; identified HPFH patients support mutation tolerance/clinical benefit 
Recreation of deletional HPFH NHEJ Low-moderate Insufficient efficiency of targeted deletion; identified HPFH patients support mutation tolerance/clinical benefit 
Other targets in β-globin cluster NHEJ — Targets unknown 
BCL11A NHEJ High HSC/B-cell dysfunction due to BCL11A requirement; haploinsufficient patients have significant HbF induction 
BCL11A enhancer NHEJ High Erythroid-specific BCL11A loss; haploinsufficient patients have significant HbF induction 
α-Globin NHEJ Moderate-high Balance α-β chains; inadvertent generation of α-thalassemia cells 
KLF1 NHEJ High Broad role in cell proliferation and cellular development 
MYB NHEJ High Broad role in cell proliferation and cellular development 
LRF/ZBTB7A NHEJ High Broad role in cell proliferation and cellular development 
EHMT1/EHMT2 NHEJ High Role in hematopoiesis unknown 
LIN28B pathway NHEJ High Role in hematopoiesis unknown 

Correction of underlying genetic defects

The most appealing and theoretically straightforward application of genome editing for monogenic disorders is correction of a mutant DNA sequence and, in that manner, preservation of all intrinsic regulatory mechanisms acting on the gene of interest. Precise gene correction relies on HDR from an extrachromosomal template containing a wild-type gene sequence. Typically, the frequency of HDR is relatively low, and particularly low in CD34+ hematopoietic stem and progenitor cells (HSPCs) as discussed in the next section. However, gene correction strategies may benefit from mixed chimerism allogeneic transplant studies suggesting that low levels of chimerism can produce clinical benefit.74  Clinical development of such strategies requires optimizing efficiency and safety of correcting the sickle mutation, whereas the diverse spectrum of β-thalassemia point mutations and deletions necessitates optimization for each unique genetic target, a significant challenge for clinical translation.

Gene editing for reactivation of HbF in SCD and β-thalassemia

Elevated HbF is beneficial in SCD and β-thalassemia. Targets for manipulation include sequences lying within the β-globin cluster or within the genes encoding transcriptional regulators of globin gene expression (Figure 1; Table 1). Depending on the target, editing would rely on HDR or NHEJ. Suitability of each target relates to the ease with which the desired gene modifications can be generated and the extent to which the modifications reactivate HbF expression. In SCD, the goal is to induce sufficient HbF to prevent sickle hemoglobin (HbS) polymerization. In β-thalassemia, the aim is to replace deficient β-globin and thereby reduce globin chain imbalance.

Genetic correction of the SCD and β-thalassemia mutations

Classical gene-targeting approaches have been used to repair the SCD mutation in embryonic stem cells,75  but this approach cannot be applied to CD34+ HSPCs due to low efficiency and the necessity to isolate and propagate faithful recombinants. Correction of genetic defects in cultured cells with an engineered nuclease and a donor repair template has been achieved for multiple disorders, including cystic fibrosis,76-79  Duchenne muscular dystrophy,80,81  ornithine transcarbamylase deficiency,82  hereditary tyrosinemia,83  and other diseases.71,84-87  Gene correction for SCD and β-thalassemia has also been accomplished in a laboratory setting.75,88-91  Of note, a recent study reported correction of an SCD allele at nearly 20% gene modification in CD34+ HSPCs upon delivery of a repair template via integration-deficient lentivirus or by DNA oligonucleotide electroporation in the presence of a β-globin–targeted ZFN. Similar levels of correction were observed in bone marrow cells isolated from SCD patients.88  Despite successful HDR in bulk cells in vitro, the levels of HDR were 10- to 20-fold reduced in the spleen and bone marrow of transplanted immunodeficient mice, suggesting that HDR within long-term engrafting hematopoietic stem cells (HSCs) was far less efficient than in downstream progenitors. Another study reported HDR rates of 17% to 43% at 2 genomic loci in fetal liver–derived or mobilized peripheral blood–derived cells via electroporation of ZFN messenger RNA (mRNA) in conjunction with an adeno-associated virus (AAV) donor repair template.92  These rates of HDR were maintained in vivo, suggesting the ability to perform HDR in primitive repopulating cells. Studies using AAV in conjunction with megaTALs,93  TAL effectors coupled to a sequence-specific homing endonuclease, demonstrated ∼14% rates of HDR in CD34+ HSPCs.94  Although megaTALs may enhance HDR through generation of 3′ DNA overhangs in HSPCs, the rate of HDR in repopulating HSCs has not been examined.

The relative efficiency of HDR vs NHEJ is critical to potential use of gene editing for gene correction. High rates of NHEJ-mediated indel formation are suboptimal for clinical translation of β-globin gene correction as the process creates the possibility of disruption of β-globin production and inadvertent generation of β-thalassemia alleles. Another consideration is that mutagenesis has also been observed in the highly homologous δ-globin gene in β-globin gene correction experiments, which may result in deletions and rearrangements affecting β-globin that may be difficult to detect by standard polymerase chain reaction (PCR)-based genotyping approaches.88 

It is possible that small molecules that enhance HDR and/or inhibit NHEJ may improve the efficiency of gene correction within CD34+ HSPCs, so long as they do not impair cell engraftment capability.95-98  Another possibility is the use of asymmetric donor template DNA to enhance rates of HDR.99  NHEJ is the dominant pathway in G1, S, and G2 phases of the cell cycle, whereas HDR preferentially occurs during late S-phase and G2 phase when sister chromatid templates become available.100  Because HSCs, the rare long-term repopulating cells within CD34+ HSPC preparations, are largely quiescent, HDR is not favored. These observations are supported by the roles of BRCA1, PALB2, and BRCA2 in DSB repair. BRCA1 creates single-strand DNA through end resection and interacts with PALB2 to recruit BRCA2 and RAD51 to mediate HDR at sites of DSB. Identification of the cell cycle’s role in suppressing BRCA1 in the G1 phase supports the dominance of NHEJ repair in quiescent cells.101  However, restoration of the BRCA1-PALB2 interaction during the G1 phase can support HDR. Therefore, it may be possible to enhance HDR in quiescent HSCs through modulation of the BRCA1-PALB2-BRCA2 pathway.101  Moreover, 1 study demonstrated enhanced rates of HDR in HEK293T and nonhematopoietic primary cells through cell cycle synchronization to achieve nuclease-mediated cleavage during the optimal portions of the cell cycle for HDR.100  However, triggering proliferation in HSCs tends to impair their ultimate repopulating potential. Whether expansion of HSPC populations with small molecules such as SR1102,103  or UM171104  will allow for improved HDR efficiencies with concomitant retention of stem cell activity in vivo is as yet unknown.

Modification of the β-globin locus to recreate hereditary persistence of fetal hemoglobin

As would be anticipated from the existence of rare hereditary persistence of fetal hemoglobin (HPFH) alleles, genome-wide association studies (GWAS) have linked the β-globin cluster itself to HbF levels.105-110  This corroborated previous human genetic studies that identified HPFH patients with elevated HbF levels resulting from large deletions within the β-globin cluster.111-113  Re-creating the larger deletional HPFH alleles is impractical given their large size.60  However, opportunities may exist for targeting discrete regions of the β-globin gene cluster by NHEJ. Comparison of large deletions in the cluster that generate either HPFH or δβ-thalassemia phenotypes has implicated sequences in the Aγ-δ intergenic region as harboring silencers of γ-gene expression. Notably, study of 3 families with overlapping deletions in the β-globin cluster identified a 3.5-kb region between the Aγ and δ genes that may be essential for γ-globin repression. Additional indirect support was derived from chromatin immunoprecipitation–PCR experiments that suggest BCL11A binding within this region.111,114-117  At present, the optimal sequences in the cluster amenable for targeted deletion by editing and NHEJ have not been identified.

Several point mutations or small deletions in the Aγ or Gγ-globin gene promoters lead to persistence of HbF into adult life. HbF levels in heterozygotes with these nondeletional HPFH mutations may be as high as 30%.116,118-120  One of the strongest HPFH alleles (−175 T>C in the Aγ promoter) was recently created in cultured K562 cells with TALENs. Increased γ-globin production resulted, most likely through de novo generation of a TAL1-binding site that facilitated increased chromatin looping between the Aγ promoter and the locus control region.121  An HPFH allele with a small deletion in the Aγ promoter was re-created in CD34+ HSPCs with sgRNA and Cas9 expression, presumably due to microdeletion of a repeated sequence.122  Therapeutic genome editing to generate HPFH mutations is an attractive strategy as the effects of these mutations are known through study of families with these rare beneficial alleles. The approach, however, faces many of the same challenges as precise gene correction, given the apparent dominance of the NHEJ pathway at the expense of HDR efficiency in HSCs.

BCL11A targeting

BCL11A gene disruption

The GWAS-implicated transcription factor BCL11A is a validated repressor of HbF.105-110,114,123  Erythroid-lineage Bcl11a knockout in a mouse model of SCD led to pancellular HbF induction and phenotypic correction of a mouse model of SCD without perturbing other hematologic parameters.124  Haploinsufficient patients with microdeletions within the BCL11A locus have significant neurocognitive phenotypes as well as elevated HbF at levels near or above therapeutic thresholds.125,126  In principle, the genetic knockout of BCL11A by targeting BCL11A coding sequence in order to create frameshift null alleles represents a potential therapeutic strategy. Roles of BCL11A in nonhematopoietic lineages, including the neural lineage,127,128  pancreatic progenitors,129  and the breast epithelium,130  would not be problematic upon modification of BCL11A in autologous CD34+ HSPCs. However, this strategy is limited by extraerythroid roles of BCL11A in the hematopoietic system, including its requirement for B-cell development127,131-133  and HSC function.134,135  These roadblocks might be circumvented by use of erythroid-restricted expression of genome-editing components. A variation of this approach involves erythroid-specific, short hairpin RNA–mediated knockdown of BCL11A expression, which is under development as a gene-therapy strategy.136  Delivery of genome-editing tools stably to CD34+ HSPCs would be inadvisable due to potential insertional mutagenesis137  as well as elevated risk of off-target mutagenesis over time. Furthermore, the effects of long-term expression of ZFNs, TALENs, or CRISPR/Cas9 on CD34+ HSPCs are unknown.

BCL11A gene enhancer

Recent fine mapping of HbF-associated GWAS variants led to the identification of a developmental stage-specific, erythroid-restricted 12-kb region bearing a characteristic enhancer chromatin signature. This enhancer region is composed of 3 DNaseI hypersensitive sites (DHS), termed +55, +58, and +62 as their distance in kilobases from the BCL11A transcriptional start site. Deletion of the orthologous element in a murine erythroid cell line resulted in a complete loss of BCL11A at both the RNA and protein levels whereas expression was spared in a B-cell line with the same deletion.67  Subsequent deletion studies demonstrated a similar requirement for this element for BCL11A expression in human erythroid cells.138,139 

BCL11A enhancer targeting has several distinct advantages over coding sequence disruption: (1) GWAS studies have demonstrated that variation in the BCL11A enhancer is associated with elevated HbF levels and is both common and well tolerated.67  (2) Targeted deletion of this element in a human erythroid cell line leads to loss of BCL11A expression and subsequent HbF induction nearly comparable to BCL11A null clones.138  (3) Targeted deletion of the murine +62 DHS within the Bcl11a erythroid enhancer results in delayed hemoglobin switching sparing expression in the brain and nonerythroid hematopoietic lineages.138  The +62 DHS knockout mice were viable and born in normal Mendelian ratios as compared with Bcl11a−/− knockout mice that are perinatal lethal likely due to neural defects.123,138  These results further highlight the erythroid specificity of this element in vivo.138  (4) Targeting the BCL11A enhancer has been shown to be better tolerated even within the erythroid lineage as compared with targeting the BCL11A coding sequence, suggesting a residual low level of BCL11A present after enhancer targeting is insufficient to repress γ-globin, but promotes cellular fitness.138,139 

Therefore, an alternative approach to targeting BCL11A coding sequence might be targeted deletion of the 12-kb BCL11A erythroid enhancer.67  However, although targeted deletions from ∼1 kb to 1 Mb have been demonstrated to occur at an appreciable frequency, these are unlikely to occur at a sufficient frequency at clinical scale with current genome-editing technologies due to competing outcomes to deletion when using a dual nuclease strategy including scarring (multifocal indels), inversions, and duplications.47,48,60,68,140  Furthermore, the heterogeneous population of cells resulting from a dual nuclease strategy would be suboptimal for clinical translation.

Functional footprinting-informed targeting by ZFNs/TALENs within the BCL11A enhancer and comprehensive functional mapping of the BCL11A enhancer by CRISPR/Cas9-mediated saturating mutagenesis has revealed an “Achilles' heel” to the BCL11A enhancer within the +58 DHS.138,139  Disruption of this minimal functional sequence at the core of the DHS +58 by CRISPR/Cas9 or ZFNs/TALENs resulted in γ-globin induction comparable to targeting coding sequence in CD34+ HSPCs subject to erythroid differentiation conditions.138,139  The core region has been fine-mapped to an ∼20-bp region including a GATA1-binding motif which appears to be essential for BCL11A expression and subsequent HbF repression.138,139  As previously discussed, the erythroid specificity of the regulatory element would not require erythroid-specific expression of the genome-editing components, as would be necessary with a BCL11A coding sequence targeting approach. Taken together, targeting of the BCL11A enhancer at the functional core of +58 DHS in autologous CD34+ HSPCs followed by BMT represents a promising therapeutic strategy to induce HbF expression in patients with the β-globin disorders (Figure 2).

Figure 2

Reversal of hemoglobin switching to induce therapeutic levels of HbF. Reversal of hemoglobin switching can be accomplished through autologous bone marrow transplantation of genome-edited CD34+ HSPCs. The gray region indicates the hypothesized levels of HbF required for clinical benefit.

Figure 2

Reversal of hemoglobin switching to induce therapeutic levels of HbF. Reversal of hemoglobin switching can be accomplished through autologous bone marrow transplantation of genome-edited CD34+ HSPCs. The gray region indicates the hypothesized levels of HbF required for clinical benefit.

Close modal

LRF/ZBTB7A gene disruption

Another transcription factor LRF/ZBTB7A (also referred to as Pokemon) has more recently been recognized as a major repressor of γ-globin.141  LRF-knockout mice exhibit elevated levels of the embryonic globin Hbb-βh1 with normal levels of Hbb-y. This contrasts from Bcl11a-null mice that exhibit elevation of both embryonic globins, Hbb-y > Hbb-βh1.123,124 Zbtb7a−/− mice are embryonic lethal due to anemia, whereas conditional knockout of Zbtb7a in adult mice leads to inefficient terminal erythropoiesis resulting in a mild macrocytic anemia.142  CRISPR/Cas9-mediated knockout of LRF in an erythroid cell line resulted in dramatic upregulation of γ-globin. LRF/BCL11A double knockout in this system was near additive and resulted in HbF of >90%, suggesting LRF’s role in γ-globin regulation is partially independent of BCL11A. Subsequent analysis demonstrated a mild delay in erythroid differentiation upon knockdown of LRF in primary human CD34+ HSPCs differentiated down the erythroid lineage with a corresponding induction of γ-globin.141  Although the effect of LRF loss on γ-globin is striking, the role of LRF in cell fate decisions in multiple hematopoietic lineages and its requirement for terminal erythropoiesis may limit its therapeutic potential.143 

Reducing chain imbalance for β-thalassemias

The physiologic hallmark of β-thalassemia is globin chain imbalance, such that deficiency of β chains leads to precipitation of unstable, free α chains, membrane damage, hemolysis, and ineffective erythropoiesis.20,144-146  α-Thalassemia serves as a genetic modifier of β-thalassemia, as chain imbalance is reduced.146  This is supported by a milder disease course in patients with the co-inheritance of α-thalassemia and β-thalassemia.146-148  In principle, therefore, α-globin genes or their regulatory elements constitute potential targets for genome editing. Targeting an α-globin gene itself could result in a heterogeneous population of cells including those null for α-globin which might not support erythropoiesis. This approach could become a viable option if technological advancements allow for the precise control of the number of α-globin null alleles generated. However, with present editing tools, the inability to control the number of α-globin null alleles created through targeting α-globin coding sequences makes targeting known regulatory elements of α-globin or RNA interference–mediated knockdown of α-globin more attractive alternatives in this context.

Other potential therapeutic targets

Transcription factors KLF1 and MYB have previously been considered potential targets for HbF reactivation, but are not attractive due to their broad roles in cell proliferation and cellular development.112,113  Other genes such as EHMT1/EHMT2 and the LIN28B pathway have been implicated in the regulation of γ-globin; however, the selectivity of these targets and roles in hematopoiesis need further investigation.149-151 

Significant obstacles to wider use of BMT for cure of patients with β-globin disorders are the availability of compatible donors and risk of GVHD. Donor availability is particularly severe for SCD patients. The most persuasive rationale for therapeutic genome editing of β-globin disorders rests with the use of autologous CD34+ HSPCs as the cellular target. Through use of the patient’s own cells for therapy, donor availability and GVHD are avoided. As with more “conventional” somatic gene therapy with modified viruses, delivery of the requisite editing components to the target cells is the principal hurdle to be overcome in achieving clinical success.152  Delivery of therapeutic genes to CD34+ HSPCs has been accomplished with integrating and nonintegrating viral vectors (such as lentiviral, adenovirus, and AAV vectors), as well as physical methods (eg, electroporation).88,153-155  The optimal method for gene editing is currently unknown but is likely related to the specific technology used. High-efficiency delivery at clinical scale, roughly >108 CD34+ HSPCs cells, presents a practical challenge. However, recent studies have taken promising steps forward with electroporation of mRNA to CD34+ HSPCs at clinical scale (>1 × 108 cells).139,156  Robust cellular delivery is required for clinical translation of any envisioned therapeutic genome-editing approaches.

Genome editing is generally more difficult in primary cells as compared with immortal cell lines for reasons that are not entirely well understood, but may reflect inefficient delivery, diminished promoter activity of constructs, interferon responses, exonuclease activity, and host mechanisms of DNA repair.152  Electroporation of ZFNs, TALENs, and CRISPRs as DNA, RNA, and/or protein is an efficient delivery strategy to CD34+ HSPCs in a laboratory setting.88,92,140,152,157  For the CRISPR/Cas9 system, mRNA or ribonucleoprotein electroporation may obviate toxicity associated with DNA delivery, as well as yield higher rates of editing in cell lines and CD34+ HSPCs.100,152  The identification of novel Cas9 proteins isolated from diverse prokaryotes or other Cas9-like nucleases that are smaller than the widely used Streptococcus pyogenes–derived Cas9 may facilitate delivery efficiency, particularly for viral vectors.53  In addition, chemical modification of sgRNAs enhances editing efficiency in primary hematopoietic cells and CD34+ HSPCs.152 

BMT poses risks to SCD and β-thalassemia patients beyond GVHD as reviewed in Lucarelli et al.158  Myeloablative or submyeloablative conditioning will be required to allow for engraftment of edited HSPCs. The inverse relationship between conditioning and engraftment rates suggests that myeloablative regimens maximize the likelihood of clinically beneficial engraftment rates. However, a myeloablative approach has elevated risk of BMT-associated morbidity and mortality.158  The optimal conditioning regimen for autologous BMT of genome-edited HSPCs requires investigation and may vary from patient to patient depending on the extent of end-organ damage resulting from vaso-occlusive events in SCD and/or iron overload in β-thalassemia patients. Given the risks associated with BMT, clinical guidelines for treatment may be stratified based on clinical disease severity for both SCD and β-thalassemia patients. BMT represents a viable therapeutic avenue in developed countries in spite of the associated risks, albeit the price of allogenic BMT exceeds $200 000 (in the United States).159  The extent of infrastructure and resources required for BMT restricts its wide use in developing countries,160,161  which have the highest prevalence of β-globin disorders.162 

Although clinical translation of therapeutic genome editing for the β-hemoglobinopathies is appealing, several steps must be taken before the vision can become a reality. (1) Target selection, (2) delivery of editing reagents to HSCs, and (3) empirical testing of off-target potential must all be addressed and optimized. Targets that may be chosen for clinical development are summarized in Figure 1 and Table 1. (1) Strategies that rely on NHEJ are likely to be the first attempted using current technologies due to the dominance of NHEJ in quiescent HSCs and overall high efficiency of NHEJ as compared with HDR. At present, disruption of the core BCL11A enhancer sequences within the +58 DHS by NHEJ appears quite favorable in terms of potency of an effect on HbF expression and sparing of consequences for nonerythroid lineages. It may also be possible to combine an NHEJ-based approach with an HDR strategy or delivery of antisickling adult hemoglobin to enhance potential clinical benefit.163,164  (2) Transient delivery (electroporation or nonintegrating viral vectors) represents a safer alternative to stable integration of genome-editing components due to reduced risk of insertional mutagenesis and risk of off-target cleavage, as well as freedom from the uncertainty of long-term expression of genome-editing tools in CD34+ HSPCs. Transient delivery also necessitates high levels of on-target editing within a shorter window of time prior to loss of the genome-editing components through cell division. One possibility would be to enrich for edited cells prior to BMT of autologous cells,60  which could be further enhanced by strategies to expand HSCs ex vivo.102-104  (3) Off-target cleavages represent a legitimate concern for therapeutic genome editing. Newly developed techniques allow for unbiased genome-wide identification of off-target mutagenesis.165,166  Various methods have been reported that aim to enhance on-target vs off-target specificity. These include use of Cas9 nickase, truncated guides, dimeric RNA-guided FokI nucleases, and rationally engineered enhanced specificity Cas9.51,52,68,167-169  In addition, alternative RNA-directed nucleases (Cpf1) or modified Cas9 derivatives with reduced off-target cleavage potential appear to be steps toward “clean” editing reagents. It will be necessary to empirically test the optimized editing reagents for off-target cleavage potential and assess the associated risk of inappropriate DSBs within the genome. As methods to predict and detect off-target cleavages continue to improve, it may become possible to screen autologous genome-edited cells prior to BMT for possible pathogenic off-target mutations. “CD34+ humanized” mice, NOD-SCID-γ mice with bone marrow–engrafted human CD34+ HSPCs, can be used to evaluate the safety of genome-editing tools as these models can demonstrate multilineage reconstitution, self-renewal, and the ability to monitor leukemogenesis. However, due to the inability to model all human hematopoietic lineages, notably the erythroid lineage, and general limitations of chimera mouse models, humanized mice have limitations in assessing safety of genome-editing treatments in vivo. Furthermore, most off-target cleavages will exhibit a neutral effect on cellular fitness, whereas only rare off-target cleavages will be pathogenic, including induction of myelodysplastic syndrome or leukemic disorders. It will be important to enhance off-target cleavage prediction and detection methods to minimize risk of these rare pathogenic mutations.

One additional challenge for clinical development is harvesting CD34+ HSPCs for autologous stem cell transplantation from patients with β-globin disorders. Sufficient numbers of CD34+ HSPCs for BMT can be harvested from 2 sources, peripheral blood or bone marrow. Harvest of CD34+ HSPCs from peripheral blood is preferred due its minimal invasiveness and higher yield of CD34+ HSPCs following mobilization by granulocyte colony-stimulating factor (G-CSF).170  Use of G-CSF as a mobilizing agent is generally well tolerated for healthy adults and cancer patients. However, there are significant risks of G-CSF administration for patients with β-globin disorders. SCD patients have significant risk of vaso-occlusive events, acute chest syndrome, multiorgan system failure, and death,171  whereas β-thalassemia patients are susceptible to splenic rupture, hyperleukocytosis, and thrombosis.170  Plerixafor is an alternative mobilizing agent that may provide a safer option to G-CSF.170,172,173  Although the effect of plerixafor in SCD patients requires investigation, it has been shown to be safe and effective in both splenectomized and nonsplenectomized β-thalassemia patients. In contrast, although G-CSF was well tolerated in nonsplenectomized patients, it resulted in hyperleukocytosis and lower yield of CD34+ HSPCs as compared with plerixafor in splenectomized β-thalassemia patients.172  Combination of plerixafor with a reduced dose of G-CSF to avoid adverse effects has been shown to be superior to either agent alone.170,173  Therefore, plerixafor or combination G-CSF/plerixafor mobilization may provide a safe avenue for peripheral blood CD34+ HSPC harvests for β-thalassemia patients. Until acceptable protocols for mobilization of CD34+ HSPCs are established for SCD patients, traditional bone marrow harvesting may be required. It may be advisable as well to test ex vivo–editing efficiencies and maintenance of modified cells upon transfer into suitable immunodeficient mice for CD34+ HSPCs obtained by different methods to ensure optimization for clinical use.

The technological advances in genome manipulation are breathtaking in terms of the speed with which they have been reported in the past several years. The potential of genome-editing approaches for clinical benefit in the β-globin disorders is immense. Besides the choice of the editing platform and its delivery to repopulating cells within CD34+ HSPC harvests, a major factor in considering application to these conditions is the target sequences to be modified. If the goal is precise gene correction, the desired sequence alteration is clear. This strategy relies on HDR, and at the moment must await improved protocols for HDR in bona fide repopulating cells for clinical implementation.

Reactivation of HbF is an attractive approach, as it might be “one size fits all” in principle, suitable for both SCD and the β-thalassemias. The precise levels of pancellular HbF necessary for clinical benefit remain elusive, but is hypothesized to be ≥20% for SCD and likely somewhat higher in β-thalassemia (Figure 2). Due to the inability to precisely model HbF control experimentally, it may be difficult to assess the minimal threshold for clinical benefit in a laboratory setting. Furthermore, it is unlikely that HSCs undergoing therapeutic genome editing based on the strategies reviewed here will have a selective advantage in vivo. However, results from mixed chimerism allogeneic transplant demonstrate that low levels of chimerism can produce clinical benefit due to the survival advantage of normal red blood cells.74 

Given the current state of genome-editing technologies, HbF induction mediated by NHEJ repair may provide a long-sought “silver bullet” for therapy. As such, harnessing the power of genome-editing tools may finally allow for therapeutic exploitation of the deep understanding of the genetics of hemoglobin and lead to a genome-editing–based therapeutic option for the β-hemoglobinopathies in the near future.

M.C.C. was supported by the National Institutes of Health (NIH) National Institute of Diabetes and Digestive and Kidney Diseases (F30DK 103359). S.H.O. was supported by the NIH National Heart, Lung, and Blood Institute (HL032259 and P01HL032262).

Contribution: M.C.C. reviewed the literature; and M.C.C. and S.H.O. wrote the manuscript.

Conflict-of-interest disclosure: S.H.O. is an inventor on a patent related to this work. M.C.C. declares no competing financial interests.

Correspondence: Stuart H. Orkin, Dana-Farber Cancer Institute, 44 Binney St, Boston, MA 02115; e-mail: stuart_orkin@dfci.harvard.edu.

1
Bauer
 
DE
Orkin
 
SH
Hemoglobin switching’s surprise: the versatile transcription factor BCL11A is a master repressor of fetal hemoglobin.
Curr Opin Genet Dev
2015
, vol. 
33
 (pg. 
62
-
70
)
2
Ingram
 
VM
A specific chemical difference between the globins of normal human and sickle-cell anaemia haemoglobin.
Nature
1956
, vol. 
178
 
4537
(pg. 
792
-
794
)
3
Orkin
 
SH
Higgs
 
DR
Medicine. Sickle cell disease at 100 years.
Science
2010
, vol. 
329
 
5989
(pg. 
291
-
292
)
4
Orkin
 
SH
Kazazian
 
HH
Antonarakis
 
SE
, et al. 
Linkage of β-thalassaemia mutations and β-globin gene polymorphisms with DNA polymorphisms in human β-globin gene cluster.
Nature
1982
, vol. 
296
 
5858
(pg. 
627
-
631
)
5
Orkin
 
SH
Kazazian
 
HH
The mutation and polymorphism of the human β-globin gene and its surrounding DNA.
Annu Rev Genet
1984
, vol. 
18
 (pg. 
131
-
171
)
6
Wong
 
C
Antonarakis
 
SE
Goff
 
SC
Orkin
 
SH
Boehm
 
CD
Kazazian
 
HH
On the origin and spread of β-thalassemia: recurrent observation of four mutations in different ethnic groups.
Proc Natl Acad Sci USA
1986
, vol. 
83
 
17
(pg. 
6529
-
6532
)
7
Kazazian
 
HH
Orkin
 
SH
Antonarakis
 
SE
, et al. 
Molecular characterization of seven β-thalassemia mutations in Asian Indians.
EMBO J
1984
, vol. 
3
 
3
(pg. 
593
-
596
)
8
Antonarakis
 
SE
Kazazian
 
HH
Orkin
 
SH
DNA polymorphism and molecular pathology of the human globin gene clusters.
Hum Genet
1985
, vol. 
69
 
1
(pg. 
1
-
14
)
9
Lie-Injo
 
LE
Cai
 
SP
Wahidijat
 
I
, et al. 
β-thalassemia mutations in Indonesia and their linkage to β haplotypes.
Am J Hum Genet
1989
, vol. 
45
 
6
(pg. 
971
-
975
)
10
Platt
 
OS
Orkin
 
SH
Dover
 
G
Beardsley
 
GP
Miller
 
B
Nathan
 
DG
Hydroxyurea enhances fetal hemoglobin production in sickle cell anemia.
J Clin Invest
1984
, vol. 
74
 
2
(pg. 
652
-
656
)
11
Letvin
 
NL
Linch
 
DC
Beardsley
 
GP
McIntyre
 
KW
Nathan
 
DG
Augmentation of fetal-hemoglobin production in anemic monkeys by hydroxyurea.
N Engl J Med
1984
, vol. 
310
 
14
(pg. 
869
-
873
)
12
Charache
 
S
Terrin
 
ML
Moore
 
RD
, et al. 
Investigators of the Multicenter Study of Hydroxyurea in Sickle Cell Anemia
Effect of hydroxyurea on the frequency of painful crises in sickle cell anemia.
N Engl J Med
1995
, vol. 
332
 
20
(pg. 
1317
-
1322
)
13
Platt
 
OS
Hydroxyurea for the treatment of sickle cell anemia.
N Engl J Med
2008
, vol. 
358
 
13
(pg. 
1362
-
1369
)
14
Bunn
 
HF
Pathogenesis and treatment of sickle cell disease.
N Engl J Med
1997
, vol. 
337
 
11
(pg. 
762
-
769
)
15
Rees
 
DC
Williams
 
TN
Gladwin
 
MT
Sickle-cell disease.
Lancet
2010
, vol. 
376
 
9757
(pg. 
2018
-
2031
)
16
Rund
 
D
Rachmilewitz
 
E
β-thalassemia.
N Engl J Med
2005
, vol. 
353
 
11
(pg. 
1135
-
1146
)
17
Platt
 
OS
Brambilla
 
DJ
Rosse
 
WF
, et al. 
Mortality in sickle cell disease. Life expectancy and risk factors for early death.
N Engl J Med
1994
, vol. 
330
 
23
(pg. 
1639
-
1644
)
18
Platt
 
OS
Thorington
 
BD
Brambilla
 
DJ
, et al. 
Pain in sickle cell disease. Rates and risk factors.
N Engl J Med
1991
, vol. 
325
 
1
(pg. 
11
-
16
)
19
Castro
 
O
Brambilla
 
DJ
Thorington
 
B
, et al. 
The Cooperative Study of Sickle Cell Disease
The acute chest syndrome in sickle cell disease: incidence and risk factors.
Blood
1994
, vol. 
84
 
2
(pg. 
643
-
649
)
20
Musallam
 
KM
Taher
 
AT
Cappellini
 
MD
Sankaran
 
VG
Clinical experience with fetal hemoglobin induction therapy in patients with β-thalassemia.
Blood
2013
, vol. 
121
 
12
(pg. 
2199
-
2212
)
21
Galanello
 
R
Sanna
 
S
Perseu
 
L
, et al. 
Amelioration of Sardinian β0 thalassemia by genetic modifiers.
Blood
2009
, vol. 
114
 
18
(pg. 
3935
-
3937
)
22
Watson
 
J
A study of sickling of young erythrocytes in sickle cell anemia.
Blood
1948
, vol. 
3
 
4
(pg. 
465
-
469
)
23
Herman
 
EC
Conley
 
CL
Hereditary persistence of fetal hemoglobin. A family study.
Am J Med
1960
, vol. 
29
 (pg. 
9
-
17
)
24
Cavazzana-Calvo
 
M
Payen
 
E
Negre
 
O
, et al. 
Transfusion independence and HMGA2 activation after gene therapy of human β-thalassaemia.
Nature
2010
, vol. 
467
 
7313
(pg. 
318
-
322
)
25
Hoban
 
MD
Orkin
 
SH
Bauer
 
DE
Genetic treatment of a molecular disorder: gene therapy approaches to sickle cell disease.
Blood
2016
, vol. 
127
 
7
(pg. 
839
-
848
)
26
Kim
 
YG
Cha
 
J
Chandrasegaran
 
S
Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain.
Proc Natl Acad Sci USA
1996
, vol. 
93
 
3
(pg. 
1156
-
1160
)
27
Smith
 
J
Berg
 
JM
Chandrasegaran
 
S
A detailed study of the substrate specificity of a chimeric restriction enzyme.
Nucleic Acids Res
1999
, vol. 
27
 
2
(pg. 
674
-
681
)
28
Bibikova
 
M
Golic
 
M
Golic
 
KG
Carroll
 
D
Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases.
Genetics
2002
, vol. 
161
 
3
(pg. 
1169
-
1175
)
29
Bibikova
 
M
Beumer
 
K
Trautman
 
JK
Carroll
 
D
Enhancing gene targeting with designed zinc finger nucleases.
Science
2003
, vol. 
300
 
5620
pg. 
764
 
30
Porteus
 
MH
Baltimore
 
D
Chimeric nucleases stimulate gene targeting in human cells.
Science
2003
, vol. 
300
 
5620
pg. 
763
 
31
Urnov
 
FD
Miller
 
JC
Lee
 
Y-L
, et al. 
Highly efficient endogenous human gene correction using designed zinc-finger nucleases.
Nature
2005
, vol. 
435
 
7042
(pg. 
646
-
651
)
32
Moehle
 
EA
Rock
 
JM
Lee
 
Y-L
, et al. 
Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases [published correction appears in Proc Natl Acad Sci USA. 2007;104(14):6090].
Proc Natl Acad Sci USA
2007
, vol. 
104
 
9
(pg. 
3055
-
3060
)
33
Urnov
 
FD
Rebar
 
EJ
Holmes
 
MC
Zhang
 
HS
Gregory
 
PD
Genome editing with engineered zinc finger nucleases.
Nat Rev Genet
2010
, vol. 
11
 
9
(pg. 
636
-
646
)
34
Miller
 
JC
Holmes
 
MC
Wang
 
J
, et al. 
An improved zinc-finger nuclease architecture for highly specific genome editing.
Nat Biotechnol
2007
, vol. 
25
 
7
(pg. 
778
-
785
)
35
Li
 
H
Haurigot
 
V
Doyon
 
Y
, et al. 
In vivo genome editing restores haemostasis in a mouse model of haemophilia.
Nature
2011
, vol. 
475
 
7355
(pg. 
217
-
221
)
36
Boch
 
J
Scholze
 
H
Schornack
 
S
, et al. 
Breaking the code of DNA binding specificity of TAL-type III effectors.
Science
2009
, vol. 
326
 
5959
(pg. 
1509
-
1512
)
37
Moscou
 
MJ
Bogdanove
 
AJ
A simple cipher governs DNA recognition by TAL effectors.
Science
2009
, vol. 
326
 
5959
pg. 
1501
 
38
Christian
 
M
Cermak
 
T
Doyle
 
EL
, et al. 
Targeting DNA double-strand breaks with TAL effector nucleases.
Genetics
2010
, vol. 
186
 
2
(pg. 
757
-
761
)
39
Li
 
T
Huang
 
S
Jiang
 
WZ
, et al. 
TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain.
Nucleic Acids Res
2011
, vol. 
39
 
1
(pg. 
359
-
372
)
40
Miller
 
JC
Tan
 
S
Qiao
 
G
, et al. 
A TALE nuclease architecture for efficient genome editing.
Nat Biotechnol
2011
, vol. 
29
 
2
(pg. 
143
-
148
)
41
Stoddard
 
BL
Homing endonucleases: from microbial genetic invaders to reagents for targeted DNA modification.
Structure
2011
, vol. 
19
 
1
(pg. 
7
-
15
)
42
Smith
 
J
Grizot
 
S
Arnould
 
S
, et al. 
A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences.
Nucleic Acids Res
2006
, vol. 
34
 
22
pg. 
e149
 
43
Silva
 
G
Poirot
 
L
Galetto
 
R
, et al. 
Meganucleases and other tools for targeted genome engineering: perspectives and challenges for gene therapy.
Curr Gene Ther
2011
, vol. 
11
 
1
(pg. 
11
-
27
)
44
Thierry
 
A
Dujon
 
B
Nested chromosomal fragmentation in yeast using the meganuclease I-Sce I: a new method for physical mapping of eukaryotic genomes.
Nucleic Acids Res
1992
, vol. 
20
 
21
(pg. 
5625
-
5631
)
45
Barrangou
 
R
Fremaux
 
C
Deveau
 
H
, et al. 
CRISPR provides acquired resistance against viruses in prokaryotes.
Science
2007
, vol. 
315
 
5819
(pg. 
1709
-
1712
)
46
Jinek
 
M
Chylinski
 
K
Fonfara
 
I
Hauer
 
M
Doudna
 
JA
Charpentier
 
E
A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.
Science
2012
, vol. 
337
 
6096
(pg. 
816
-
821
)
47
Cong
 
L
Ran
 
FA
Cox
 
D
, et al. 
Multiplex genome engineering using CRISPR/Cas systems.
Science
2013
, vol. 
339
 
6121
(pg. 
819
-
823
)
48
Mali
 
P
Yang
 
L
Esvelt
 
KM
, et al. 
RNA-guided human genome engineering via Cas9.
Science
2013
, vol. 
339
 
6121
(pg. 
823
-
826
)
49
Hsu
 
PD
Lander
 
ES
Zhang
 
F
Development and applications of CRISPR-Cas9 for genome engineering.
Cell
2014
, vol. 
157
 
6
(pg. 
1262
-
1278
)
50
Zetsche
 
B
Gootenberg
 
JS
Abudayyeh
 
OO
, et al. 
Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.
Cell
2015
, vol. 
163
 
3
(pg. 
759
-
771
)
51
Tsai
 
SQ
Wyvekens
 
N
Khayter
 
C
, et al. 
Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing.
Nat Biotechnol
2014
, vol. 
32
 
6
(pg. 
569
-
576
)
52
Wyvekens
 
N
Topkar
 
VV
Khayter
 
C
Joung
 
JK
Tsai
 
SQ
Dimeric CRISPR RNA-guided FokI-dCas9 nucleases directed by truncated gRNAs for highly specific genome editing.
Hum Gene Ther
2015
, vol. 
26
 
7
(pg. 
425
-
431
)
53
Ran
 
FA
Cong
 
L
Yan
 
WX
, et al. 
In vivo genome editing using Staphylococcus aureus Cas9.
Nature
2015
, vol. 
520
 
7546
(pg. 
186
-
191
)
54
Esvelt
 
KM
Mali
 
P
Braff
 
JL
Moosburner
 
M
Yaung
 
SJ
Church
 
GM
Orthogonal Cas9 proteins for RNA-guided gene regulation and editing.
Nat Methods
2013
, vol. 
10
 
11
(pg. 
1116
-
1121
)
55
Shi
 
J
Wang
 
E
Milazzo
 
JP
Wang
 
Z
Kinney
 
JB
Vakoc
 
CR
Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains.
Nat Biotechnol
2015
, vol. 
33
 
6
(pg. 
661
-
667
)
56
Ding
 
Q
Regan
 
SN
Xia
 
Y
Oostrom
 
LA
Cowan
 
CA
Musunuru
 
K
Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs.
Cell Stem Cell
2013
, vol. 
12
 
4
(pg. 
393
-
394
)
57
Tebas
 
P
Stein
 
D
Tang
 
WW
, et al. 
Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV.
N Engl J Med
2014
, vol. 
370
 
10
(pg. 
901
-
910
)
58
Sander
 
JD
Joung
 
JK
CRISPR-Cas systems for editing, regulating and targeting genomes.
Nat Biotechnol
2014
, vol. 
32
 
4
(pg. 
347
-
355
)
59
Bibikova
 
M
Carroll
 
D
Segal
 
DJ
, et al. 
Stimulation of homologous recombination through targeted cleavage by chimeric nucleases.
Mol Cell Biol
2001
, vol. 
21
 
1
(pg. 
289
-
297
)
60
Canver
 
MC
Bauer
 
DE
Dass
 
A
, et al. 
Characterization of genomic deletion efficiency mediated by clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells.
J Biol Chem
2014
, vol. 
289
 
31
(pg. 
21312
-
21324
)
61
Maddalo
 
D
Manchado
 
E
Concepcion
 
CP
, et al. 
In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system.
Nature
2014
, vol. 
516
 
7531
(pg. 
423
-
427
)
62
Choi
 
PS
Meyerson
 
M
Targeted genomic rearrangements using CRISPR/Cas technology.
Nat Commun
2014
, vol. 
5
 pg. 
3728
 
63
Blasco
 
RB
Karaca
 
E
Ambrogio
 
C
, et al. 
Simple and rapid in vivo generation of chromosomal rearrangements using CRISPR/Cas9 technology.
Cell Reports
2014
, vol. 
9
 
4
(pg. 
1219
-
1227
)
64
Xiao
 
A
Wang
 
Z
Hu
 
Y
, et al. 
Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish.
Nucleic Acids Res
2013
, vol. 
41
 
14
pg. 
e141
 
65
Gupta
 
A
Hall
 
VL
Kok
 
FO
, et al. 
Targeted chromosomal deletions and inversions in zebrafish.
Genome Res
2013
, vol. 
23
 
6
(pg. 
1008
-
1017
)
66
Lee
 
HJ
Kim
 
E
Kim
 
JS
Targeted chromosomal deletions in human cells using zinc finger nucleases.
Genome Res
2010
, vol. 
20
 
1
(pg. 
81
-
89
)
67
Bauer
 
DE
Kamran
 
SC
Lessard
 
S
, et al. 
An erythroid enhancer of BCL11A subject to genetic variation determines fetal hemoglobin level.
Science
2013
, vol. 
342
 
6155
(pg. 
253
-
257
)
68
Ran
 
FA
Hsu
 
PD
Lin
 
CY
, et al. 
Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity [published correction appears in Cell. 2013;155(2):479-480].
Cell
2013
, vol. 
154
 
6
(pg. 
1380
-
1389
)
69
Ran
 
FA
Hsu
 
PD
Wright
 
J
Agarwala
 
V
Scott
 
DA
Zhang
 
F
Genome engineering using the CRISPR-Cas9 system.
Nat Protoc
2013
, vol. 
8
 
11
(pg. 
2281
-
2308
)
70
Andrey
 
G
Kraft
 
K
Geuer
 
S
, et al. 
Deletions, inversions, duplications: engineering of structural variants using CRISPR/Cas in mice.
Cell Reports
2015
, vol. 
10
 (pg. 
833
-
839
)
71
Park
 
C-Y
Kim
 
DH
Son
 
JS
, et al. 
Functional correction of large factor VIII gene chromosomal inversions in hemophilia A patient-derived iPSCs using CRISPR-Cas9.
Cell Stem Cell
2015
, vol. 
17
 
2
(pg. 
213
-
220
)
72
Li
 
J
Shou
 
J
Guo
 
Y
, et al. 
Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9.
J Mol Cell Biol
2015
, vol. 
7
 
4
(pg. 
284
-
298
)
73
Zhang
 
L
Jia
 
R
Palange
 
NJ
, et al. 
Large genomic fragment deletions and insertions in mouse using CRISPR/Cas9.
PLoS One
2015
, vol. 
10
 
3
pg. 
e0120396
 
74
Andreani
 
M
Testi
 
M
Gaziev
 
J
, et al. 
Quantitatively different red cell/nucleated cell chimerism in patients with long-term, persistent hematopoietic mixed chimerism after bone marrow transplantation for thalassemia major or sickle cell disease.
Haematologica
2011
, vol. 
96
 
1
(pg. 
128
-
133
)
75
Chang
 
JC
Ye
 
L
Kan
 
YW
Correction of the sickle cell mutation in embryonic stem cells.
Proc Natl Acad Sci USA
2006
, vol. 
103
 
4
(pg. 
1036
-
1040
)
76
Schwank
 
G
Koo
 
B-K
Sasselli
 
V
, et al. 
Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients.
Cell Stem Cell
2013
, vol. 
13
 
6
(pg. 
653
-
658
)
77
Wu
 
Y
Liang
 
D
Wang
 
Y
, et al. 
Correction of a genetic disease in mouse via use of CRISPR-Cas9.
Cell Stem Cell
2013
, vol. 
13
 
6
(pg. 
659
-
662
)
78
Firth
 
AL
Menon
 
T
Parker
 
GS
, et al. 
Functional gene correction for cystic fibrosis in lung epithelial cells generated from patient iPSCs.
Cell Reports
2015
, vol. 
12
 
9
(pg. 
1385
-
1390
)
79
Lokody
 
I
Genetic therapies: correcting genetic defects with CRISPR-Cas9.
Nat Rev Genet
2014
, vol. 
15
 
2
pg. 
63
 
80
Long
 
C
McAnally
 
JR
Shelton
 
JM
Mireault
 
AA
Bassel-Duby
 
R
Olson
 
EN
Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA.
Science
2014
, vol. 
345
 
6201
(pg. 
1184
-
1188
)
81
Ousterout
 
DG
Kabadi
 
AM
Thakore
 
PI
Majoros
 
WH
Reddy
 
TE
Gersbach
 
CA
Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy.
Nat Commun
2015
, vol. 
6
 pg. 
6244
 
82
Yang
 
Y
Wang
 
L
Bell
 
P
, et al. 
A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice.
Nat Biotechnol
2016
, vol. 
34
 
3
(pg. 
334
-
338
)
83
Yin
 
H
Song
 
C-Q
Dorkin
 
JR
, et al. 
Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo.
Nat Biotechnol
2016
, vol. 
34
 
3
(pg. 
328
-
333
)
84
Wu
 
Y
Zhou
 
H
Fan
 
X
, et al. 
Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells.
Cell Res
2015
, vol. 
25
 
1
(pg. 
67
-
79
)
85
Osborn
 
MJ
Gabriel
 
R
Webber
 
BR
, et al. 
Fanconi anemia gene editing by the CRISPR/Cas9 system.
Hum Gene Ther
2015
, vol. 
26
 
2
(pg. 
114
-
126
)
86
Chang
 
C-W
Lai
 
Y-S
Westin
 
E
, et al. 
Modeling human severe combined immunodeficiency and correction by CRISPR/Cas9-enhanced gene targeting.
Cell Reports
2015
, vol. 
12
 
10
(pg. 
1668
-
1677
)
87
Flynn
 
R
Grundmann
 
A
Renz
 
P
, et al. 
CRISPR-mediated genotypic and phenotypic correction of a chronic granulomatous disease mutation in human iPS cells.
Exp Hematol
2015
, vol. 
43
 
10
(pg. 
838
-
848
)
88
Hoban
 
MD
Cost
 
GJ
Mendel
 
MC
, et al. 
Correction of the sickle cell disease mutation in human hematopoietic stem/progenitor cells.
Blood
2015
, vol. 
125
 
17
(pg. 
2597
-
2604
)
89
Zou
 
J
Mali
 
P
Huang
 
X
Dowey
 
SN
Cheng
 
L
Site-specific gene correction of a point mutation in human iPS cells derived from an adult patient with sickle cell disease.
Blood
2011
, vol. 
118
 
17
(pg. 
4599
-
4608
)
90
Sun
 
N
Zhao
 
H
Seamless correction of the sickle cell disease mutation of the HBB gene in human induced pluripotent stem cells using TALENs.
Biotechnol Bioeng
2014
, vol. 
111
 
5
(pg. 
1048
-
1053
)
91
Xie
 
F
Ye
 
L
Chang
 
JC
, et al. 
Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac.
Genome Res
2014
, vol. 
24
 
9
(pg. 
1526
-
1533
)
92
Wang
 
J
Exline
 
CM
DeClercq
 
JJ
, et al. 
Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors.
Nat Biotechnol
2015
, vol. 
33
 
12
(pg. 
1256
-
1263
)
93
Boissel
 
S
Jarjour
 
J
Astrakhan
 
A
, et al. 
megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering.
Nucleic Acids Res
2014
, vol. 
42
 
4
(pg. 
2591
-
2601
)
94
Sather
 
BD
Romano Ibarra
 
GS
Sommer
 
K
, et al. 
Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template.
Sci Transl Med
2015
, vol. 
7
 
307
pg. 
307ra156
 
95
Yu
 
C
Liu
 
Y
Ma
 
T
, et al. 
Small molecules enhance CRISPR genome editing in pluripotent stem cells.
Cell Stem Cell
2015
, vol. 
16
 
2
(pg. 
142
-
147
)
96
Pinder
 
J
Salsman
 
J
Dellaire
 
G
Nuclear domain ‘knock-in’ screen for the evaluation and identification of small molecule enhancers of CRISPR-based genome editing.
Nucleic Acids Res
2015
, vol. 
43
 
19
(pg. 
9379
-
9392
)
97
Maruyama
 
T
Dougan
 
SK
Truttmann
 
MC
Bilate
 
AM
Ingram
 
JR
Ploegh
 
HL
Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining.
Nat Biotechnol
2015
, vol. 
33
 
5
(pg. 
538
-
542
)
98
Chu
 
VT
Weber
 
T
Wefers
 
B
, et al. 
Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells.
Nat Biotechnol
2015
, vol. 
33
 
5
(pg. 
543
-
548
)
99
Richardson
 
CD
Ray
 
GJ
DeWitt
 
MA
Curie
 
GL
Corn
 
JE
Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA.
Nat Biotechnol
2016
, vol. 
34
 
3
(pg. 
339
-
344
)
100
Lin
 
S
Staahl
 
BT
Alla
 
RK
Doudna
 
JA
Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery.
eLife
2014
, vol. 
3
 pg. 
e04766
 
101
Orthwein
 
A
Noordermeer
 
SM
Wilson
 
MD
, et al. 
A mechanism for the suppression of homologous recombination in G1 cells.
Nature
2015
, vol. 
528
 
7582
(pg. 
422
-
426
)
102
Boitano
 
AE
Wang
 
J
Romeo
 
R
, et al. 
Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells.
Science
2010
, vol. 
329
 
5997
(pg. 
1345
-
1348
)
103
Wagner
 
JE
Brunstein
 
CG
Boitano
 
AE
, et al. 
Phase I/II trial of StemRegenin-1 expanded umbilical cord blood hematopoietic stem cells supports testing as a stand-alone graft.
Cell Stem Cell
2016
, vol. 
18
 
1
(pg. 
144
-
155
)
104
Fares
 
I
Chagraoui
 
J
Gareau
 
Y
, et al. 
Cord blood expansion. Pyrimidoindole derivatives are agonists of human hematopoietic stem cell self-renewal.
Science
2014
, vol. 
345
 
6203
(pg. 
1509
-
1512
)
105
Menzel
 
S
Garner
 
C
Gut
 
I
, et al. 
A QTL influencing F cell production maps to a gene encoding a zinc-finger protein on chromosome 2p15.
Nat Genet
2007
, vol. 
39
 
10
(pg. 
1197
-
1199
)
106
Uda
 
M
Galanello
 
R
Sanna
 
S
, et al. 
Genome-wide association study shows BCL11A associated with persistent fetal hemoglobin and amelioration of the phenotype of beta-thalassemia.
Proc Natl Acad Sci USA
2008
, vol. 
105
 
5
(pg. 
1620
-
1625
)
107
Lettre
 
G
Sankaran
 
VG
Bezerra
 
MA
, et al. 
DNA polymorphisms at the BCL11A, HBS1L-MYB, and beta-globin loci associate with fetal hemoglobin levels and pain crises in sickle cell disease.
Proc Natl Acad Sci USA
2008
, vol. 
105
 
33
(pg. 
11869
-
11874
)
108
Nuinoon
 
M
Makarasara
 
W
Mushiroda
 
T
, et al. 
A genome-wide association identified the common genetic variants influence disease severity in β0-thalassemia/hemoglobin E.
Hum Genet
2010
, vol. 
127
 
3
(pg. 
303
-
314
)
109
Solovieff
 
N
Milton
 
JN
Hartley
 
SW
, et al. 
Fetal hemoglobin in sickle cell anemia: genome-wide association studies suggest a regulatory region in the 5′ olfactory receptor gene cluster.
Blood
2010
, vol. 
115
 
9
(pg. 
1815
-
1822
)
110
Bhatnagar
 
P
Purvis
 
S
Barron-Casella
 
E
, et al. 
Genome-wide association study identifies genetic variants influencing F-cell levels in sickle-cell patients.
J Hum Genet
2011
, vol. 
56
 
4
(pg. 
316
-
323
)
111
Sankaran
 
VG
Xu
 
J
Byron
 
R
, et al. 
A functional element necessary for fetal hemoglobin silencing.
N Engl J Med
2011
, vol. 
365
 
9
(pg. 
807
-
814
)
112
Sankaran
 
VG
Orkin
 
SH
The switch from fetal to adult hemoglobin.
Cold Spring Harb Perspect Med
2013
, vol. 
3
 
1
pg. 
a011643
 
113
Bauer
 
DE
Kamran
 
SC
Orkin
 
SH
Reawakening fetal hemoglobin: prospects for new therapies for the β-globin disorders.
Blood
2012
, vol. 
120
 
15
(pg. 
2945
-
2953
)
114
Sankaran
 
VG
Menne
 
TF
Xu
 
J
, et al. 
Human fetal hemoglobin expression is regulated by the developmental stage-specific repressor BCL11A.
Science
2008
, vol. 
322
 
5909
(pg. 
1839
-
1842
)
115
Bank
 
A
Regulation of human fetal hemoglobin: new players, new complexities.
Blood
2006
, vol. 
107
 
2
(pg. 
435
-
443
)
116
Forget
 
BG
Molecular basis of hereditary persistence of fetal hemoglobin.
Ann N Y Acad Sci
1998
, vol. 
850
 (pg. 
38
-
44
)
117
Chakalova
 
L
Osborne
 
CS
Dai
 
Y-F
, et al. 
The Corfu deltabeta thalassemia deletion disrupts γ-globin gene silencing and reveals post-transcriptional regulation of HbF expression.
Blood
2005
, vol. 
105
 
5
(pg. 
2154
-
2160
)
118
Comi
 
P
Giglioni
 
B
Ottolenghi
 
S
, et al. 
Globin chain synthesis in single erythroid bursts from cord blood: studies on gamma leads to beta and G gamma leads to A gamma switches.
Proc Natl Acad Sci USA
1980
, vol. 
77
 
1
(pg. 
362
-
365
)
119
Ottolenghi
 
S
Nicolis
 
S
Taramelli
 
R
, et al. 
Sardinian G gamma-HPFH: a T----C substitution in a conserved “octamer” sequence in the G gamma-globin promoter.
Blood
1988
, vol. 
71
 
3
(pg. 
815
-
817
)
120
Martin
 
DI
Tsai
 
S-F
Orkin
 
SH
Increased γ-globin expression in a nondeletion HPFH mediated by an erythroid-specific DNA-binding factor.
Nature
1989
, vol. 
338
 
6214
(pg. 
435
-
438
)
121
Wienert
 
B
Funnell
 
APW
Norton
 
LJ
, et al. 
Editing the genome to introduce a beneficial naturally occurring mutation associated with increased fetal globin.
Nat Commun
2015
, vol. 
6
 pg. 
7085
 
122
Traxler
 
E
Yao
 
Y
Li
 
C
, et al. 
 
Genome editing recreates hereditary persistence of fetal hemoglobin in primary human erythroblasts [abstract]. Blood. 2015;126(23). Abstract 640
123
Sankaran
 
VG
Xu
 
J
Ragoczy
 
T
, et al. 
Developmental and species-divergent globin switching are driven by BCL11A.
Nature
2009
, vol. 
460
 
7259
(pg. 
1093
-
1097
)
124
Xu
 
J
Peng
 
C
Sankaran
 
VG
, et al. 
Correction of sickle cell disease in adult mice by interference with fetal hemoglobin silencing.
Science
2011
, vol. 
334
 
6058
(pg. 
993
-
996
)
125
Funnell
 
APW
Prontera
 
P
Ottaviani
 
V
, et al. 
2p15-p16.1 microdeletions encompassing and proximal to BCL11A are associated with elevated HbF in addition to neurologic impairment.
Blood
2015
, vol. 
126
 
1
(pg. 
89
-
93
)
126
Basak
 
A
Hancarova
 
M
Ulirsch
 
JC
, et al. 
BCL11A deletions result in fetal hemoglobin persistence and neurodevelopmental alterations.
J Clin Invest
2015
, vol. 
125
 
6
(pg. 
2363
-
2368
)
127
John
 
A
Brylka
 
H
Wiegreffe
 
C
, et al. 
Bcl11a is required for neuronal morphogenesis and sensory circuit formation in dorsal spinal cord development.
Development
2012
, vol. 
139
 
10
(pg. 
1831
-
1841
)
128
Kuo
 
TY
Chen
 
CY
Hsueh
 
YP
Bcl11A/CTIP1 mediates the effect of the glutamate receptor on axon branching and dendrite outgrowth.
J Neurochem
2010
, vol. 
114
 
5
(pg. 
1381
-
1392
)
129
Benitez
 
CM
Qu
 
K
Sugiyama
 
T
, et al. 
An integrated cell purification and genomics strategy reveals multiple regulators of pancreas development.
PLoS Genet
2014
, vol. 
10
 
10
pg. 
e1004645
 
130
Khaled
 
WT
Choon Lee
 
S
Stingl
 
J
, et al. 
BCL11A is a triple-negative breast cancer gene with critical functions in stem and progenitor cells.
Nat Commun
2015
, vol. 
6
 pg. 
5987
 
131
Liu
 
P
Keller
 
JR
Ortiz
 
M
, et al. 
Bcl11a is essential for normal lymphoid development.
Nat Immunol
2003
, vol. 
4
 
6
(pg. 
525
-
532
)
132
Yu
 
Y
Wang
 
J
Khaled
 
W
, et al. 
Bcl11a is essential for lymphoid development and negatively regulates p53.
J Exp Med
2012
, vol. 
209
 
13
(pg. 
2467
-
2483
)
133
Lin
 
Y
Zhang
 
Q
Zhang
 
HM
, et al. 
Transcription factor and miRNA co-regulatory network reveals shared and specific regulators in the development of B cell and T cell.
Sci Rep
2015
, vol. 
5
 pg. 
15215
 
134
Powers
 
AN
Satija
 
R
Single-cell analysis reveals key roles for Bcl11a in regulating stem cell fate decisions.
Genome Biol
2015
, vol. 
16
 pg. 
199
 
135
Tsang
 
JCH
Yu
 
Y
Burke
 
S
, et al. 
Single-cell transcriptomic reconstruction reveals cell cycle and multi-lineage differentiation defects in Bcl11a-deficient hematopoietic stem cells.
Genome Biol
2015
, vol. 
16
 
1
pg. 
178
 
136
Guda
 
S
Brendel
 
C
Renella
 
R
, et al. 
miRNA-embedded shRNAs for lineage-specific BCL11A knockdown and hemoglobin F induction.
Mol Ther
2015
, vol. 
23
 
9
(pg. 
1465
-
1474
)
137
Howe
 
SJ
Mansour
 
MR
Schwarzwaelder
 
K
, et al. 
Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients.
J Clin Invest
2008
, vol. 
118
 
9
(pg. 
3143
-
3150
)
138
Canver
 
MC
Smith
 
EC
Sher
 
F
, et al. 
BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis.
Nature
2015
, vol. 
527
 
7577
(pg. 
192
-
197
)
139
Vierstra
 
J
Reik
 
A
Chang
 
K-H
, et al. 
Functional footprinting of regulatory DNA.
Nat Methods
2015
, vol. 
12
 
10
(pg. 
927
-
930
)
140
Mandal
 
PK
Ferreira
 
LMR
Collins
 
R
, et al. 
Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9.
Cell Stem Cell
2014
, vol. 
15
 
5
(pg. 
643
-
652
)
141
Masuda
 
T
Wang
 
X
Maeda
 
M
, et al. 
Transcription factors LRF and BCL11A independently repress expression of fetal hemoglobin.
Science
2016
, vol. 
351
 
6270
(pg. 
285
-
289
)
142
Maeda
 
T
Ito
 
K
Merghoub
 
T
, et al. 
LRF is an essential downstream target of GATA1 in erythroid development and regulates BIM-dependent apoptosis.
Dev Cell
2009
, vol. 
17
 
4
(pg. 
527
-
540
)
143
Lunardi
 
A
Guarnerio
 
J
Wang
 
G
Maeda
 
T
Pandolfi
 
PP
Role of LRF/Pokemon in lineage fate decisions.
Blood
2013
, vol. 
121
 
15
(pg. 
2845
-
2853
)
144
Musallam
 
KM
Sankaran
 
VG
Cappellini
 
MD
Duca
 
L
Nathan
 
DG
Taher
 
AT
Fetal hemoglobin levels and morbidity in untransfused patients with β-thalassemia intermedia.
Blood
2012
, vol. 
119
 
2
(pg. 
364
-
367
)
145
Wilber
 
A
Hargrove
 
PW
Kim
 
Y-S
, et al. 
Therapeutic levels of fetal hemoglobin in erythroid progeny of β-thalassemic CD34+ cells after lentiviral vector-mediated gene transfer.
Blood
2011
, vol. 
117
 
10
(pg. 
2817
-
2826
)
146
Mettananda
 
S
Gibbons
 
RJ
Higgs
 
DR
 
α-Globin as a molecular target in the treatment of β-thalassemia. Blood. 2015;125(24):3694-3701
147
Kan
 
YW
Nathan
 
DG
Mild thalassemia: the result of interactions of alpha and beta thalassemia genes.
J Clin Invest
1970
, vol. 
49
 
4
(pg. 
635
-
642
)
148
Thein
 
SL
Genetic modifiers of the β-haemoglobinopathies.
Br J Haematol
2008
, vol. 
141
 
3
(pg. 
357
-
366
)
149
Renneville
 
A
Van Galen
 
P
Canver
 
MC
, et al. 
EHMT1 and EHMT2 inhibition induces fetal hemoglobin expression.
Blood
2015
, vol. 
126
 
16
(pg. 
1930
-
1939
)
150
Krivega
 
I
Byrnes
 
C
de Vasconcellos
 
JF
, et al. 
Inhibition of G9a methyltransferase stimulates fetal hemoglobin production by facilitating LCR/γ-globin looping.
Blood
2015
, vol. 
126
 
5
(pg. 
665
-
672
)
151
Lee
 
YT
de Vasconcellos
 
JF
Yuan
 
J
, et al. 
LIN28B-mediated expression of fetal hemoglobin and production of fetal-like erythrocytes from adult human erythroblasts ex vivo.
Blood
2013
, vol. 
122
 
6
(pg. 
1034
-
1041
)
152
Hendel
 
A
Bak
 
RO
Clark
 
JT
, et al. 
Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells.
Nat Biotechnol
2015
, vol. 
33
 
9
(pg. 
985
-
989
)
153
Nienhuis
 
AW
Persons
 
DA
Development of gene therapy for thalassemia.
Cold Spring Harb Perspect Med
2012
, vol. 
2
 
11
pg. 
a011833
 
154
Nienhuis
 
AW
Development of gene therapy for blood disorders: an update.
Blood
2013
, vol. 
122
 
9
(pg. 
1556
-
1564
)
155
Papapetrou
 
EP
Zoumbos
 
NC
Athanassiadou
 
A
Genetic modification of hematopoietic stem cells with nonviral systems: past progress and future prospects.
Gene Ther
2005
, vol. 
12
 
suppl 1
(pg. 
S118
-
S130
)
156
Urnov
 
FD
Reik
 
A
Vierstra
 
J
, et al. 
Clinical-scale genome editing of the human BCL11A erythroid enhancer for treatment of the hemoglobinopathies [abstract].
Blood
2015
, vol. 
126
 
23
 
Abstract 204
157
Buechele
 
C
Breese
 
EH
Schneidawind
 
D
, et al. 
MLL leukemia induction by genome editing of human CD34+ hematopoietic cells.
Blood
2015
, vol. 
126
 
14
(pg. 
1683
-
1694
)
158
Lucarelli
 
G
Isgrò
 
A
Sodani
 
P
Gaziev
 
J
Hematopoietic stem cell transplantation in thalassemia and sickle cell anemia.
Cold Spring Harb Perspect Med
2012
, vol. 
2
 
5
pg. 
a011825
 
159
Cupit
 
MC
Duncan
 
C
Savani
 
BN
Hashmi
 
SK
Childhood to adult transition and long-term follow-up after blood and marrow transplantation.
Bone Marrow Transplant
2016
, vol. 
51
 
2
(pg. 
176
-
181
)
160
Faulkner
 
LB
Setting up low-risk bone marrow transplantation for children with thalassemia may facilitate pediatric cancer care.
South Asian J Cancer
2013
, vol. 
2
 
3
(pg. 
109
-
112
)
161
Mahmoud
 
H
El-Haddad
 
A
Fahmy
 
O
, et al. 
Hematopoietic stem cell transplantation in Egypt.
Bone Marrow Transplant
2008
, vol. 
42
 
suppl 1
(pg. 
S76
-
S80
)
162
Piel
 
FB
Hay
 
SI
Gupta
 
S
Weatherall
 
DJ
Williams
 
TN
Global burden of sickle cell anaemia in children under five, 2010-2050: modelling based on demographics, excess mortality, and interventions.
PLoS Med
2013
, vol. 
10
 
7
pg. 
e1001484
 
163
Zuccato
 
C
Breda
 
L
Salvatori
 
F
, et al. 
A combined approach for β-thalassemia based on gene therapy-mediated adult hemoglobin (HbA) production and fetal hemoglobin (HbF) induction.
Ann Hematol
2012
, vol. 
91
 
8
(pg. 
1201
-
1213
)
164
Breda
 
L
Rivella
 
S
Zuccato
 
C
Gambari
 
R
Combining gene therapy and fetal hemoglobin induction for treatment of β-thalassemia.
Expert Rev Hematol
2013
, vol. 
6
 
3
(pg. 
255
-
264
)
165
Tsai
 
SQ
Zheng
 
Z
Nguyen
 
NT
, et al. 
GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases.
Nat Biotechnol
2015
, vol. 
33
 
2
(pg. 
187
-
197
)
166
Frock
 
RL
Hu
 
J
Meyers
 
RM
Ho
 
YJ
Kii
 
E
Alt
 
FW
Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases.
Nat Biotechnol
2015
, vol. 
33
 
2
(pg. 
179
-
186
)
167
Fu
 
Y
Sander
 
JD
Reyon
 
D
Cascio
 
VM
Joung
 
JK
Improving CRISPR-Cas nuclease specificity using truncated guide RNAs.
Nat Biotechnol
2014
, vol. 
32
 
3
(pg. 
279
-
284
)
168
Slaymaker
 
IM
Gao
 
L
Zetsche
 
B
Scott
 
DA
Yan
 
WX
Zhang
 
F
Rationally engineered Cas9 nucleases with improved specificity.
Science
2016
, vol. 
351
 
6268
(pg. 
84
-
88
)
169
Kleinstiver
 
BP
Pattanayak
 
V
Prew
 
MS
, et al. 
High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.
Nature
2016
, vol. 
529
 
7587
(pg. 
490
-
495
)
170
Yannaki
 
E
Karponi
 
G
Zervou
 
F
, et al. 
Hematopoietic stem cell mobilization for gene therapy: superior mobilization by the combination of granulocyte-colony stimulating factor plus plerixafor in patients with β-thalassemia major.
Hum Gene Ther
2013
, vol. 
24
 
10
(pg. 
852
-
860
)
171
Fitzhugh
 
CD
Hsieh
 
MM
Bolan
 
CD
Saenz
 
C
Tisdale
 
JF
Granulocyte colony-stimulating factor (G-CSF) administration in individuals with sickle cell disease: time for a moratorium?
Cytotherapy
2009
, vol. 
11
 
4
(pg. 
464
-
471
)
172
Yannaki
 
E
Papayannopoulou
 
T
Jonlin
 
E
, et al. 
Hematopoietic stem cell mobilization for gene therapy of adult patients with severe β-thalassemia: results of clinical trials using G-CSF or plerixafor in splenectomized and nonsplenectomized subjects.
Mol Ther
2012
, vol. 
20
 
1
(pg. 
230
-
238
)
173
Karponi
 
G
Psatha
 
N
Lederer
 
CW
, et al. 
Plerixafor+G-CSF-mobilized CD34+ cells represent an optimal graft source for thalassemia gene therapy.
Blood
2015
, vol. 
126
 
5
(pg. 
616
-
619
)

Sign in via your Institution