Engineering of the endogenous HBD promoter increases HbA2
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eLife assessment
This study presents the important finding that gene editing could be used to activate delta-globin expression to treat disorders of red blood cell synthesis. The evidence supporting the claims of the authors is solid, particularly in the immortalized cell lines, although inclusion of a larger number of donor patient samples may have strengthened the conclusions that were able to be drawn from the primary cell experiments. The data show this approach to have promise and identify avenues of effort that could be pursued to advance it to a clinical strategy for hemoglobinopathy treatment.
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Abstract
The β-hemoglobinopathies, such as sickle cell disease and β-thalassemia, are one of the most common genetic diseases worldwide and are caused by mutations affecting the structure or production of β-globin subunits in adult hemoglobin. Many gene editing efforts to treat the β-hemoglobinopathies attempt to correct β-globin mutations or increase γ-globin for fetal hemoglobin production. δ-globin, the subunit of adult hemoglobin A2, has high homology to β-globin and is already pan-cellularly expressed at low levels in adult red blood cells. However, upregulation of δ-globin is a relatively unexplored avenue to increase the amount of functional hemoglobin. Here, we use CRISPR-Cas9 to repair non-functional transcriptional elements in the endogenous promoter region of δ-globin to increase overall expression of adult hemoglobin 2 (HbA2). We find that insertion of a KLF1 site alone is insufficient to upregulate δ-globin. Instead, multiple transcription factor elements are necessary for robust upregulation of δ-globin from the endogenous locus. Promoter edited HUDEP-2 immortalized erythroid progenitor cells exhibit striking increases of HBD transcript, from less than 5% to over 20% of total β-like globins in clonal populations. Edited CD34 +hematopoietic stem and progenitors (HSPCs) differentiated to primary human erythroblasts express up to 46% HBD in clonal populations. These findings add mechanistic insight to globin gene regulation and offer a new therapeutic avenue to treat β-hemoglobinopathies.
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Author Response
Reviewer #2 (Public Review):
Targeted genetic engineering with programmable nucleases and other targetable enzymes (aka "genome editing") has emerged as a technology with curative potential in hemoglobinopathies, sickle cell disease, and beta-thalassemia. Multiple ongoing clinical trials are evaluating such editing using distinct approaches: elevation of fetal hemoglobin (HbF), direct repair of the mutation causing SCD, and engineering of a Hb variant. The present work explores a different strategy: the targeted engineering of the promoter of a paralog of adult beta-globin known as HBD. This is a timely effort because there has emerged, over the past decade, a clear and charted path for advancing any such approach to human clinical trials. The study identifies three transcription factor binding sites as divergent in …
Author Response
Reviewer #2 (Public Review):
Targeted genetic engineering with programmable nucleases and other targetable enzymes (aka "genome editing") has emerged as a technology with curative potential in hemoglobinopathies, sickle cell disease, and beta-thalassemia. Multiple ongoing clinical trials are evaluating such editing using distinct approaches: elevation of fetal hemoglobin (HbF), direct repair of the mutation causing SCD, and engineering of a Hb variant. The present work explores a different strategy: the targeted engineering of the promoter of a paralog of adult beta-globin known as HBD. This is a timely effort because there has emerged, over the past decade, a clear and charted path for advancing any such approach to human clinical trials. The study identifies three transcription factor binding sites as divergent in the HBD promoter vs the HBB one. A homology-directed repair (HDR)-based scheme using oligonucleotide repair templates in combination with a CRISPR-Cas9-induced double-strand break (DSB) is designed and used to generate pools of human immortalized cells bearing one, two, or all three such de novo introduced TF binding sites at the HBD promoter. Only the latter scheme is shown to measurably increase HBD (following erythroid differentiation) in pools of cells and single-cell-derived clones as gauged by qPCR and HPLC. A similar analysis is performed on pools of erythroid-like cells generated from genome-edited human hematopoietic stem and progenitor cells (HSPCs), as well as genetically clonal erythroid colonies bearing the edits of interest; trends in these data support the observations made on the immortalized cells. Overall the data support the notion that HBD promoter genome editing has the potential as a strategy to normalize hemoglobin synthesis in hemoglobinopathies. Further, the data support an advance of this approach down a well-established path of preclinical development in such cases: increasing the efficiency of genome editing in HSPCs to what would be deemed therapeutically useful, assessing the genotoxic burden from the editing, evaluating the potential negative impact on stemness, and determining whether this approach would normalize hemoglobin synthesis in the erythroid progeny of patient HSPCs.
We thank reviewer 2 for their input on our manuscript, especially sharing their insight from a clinical path perspective.
The genome editing scheme for the "KDT" strategy in Fig 1B involves the introduction of three binding sites for transcription factors at progressively increasing distances from the site of the DSB induced by Cas9. It would be of interest to determine from the next-generation-sequencing data whether partial gene conversion tracks are observed at the edited locus (Elliott and Jasin MCB 18: 93), and if yes, whether these affect in some way the pool-level measurement by qPCR on HBD mRNA levels (Fig 1D).
For the analysis of our NGS reads, we utilized the CRISPResso2 analysis pipeline. After CRISPResso2 aligns the reads and makes allelic calls of either unmodified, NHEJ, or HDR. It is important to note that the KDT of our HBD knock-in construct, is not identical to the HBB promoter. Through simple searching of the CRISPResso aligned-reads, we did not find any HBB promoter sequence present. In this regard, our CRISPResso analysis does not seem to find any gene-conversions between HBB and HBD. However, we cannot rule out the possibility of gene conversions altogether – it can be that since our primers for NGS anneal specifically to HBD, we are unable to amplify, and therefore unable to see, the alleles in which these gene conversion events occurred.
The data in Fig 2A show an analysis of transcription factor and RNA pol II occupancy following genome editing at HBD. The figure legend refers to these data as having been obtained on single-cell-derived clones bearing the edits in homozygous or heterozygous form, but it is unclear from fig 2A, which clones were used for which analysis.
We have now clarified this point in the figure legend.
The data in Fig 3C present an analysis of HBD levels in erythroid colonies derived from genome-edited HSPCs. It would be helpful to clarify whether an individual dot represents a single such colony (this would seem to be the case from the cognate figure legend). If so, what number of such colonies would one need to obtain to gain a clearer sense of the effect on HBD levels from the various genome editing strategies used?
Indeed, each dot represents a singular colony. We have now expanded this dataset from colonies derived from n=2 HSPC donors to n=4 HSPC donors. Figure 3C and Supp Fig 3 have been updated accordingly.
It would be helpful to comment, in the Discussion, on potential genome editing strategies to obtain high-efficiency pool-level uniform long-track gene conversion that is necessary to obtain high HBD levels in the progeny of edited CD34 cells. Would this be a good application of the AAV6 strategy developed by the Sangamo and Porteus groups? Would prime editing as developed by Liu be an option here?
Prime editing can introduce small insertions, but still has limitations of low-editing efficiency (https://doi.org/10.1016/j.tibtech.2023.03.004). Additionally, our KDT construct would require a larger insertion that prime editing would not be able to facilitate easily. In light of the adverse effects using AAV6 for biotech company Graphite Bio, we will not suggest this in the discussion.
It would be equally helpful, in the Discussion, to place the level of HbA2 obtained via the strategy shown in the manuscript in the context of other genome-editing-based approaches for normalizing Hb synthesis in the hemoglobinopathies (ie HbF elevation by editing the BCL11A enhancer, or the gammaglobin promoter; or direct repair of the SCD mutation; or engineering of Hb Makassar).
We have now added a new section in the discussion summarizing some of the recent genome editing approaches for hemoglobinopathies. Specifically, we mention CRISPR Therapeutics’ clinical trial on the BCL11A enhancer, David Liu’s most recent paper on base-editing to correct the SCD mutation, and Annarita Miccio’s recent paper on disrupting a repressor binding site on the gamma-globin promoter.
Reviewer #3 (Public Review):
This is a well-written and referenced paper from the laboratory of an outstanding senior investigator. Dr. Corn and colleagues demonstrate convincingly that correction of three transcription factor binding sites in the delta-globin gene promoter results in high levels of delta-globin expression in HUDEP-2 clonal cell populations (Fig. 2B and C) and in CD34+ HSPC (hematopoietic stem and progenitor cells) clonal cell expansions (Fig. 3C). Although correction of the mutant KLF1 binding site has previously been shown to upregulate delta-globin gene transgenes, this new data demonstrate that correction of multiple factor binding sites is required to achieve high-level expression of the delta-globin gene in the endogenous beta-globin gene locus. The results are important because high delta-globin protein levels inhibit the formation of sickle hemoglobin (HbS) polymers that cause sickle cell disease.
We thank reviewer 3 for their feedback on our manuscript.
Unfortunately, high levels of delta-globin gene expression were not observed after editing of pooled (non-clonal) populations of HUDEP-2 cells (Fig. 1D) or CD34+ HSPC pooled cell populations (Fig. 3B). This result suggests that correction of all 3 promoter elements on individual alleles in CD34+ HSPC populations is far below the level required to be clinically relevant.
We have added to the discussion on ways to improve HDR efficiency. Additionally, we show new data where we utilize an HDR enhancer drug and show that we can increase HDR and overall HBD in edited pooled populations of HSPCs (Fig 3 C and D)
Also, NHEJ is high in CD34+ HSPC (Fig. 3A); therefore, promoter deletions will inactivate many alleles, and total hemoglobin levels in erythrocytes derived from populations of edited CD34+ HSPC will be much less than normal (29 pg/cell). These cells would be extremely beta-thalassemic.
We were not completely sure about the origin of this point, since our edits are aimed at HBD, which makes up less than 5% of total hemoglobins under normal conditions. NHEJ occurring in HBB (e.g. when doing HDR for direct correction) would potentially yield thalassemic cells. But indels in the HBD promoter might at most cause a 5% decrease in total globin levels (if delta expression was completely destroyed). We have performed a new experiment to explicitly address this point. We edited n=4 CD34+ HSPCs donors and compared unedited populations to populations edited with Cas9+HBD gRNA but no repair template. This represents a “worst case” scenario, in which there can be no HDR-based promoter engineering and only NHEJ. These data are included this in Supplementary Figure 3. We observed high editing efficiency of 61 – 78% in the HBD promoter. We performed qRT-PCR of the beta-like globins in edited pools and normalized to HBA, reasoning that HBA is a neutral control for absolute levels of each globin in the beta locus because HBA is located in a different locus. By qRT-PCR, HBD transcripts were decreased by half compared to mock treated cells, while HBB and HBG1/2 were non-significantly affected. But as mentioned above, HBD expression makes up less than 5% of total hemoglobins, and therefore a half reduction in HBD represents a total reduction of 2.5% of globins. We do acknowledge that this experiment does not specifically quantify the rates of large deletions that might span from delta to beta, and further studies would be needed to address this point. But if such large deletions do exist, they do not greatly affect beta expression. We have included this in the results and the discussion section.
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eLife assessment
This study presents the important finding that gene editing could be used to activate delta-globin expression to treat disorders of red blood cell synthesis. The evidence supporting the claims of the authors is solid, particularly in the immortalized cell lines, although inclusion of a larger number of donor patient samples may have strengthened the conclusions that were able to be drawn from the primary cell experiments. The data show this approach to have promise and identify avenues of effort that could be pursued to advance it to a clinical strategy for hemoglobinopathy treatment.
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**Reviewer #1 (Public Review):
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Beta-hemoglobinopathies, such as sickle cell disease and beta-thalassemia, are common and debilitating genetic diseases caused by mutations in the adult beta-globin gene. Many in the field are pursuing various strategies to therapeutically upregulate fetal gamma-globin to treat these diseases. In this paper, the authors aimed to instead edit the promoter of the delta-globin gene to cause upregulation of delta-globin expression. Delta-globin is highly homologous to adult beta-globin and is pan-cellularly expressed in adult red blood cells, albeit at low levels due to the low activity of its promoter. Gene editing to activate the promoter of delta-globin could allow delta-globin expression to be elevated which could compensate for defective beta-globin in patients with beta-hemoglobinopathies. This is an …**Reviewer #1 (Public Review):
**
Beta-hemoglobinopathies, such as sickle cell disease and beta-thalassemia, are common and debilitating genetic diseases caused by mutations in the adult beta-globin gene. Many in the field are pursuing various strategies to therapeutically upregulate fetal gamma-globin to treat these diseases. In this paper, the authors aimed to instead edit the promoter of the delta-globin gene to cause upregulation of delta-globin expression. Delta-globin is highly homologous to adult beta-globin and is pan-cellularly expressed in adult red blood cells, albeit at low levels due to the low activity of its promoter. Gene editing to activate the promoter of delta-globin could allow delta-globin expression to be elevated which could compensate for defective beta-globin in patients with beta-hemoglobinopathies. This is an underexplored and very interesting approach, and this study represents the first time that delta-globin upregulation has been attempted using gene editing in adult-like human erythroid immortalised and primary cells.The first major finding from this study was that gene editing to insert KLF1, beta-DRF, and TFIIb sites into the delta-globin promoter was sufficient to cause upregulation of delta-globin expression at the mRNA and protein levels in immortalized HUDEP-2 cells. Modest upregulation was seen in pooled populations of HUDEP-2 cells (where ~25% of cells were HDR edited). Robust expression of delta-globin was seen in homozygously edited clonal populations of HUDEP-2 cells, with delta-globin constituting ~25% of total beta-like globin expression at the mRNA level in these cells. The results presented thus strongly support this finding.
The second major finding was that gene editing to insert KLF1, beta-DRF, and TFIIb sites into the delta globin promoter was sufficient to cause upregulation of delta-globin in primary human CD34+ cells. Despite HDR editing efficiencies of ~25% in these primary cells, and possibly due to only two donor cell populations being used, significant upregulation of delta-globin was not detected in pooled populations of edited primary CD34+ cells. Encouraging evidence of upregulation was seen in the clonal population of edited cells from the two donors. As such the results provide moderate support for this finding.
In combination, the HUDEP-2 cell and CD34+ cell data provide compelling evidence that gene editing of the delta-globin promoter is a promising line of enquiry for the treatment of beta-hemoglobinopathies.
This important study establishes and provides a proof-of-principle for this alternative therapeutic approach for those with beta-hemoglobinopathies. Future studies based on this work may enable delta-globin to be upregulated to therapeutically relevant levels in patient cells, including in cells from patients with beta-hemoglobinopathies. The therapeutic benefits of delta-globin upregulation will then be able to be assessed. This finding will be of interest to those in the globin switching and gene editing fields.
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Reviewer #2 (Public Review):
Targeted genetic engineering with programmable nucleases and other targetable enzymes (aka "genome editing") has emerged as a technology with curative potential in hemoglobinopathies, sickle cell disease, and beta-thalassemia. Multiple ongoing clinical trials are evaluating such editing using distinct approaches: elevation of fetal hemoglobin (HbF), direct repair of the mutation causing SCD, and engineering of a Hb variant. The present work explores a different strategy: the targeted engineering of the promoter of a paralog of adult beta-globin known as HBD. This is a timely effort because there has emerged, over the past decade, a clear and charted path for advancing any such approach to human clinical trials. The study identifies three transcription factor binding sites as divergent in the HBD promoter vs …
Reviewer #2 (Public Review):
Targeted genetic engineering with programmable nucleases and other targetable enzymes (aka "genome editing") has emerged as a technology with curative potential in hemoglobinopathies, sickle cell disease, and beta-thalassemia. Multiple ongoing clinical trials are evaluating such editing using distinct approaches: elevation of fetal hemoglobin (HbF), direct repair of the mutation causing SCD, and engineering of a Hb variant. The present work explores a different strategy: the targeted engineering of the promoter of a paralog of adult beta-globin known as HBD. This is a timely effort because there has emerged, over the past decade, a clear and charted path for advancing any such approach to human clinical trials. The study identifies three transcription factor binding sites as divergent in the HBD promoter vs the HBB one. A homology-directed repair (HDR)-based scheme using oligonucleotide repair templates in combination with a CRISPR-Cas9-induced double-strand break (DSB) is designed and used to generate pools of human immortalized cells bearing one, two, or all three such de novo introduced TF binding sites at the HBD promoter. Only the latter scheme is shown to measurably increase HBD (following erythroid differentiation) in pools of cells and single-cell-derived clones as gauged by qPCR and HPLC. A similar analysis is performed on pools of erythroid-like cells generated from genome-edited human hematopoietic stem and progenitor cells (HSPCs), as well as genetically clonal erythroid colonies bearing the edits of interest; trends in these data support the observations made on the immortalized cells. Overall the data support the notion that HBD promoter genome editing has the potential as a strategy to normalize hemoglobin synthesis in hemoglobinopathies. Further, the data support an advance of this approach down a well-established path of preclinical development in such cases: increasing the efficiency of genome editing in HSPCs to what would be deemed therapeutically useful, assessing the genotoxic burden from the editing, evaluating the potential negative impact on stemness, and determining whether this approach would normalize hemoglobin synthesis in the erythroid progeny of patient HSPCs.
The genome editing scheme for the "KDT" strategy in Fig 1B involves the introduction of three binding sites for transcription factors at progressively increasing distances from the site of the DSB induced by Cas9. It would be of interest to determine from the next-generation-sequencing data whether partial gene conversion tracks are observed at the edited locus (Elliott and Jasin MCB 18: 93), and if yes, whether these affect in some way the pool-level measurement by qPCR on HBD mRNA levels (Fig 1D).
The data in Fig 2A show an analysis of transcription factor and RNA pol II occupancy following genome editing at HBD. The figure legend refers to these data as having been obtained on single-cell-derived clones bearing the edits in homozygous or heterozygous form, but it is unclear from fig 2A, which clones were used for which analysis.
The data in Fig 3C present an analysis of HBD levels in erythroid colonies derived from genome-edited HSPCs. It would be helpful to clarify whether an individual dot represents a single such colony (this would seem to be the case from the cognate figure legend). If so, what number of such colonies would one need to obtain to gain a clearer sense of the effect on HBD levels from the various genome editing strategies used?
It would be helpful to comment, in the Discussion, on potential genome editing strategies to obtain high-efficiency pool-level uniform long-track gene conversion that is necessary to obtain high HBD levels in the progeny of edited CD34 cells. Would this be a good application of the AAV6 strategy developed by the Sangamo and Porteus groups? Would prime editing as developed by Liu be an option here?
It would be equally helpful, in the Discussion, to place the level of HbA2 obtained via the strategy shown in the manuscript in the context of other genome-editing-based approaches for normalizing Hb synthesis in the hemoglobinopathies (ie HbF elevation by editing the BCL11A enhancer, or the gamma-globin promoter; or direct repair of the SCD mutation; or engineering of Hb Makassar).
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Reviewer #3 (Public Review):
This is a well-written and referenced paper from the laboratory of an outstanding senior investigator. Dr. Corn and colleagues demonstrate convincingly that correction of three transcription factor binding sites in the delta-globin gene promoter results in high levels of delta-globin expression in HUDEP-2 clonal cell populations (Fig. 2B and C) and in CD34+ HSPC (hematopoietic stem and progenitor cells) clonal cell expansions (Fig. 3C). Although correction of the mutant KLF1 binding site has previously been shown to upregulate delta-globin gene transgenes, this new data demonstrate that correction of multiple factor binding sites is required to achieve high-level expression of the delta-globin gene in the endogenous beta-globin gene locus. The results are important because high delta-globin protein levels …
Reviewer #3 (Public Review):
This is a well-written and referenced paper from the laboratory of an outstanding senior investigator. Dr. Corn and colleagues demonstrate convincingly that correction of three transcription factor binding sites in the delta-globin gene promoter results in high levels of delta-globin expression in HUDEP-2 clonal cell populations (Fig. 2B and C) and in CD34+ HSPC (hematopoietic stem and progenitor cells) clonal cell expansions (Fig. 3C). Although correction of the mutant KLF1 binding site has previously been shown to upregulate delta-globin gene transgenes, this new data demonstrate that correction of multiple factor binding sites is required to achieve high-level expression of the delta-globin gene in the endogenous beta-globin gene locus. The results are important because high delta-globin protein levels inhibit the formation of sickle hemoglobin (HbS) polymers that cause sickle cell disease.
Unfortunately, high levels of delta-globin gene expression were not observed after editing of pooled (non-clonal) populations of HUDEP-2 cells (Fig. 1D) or CD34+ HSPC pooled cell populations (Fig. 3B). This result suggests that correction of all 3 promoter elements on individual alleles in CD34+ HSPC populations is far below the level required to be clinically relevant. Also, NHEJ is high in CD34+ HSPC (Fig. 3A); therefore, promoter deletions will inactivate many alleles, and total hemoglobin levels in erythrocytes derived from populations of edited CD34+ HSPC will be much less than normal (29 pg/cell). These cells would be extremely beta-thalassemic.
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