"In an attempt to recapitulate the phenotype of hereditary persistence of fetal hemoglobin, we used CRISPR-Cas9 gene-editing techniques in hematopoietic stem and progenitor cells (HSPCs) at the erythroid-specific enhancer region of BCL11A to reduce BCL11A expression in erythroid-lineage cells, restore γ-globin synthesis, and reactivate production of fetal hemoglobin." The paper describes CTX001 as "autologous CRISPR-Cas9–edited CD34+ HSPCs that were genetically edited to reactivate the production of fetal hemoglobin" and notes that editing occurred at the BCL11A enhancer, not the HBB mutation itself.

Vertex Pharmaceuticals and CRISPR Therapeutics have obtained promising results in their Phase-1 clinical trial (CTX001, clinicaltrials.gov) using CRISPR/Cas9 to edit the BCL11A erythroid enhancer to induce HbF expression. Together, this work demonstrated that editing the BCL11A enhancer by CRISPR/Cas9 is a practical therapeutic strategy to produce a durable therapeutic level of HbF induction in engrafting HSCs. BCL11A is the chief regulator of HbF level and suppresses fetal hemoglobin expression…Numerous studies have validated BCL11A erythroid enhancer as a target for HbF induction, offering a framework for erythroid-specific therapeutic genome editing by targeting the core sequences of BCL11A enhancer in HSPCs.

Expression of BCL11A in erythroid cells is governed by a cell-type-specific enhancer that contains a critical GATA1 binding site. Chang et al. demonstrate that ZFN-mediated disruption of the erythroid-specific enhancer in the BCL11A gene locus by NHEJ led to increased expression of γ-globin without impairing the differentiation capacity of the edited HSPCs. These data demonstrate that reducing, but not eliminating, BCL11A expression represents a viable approach for the treatment of sickle cell disease and other β-hemoglobinopathies.

"Pre-clinical evaluation led to the opening of clinical trials evaluating CRISPR-Cas9 editing to disrupt the GATA1 binding site in the BCL11A enhancer to reactivate HbF expression in adult RBCs of patients with transfusion-dependent β-thalassemia (ClinicalTrials.gov: NCT03655678) and severe SCD (ClinicalTrials.gov: NCT03745287)." The review further states: "Current clinical trials (ClinicalTrials.gov: NCT03653247, NCT03432364, NCT03745287, and NCT03655678) are using genome-editing tools to create DSBs in the erythroid-specific BCL11A enhancer to induce HbF levels as a potential treatment option for SCD and β-thalassemia."

"Recently approved gene therapies for these disorders employ CRISPR-based genome editing to target the BCL11A enhancer in blood stem cells, achieving transformative outcomes for patients." The release explains: "In these therapies, CRISPR-Cas9 targets a regulatory DNA element, called an enhancer, which controls expression of BCL11A, a key gene responsible for switching hemoglobin production from fetal to adult forms… As a result, BCL11A is silenced, leading to reactivation of fetal hemoglobin, which compensates for defective adult hemoglobin in sickle cell disease and β-thalassemia."

To date, gene therapies for SCD have been based on either gene addition or gene editing approaches to induce fetal hemoglobin (HbF) (for example by targeting transcriptional regulators such as BCL11A, as shown for the Casgevy gene therapy), or by downregulating the expression of BCL11A, a major HbF transcriptional repressor, or to revert the SCD-causing mutation. Importantly, these DSB-free strategies generate far fewer InDels and large chromosomal rearrangements, such as translocations and deletions. CRISPR-Cas9 treatment in healthy donor and patient-derived HSPCs resulted in a high frequency of LRF BS disruption and potent HbF synthesis in their erythroid progeny, further expanding the spectrum of HbF-based therapeutic gene-editing approaches for SCD.

CRISPR-Cas9-mediated disruption of the BCL11A erythroid enhancer results in the reduction of BCL11A expression and the induction of fetal γ-globin, which is a practicable therapeutic strategy for treating transfusion-dependent β-thalassemia (TDT). We obtained mobilized autologous CD34+ cells from 4 TDT patients sponsor initiated in phase I/II clinical trial (NCT05577312) and 6 TDT patients in investigator initiated clinical study (NCT04211480, NCT04205435) respectively. All patients were treated with Brl-101, in which the BCL11A erythroid enhancer was edited by CRISPR-Cas9.

EDIT-301 is the first experimental medicine developed using CRISPR-Cas12a gene-editing technology, and it is specifically edited using a CRISPR-Cas12a ribonucleoprotein to enhance the HBG1/2 promoter region in the beta-globin locus of patient-derived HSPCs. BIVV003 (also known as SAR445136) is an ex vivo-engineered autologous HSPC therapy that was designed to express HbF through BCL11A disruption. Specifically, CRISPR-Cas9 is used to disrupt the BCL11A gene in a patient's own haematopoietic stem cells (HSCs) ex vivo, and the engineered cells are then returned to the patient.

Discussing the Casgevy therapy, the article states: "The newly-approved Casgevy gene therapy restarts fetal hemoglobin production by using CRISPR gene editing to dial down a gene called BCL11A." It notes that Orkin and Bauer "find a region of DNA within the BCL11A gene, a so-called 'enhancer' region, that is required for production of BCL11A, but only in red blood cells, providing a safer target for inducing fetal hemoglobin production" and that the treatment "uses CRISPR-Cas9 to edit the BCL11A enhancer at a particularly vulnerable site."

In this study, we compared 3 gene therapy approaches: CRISPR-Cas9 editing of the BCL11A enhancer to reactivate fetal hemoglobin (HbF), lentiviral addition of a β-globin gene, and CRISPR-Cas9 correction of the sickle mutation in HBB. The BCL11A enhancer editing strategy, similar to that used in the Casgevy trial, resulted in robust HbF induction and effective amelioration of the sickling phenotype. By contrast, direct correction of the HBB mutation required precise homology-directed repair and showed lower editing efficiencies, highlighting technical challenges for widespread clinical application.

This is a non-randomized, single center, open-label, pilot safety and feasibility study involving a single infusion of autologous bone marrow derived CD34+ hematopoietic stem cells (HSPCs) electroporated with BCL11A enhancer targeting Cas9 ribonucleoprotein. Accrual will be a maximum of 14 evaluable subjects with sickle cell disease (SCD) or β-thalassemia. The study aims to edit the BCL11A enhancer in HSPCs to increase fetal hemoglobin as a treatment strategy.

"BCL11A gene is a transcription factor that has been shown to repress fetal hemoglobin expression. The function of BCL11A serves as the molecular switch for transitioning fetal hemoglobin (HbF) to beta hemoglobin also referred to as adult hemoglobin (HbA). Thus, knocking out BCL11A increases HbF and represses the sickle hemoglobin." The article describes CTX001: "CTX001 is an autologous, ex vivo CRISPR- Cas9 gene editing therapy which successfully induces HbF in the RBCs using the patient’s hematopoietic stem cells… The discovery of an enhancer region of BCL11A specific to the erythroid lineage allowed researchers to design guide RNAs with a target sequence for BCL11A."

And so Casgevy doesn’t work by correcting the disease-causing, single base-pair mutation in the β-hemoglobin gene directly. Instead, this particular gene therapy is a bank shot: CRISPR is used to reactivate a different hemoglobin gene, fetal γ-hemoglobin…The Casgevy strategy is to switch the fetal γ-hemoglobin genes back on by switching off another gene, BCL11A, which represses fetal γ-hemoglobin. Casgevy does this via CRISPR deletion of a regulatory DNA region (called an “enhancer”) that controls the expression of BCL11A.

Bone marrow stem cells are extracted from patients and CRISPR-edited to inactivate BCL11A, a repressor of fetal hemoglobin production. The second approach to CRISPR sickle cell gene therapy involves a gene knockout, switching off the gene that suppresses fetal hemoglobin… This method causes fetal hemoglobin (hemoglobin F) to be expressed, replacing the mutated adult hemoglobin. GPH101 uses gene editing to correct the mutation present in the HBB gene of sickle cell disease patients and restore normal hemoglobin expression… In early 2023, Graphite Bio voluntarily paused GPH101 the phase I/II clinical trials because patients developed adverse side effects. NCT04774536 | CRISPR_SCD001 | … replacing mutated beta-globin gene via CRISPR-Cas9 knock-in… NCT04819841 (CEDAR Trial) | GPH101 | … correcting the mutation in HBB to restore normal hemoglobin expression.

Reporting on CTX001 early clinical data, the article explains: "CTX001 is designed to suppress BCL11A, which results in an upregulation of fetal hemoglobin (HbF)… elevated HbF is associated with reduced morbidity and mortality in patients with sickle cell disease and β-thalassemia." It describes the approach as editing hematopoietic stem cells to alter BCL11A regulation, rather than correcting the β-globin mutation directly.

Similarly, a different research study reinstated HbF expression in rhesus macaques by disrupting the erythroid-specific BCL11A enhancer using CRISPR-Cas9. The therapeutic strategy involved targeting the BCL11A enhancer to decrease BCL11A expression, thereby increasing fetal hemoglobin levels to ameliorate sickle cell disease manifestations. These approaches focus on reactivating HbF rather than directly correcting the causative mutation in the HBB gene.

This review notes that "BCL11A is a critical silencer of γ-globin and fetal hemoglobin in adult erythroid cells" and that "a common genetic variant in an erythroid-specific enhancer of BCL11A is associated with elevated HbF and reduced severity of sickle cell disease." It further highlights that therapeutic CRISPR strategies "target the enhancer of BCL11A" to disrupt its function, thereby reactivating fetal hemoglobin, as an alternative to directly repairing HBB mutations.

In this experimental study, the authors write: "Here, we performed single-gene editing and multiplex gene editing via CRISPR/Cas9 technology to edit BCL11A erythroid-specific enhancer and BCL11A binding site in the HBG promoter." They report that "CRISPR/Cas9-based multiplex genome editing of BCL11A and HBG efficiently induces fetal hemoglobin expression," illustrating a strategy focused on regulatory elements controlling HbF rather than on correcting HBB mutations.

The process works by disrupting an enhancer sequence within BCL11A, a key regulator of the fetal-to-adult haemoglobin switch. By targeting this erythroid-specific enhancer in haematopoietic stem and progenitor cells, exagamglogene autotemcel (exa-cel, Casgevy) reactivates fetal haemoglobin, which can compensate for the defective adult haemoglobin characteristic of sickle cell disease. This indirect editing approach, sometimes described as ‘switch editing’, differs from strategies that attempt to repair the HBB mutation directly.

Studies revealed the gene that would hold the ticket: BCL11A. Sankaran and Orkin showed that BCL11A suppresses production of fetal hemoglobin. Targeting this gene or its regulatory elements could potentially reactivate fetal hemoglobin in adults with sickle cell disease, providing an alternative to directly repairing the mutation in the β-globin gene. This insight underpins the development of the CRISPR-based therapy that edits the BCL11A enhancer in hematopoietic stem cells.

This study is being done to test the safety of a new treatment called gene editing in Sickle Cell Disease (SCD) patients. Intervention: Gene Editing… Genetic: Gene Editing for SCD – This experimental therapy aims to correct the pathogenic variant in the HBB gene in autologous hematopoietic stem cells using CRISPR-based technology, with the goal of restoring production of normal adult hemoglobin. Phase 1/2 study will assess feasibility, safety, and preliminary efficacy of direct HBB repair compared to existing HbF-induction strategies.

Hanna and colleagues evaluated renizgamglogene autogedtemcel (reni-cel), an autologous hematopoietic stem-cell therapy engineered using AsCas12a to disrupt BCL11A binding sites within the HBG1 and HBG2 promoters. By targeting the γ-globin promoters directly, reni-cel operates at the most proximal regulatory node of HbF repression…CRISPR-Cas12a–mediated disruption of BCL11A binding at the HBG1 and HBG2 promoters produces deep, durable HbF reactivation—normalizing hemoglobin and effectively eliminating vaso-occlusive events in the vast majority of patients. Direct promoter editing offers a more proximal, mechanistically elegant solution: disable the switch itself, rather than modulating its regulator.

This research article states: "Targeted deletion of a 200bp region in BCL11A enhances γ-globin expression in K562 cells. CRISPR-Cas9 effectively induces fetal hemoglobin reactivation… In our study, deletion of 200bp of BCL11A erythroid enhancer including GATAA motif leads to strong induction of γ-hemoglobin expression in K562 cells." The authors conclude that "The targeted deletion of BCL11A gene can significantly induce γ-globin expression, providing a promising therapeutic strategy for β-thalassemia," supporting the concept of enhancer-targeting to reactivate HbF.

Two people with beta-thalassemia and one with sickle cell disease no longer require blood transfusions… after their bone marrow stem cells were gene-edited with CRISPR. This new FDA approval comes after CRISPR-Cas9 gene-editing therapy was approved to treat patients with sickle cell disease, in December. The approved therapy uses CRISPR to edit the BCL11A enhancer in patients’ hematopoietic stem cells to increase fetal hemoglobin production, rather than directly modifying the HBB mutation.

This is a non-randomized, single center, open-label, pilot safety and feasibility study involving a single infusion of autologous bone marrow derived CD34+ hematopoietic stem cells (HSPCs) electroporated with BCL11A enhancer targeting Cas9 ribonucleoprotein. The study will enroll 7 evaluable subjects within each disease group: SCD and β-thalassemia. The edited cells are intended to increase fetal hemoglobin via BCL11A enhancer disruption rather than directly altering the HBB gene.

A description of an ongoing trial notes: "Combining CRISPR-Cas9 editing of the BCL11A enhancers significantly enhances fetal hemoglobin (HbF) induction, which is crucial for treating β-hemoglobinopathies like sickle cell disease and β-thalassemia." It explains that "This gene therapy stands out by directly modifying the BCL11A gene enhancer, which could lead to the production of healthier red blood cells by reactivating fetal hemoglobin production… one study found that 86% of blood stem cells were successfully edited, resulting in at least 40% fetal hemoglobin in patients after six months."

From background knowledge on current gene therapy approaches: the CRISPR-based sickle cell therapy Casgevy/CTX001 (developed by CRISPR Therapeutics and Vertex) and the similar exagamglogene autotemcel (exa-cel) use ex vivo CRISPR-Cas9 editing of the erythroid-specific BCL11A enhancer in patients’ hematopoietic stem cells to downregulate BCL11A and reactivate fetal hemoglobin (HbF), rather than repairing the underlying Glu6Val mutation in the HBB gene. These strategies mimic hereditary persistence of fetal hemoglobin and are the dominant CRISPR clinical trial approach for sickle cell disease to date.