Combined BCL11A Enhancer Editing and CD45-ADC Preconditioning for Long-Term HbF Induction in β-Hemoglobinopathies

β-Hemoglobinopathies, including sickle cell disease (SCD) and β-thalassemia, are prevalent monogenic disorders caused by defective β-globin synthesis. Current treatments like busulfan require lifelong use with limited efficacy, while hematopoietic stem cell transplantation is limited by donor availability. Fetal γ-globin (HbF) can compensate for β-globin defects, but its expression diminishes post-birth. This study explores reactivating HbF via BCL11A enhancer editing and CD45-ADC preconditioning for a safer, long-term gene therapy solution.

BCL11A regulates γ-globin expression through enhancers (+58, +55, +62) in its second intron. While +58 is the primary target in clinical trials, variability in HbF response necessitates multi-site editing. This study tests combined +58/+55 editing and replaces toxic chemotherapy with CD45 antibody-drug conjugates (ADC) for preconditioning, aiming for efficient, sustained HbF induction.

1. Combined BCL11A +55/+58 Enhancer Editing Efficiently Induces HbF

The study targeted BCL11A +58 and +55 enhancers using CRISPR-Cas9, comparing single-site and combined editing in rhesus macaque CD34⁺ cells. Combined editing significantly increased HbF expression without affecting cell viability or differentiation, outperforming single-site approaches.

Figure 2. Long-term editing frequency and γ-globin expression in rhesus macaques following transplantation of autologous CD34+ HSPCs with combined +58 and +55 BCL11A enhancer editing

In vivo tests showed stable engraftment of edited CD34+ cells in rhesus macaques over 4 years, with no toxicity. HbF levels peaked at 2 months and remained stable. Phlebotomy and hydroxyurea (HU) further boosted HbF in edited cells, unlike controls, enhancing therapeutic flexibility.

Figure 3. Combined edited autologous CD34+ cells stably engraft in multiple blood cell lineages

2. Phlebotomy and Hydroxyurea (HU) Enhance HbF Levels

Stress erythropoiesis via phlebotomy and HU administration significantly increased HbF in edited cells, demonstrating heightened sensitivity to γ-globin induction.

Figure 4. Stress erythropoiesis significantly increases F-cell proportion and γ-globin levels, with no significant change in the control group.

3. Targeted, High-Quality Mutations

Analysis via ddPCR and deep sequencing showed combined editing induced precise 3.1 kb deletions/inversions at GATA1 binding sites, with minimal (<2.2%) aberrant edits in vivo, ensuring predictability.

Figure 5. Combined editing of +58 and +55 enhancers disrupts critical GATA binding sites in engrafted rhesus macaque CD34⁺ HSPCs.

4. High Off-Target Safety

CIRCLE-seq and deep sequencing detected minimal off-target edits (<0.24%), confirming high biosafety of the editing strategy.

Figure 6. No off-target activity detected in engrafted CD34⁺ HSPCs after combined +58 and +55 enhancer editing.

5. CD45-ADC Preconditioning

CD45-ADC replaced toxic busulfan for preconditioning, achieving comparable engraftment with reduced side effects, advancing chemotherapy-free gene therapy.

Figure 7. Autologous CD34+ cells with combined BCL11A enhancer editing, preconditioned with CD45-ADC, maintain high editing rates and significantly upregulate γ-globin in rhesus macaques.

This study validates dual-enhancer BCL11A editing with CD45-ADC preconditioning as a safe, effective gene therapy for β-hemoglobinopathies, offering a robust foundation for clinical applications.

Key Targets Overview:

BCL11A: A Critical Suppressor of HbF Expression

BCL11A (B-cell leukemia/lymphoma 11A) is a zinc-finger transcription factor that plays a pivotal role in hematopoiesis, particularly in the fetal-to-adult hemoglobin switch by suppressing γ-globin (HbF) expression. It is highly expressed in erythroid cells, where it binds to the HbF promoter or recruits chromatin-remodeling complexes to downregulate γ-globin transcription.

Recent studies have identified that BCL11A expression in erythroid cells is regulated by multiple enhancer elements within its second intron, with the +58 site being the most active, followed by +55. Targeting these enhancer regions achieves erythroid-specific BCL11A suppression, relieving HbF repression without systemic inactivation, thus minimizing off-target risks. Clinical trials, such as Vertex/CRISPR Therapeutics’ exa-cel (CTX001), have employed CRISPR or shRNA to target BCL11A enhancers, yielding positive results for sickle cell disease and β-thalassemia treatment.

CD45: A Target for Non-Chemotherapy Myeloablation

CD45 (also known as PTPRC) is a transmembrane protein tyrosine phosphatase expressed on all leukocytes, essential for immune cell development and signaling. Its widespread expression on hematopoietic stem/progenitor cells, lymphocytes, and myeloid cells makes it an ideal target for clearing bone marrow hematopoietic cells. The CD45 antibody-drug conjugate (CD45-ADC) used in this study combines an anti-CD45 antibody with a cytotoxic PBD dimer, serie killing of CD45⁺ cells while avoiding the nonspecific toxicity of traditional chemotherapy (e.g., hair loss, infertility, DNA damage). Compared to chemotherapy, CD45-ADC offers superior specificity, lower side effects, and dose-dependent control, validated in humanized mouse and non-human primate models for myeloablative capacity.

CAT: An Antioxidant Enzyme for Oxidative Stress Mitigation

CAT (Catalase) is an enzyme that catalyzes the decomposition of hydrogen peroxide into water and oxygen, protecting cells from oxidative damage caused by reactive oxygen species. In β-hemoglobinopathies like sickle cell disease and β-thalassemia, increased oxidative stress contributes to red blood cell damage and clinical symptoms. Enhancing CAT activity could complement gene therapies by reducing oxidative burden on hematopoietic cells, improving overall therapeutic outcomes.

abinScience is a France-based biotechnology company specializing in the development and production of high-quality research reagents. We are committed to providing innovative, reliable tools and technical solutions for life science researchers worldwide. Leveraging advanced technology platforms and stringent quality control systems, abinScience has established a comprehensive product portfolio in research areas such as autoimmune diseases, bacteria and viruses, neuroscience, and immune targets, covering antibodies, recombinant proteins, assay kits, and functional research tools. Our products, with high sensitivity and specificity, provide robust support for translational research.

List of BCL11A, CD45, and CAT-Related Proteins and Antibodies from abinScience: