Gene therapy and gene editing strategies in inherited blood disorders.

Gene therapy has shown significant potential in treating various diseases, particularly inherited blood disorders such as hemophilia, sickle cell disease, and thalassemia. Advances in understanding the regulatory network of disease-associated genes have led to the identification of additional therapeutic targets for treatment, especially for ß-hemoglobinopathies. Erythroid regulatory factor BCL11A offers the most promising therapeutic target for ß-hemoglobinopathies and reduction of its expression using the commercialized gene therapy product Casgevy was approved for use in the UK and USA in 2023. Notably, the emergence of innovative gene editing technologies has further broadened the gene therapy landscape, presenting new possibilities for treatment. Intensive studies indicate that base editing and prime editing, built upon CRISPR technology, enable precise single-base modification in hematopoietic stem cells for addressing inherited blood disorders ex vivo and in vivo. In this review, we present an overview of the current landscape of gene therapies, focusing on clinical research and gene therapy products for inherited blood disorders, evaluation of potential gene targets, and the gene editing tools employed in current gene therapy practices, which provides an insight for the establishment of safer and more effective gene therapy methods for a wider range of diseases in the future.

CRISPR-Based Gene Therapies: From Preclinical to Clinical Treatments.

In recent years, clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) protein have emerged as a revolutionary gene editing tool to treat inherited disorders affecting different organ systems, such as blood and muscles. Both hematological and neuromuscular genetic disorders benefit from genome editing approaches but face different challenges in their clinical translation. The ability of CRISPR/Cas9 technologies to modify hematopoietic stem cells ex vivo has greatly accelerated the development of genetic therapies for blood disorders. In the last decade, many clinical trials were initiated and are now delivering encouraging results. The recent FDA approval of Casgevy, the first CRISPR/Cas9-based drug for severe sickle cell disease and transfusion-dependent ß-thalassemia, represents a significant milestone in the field and highlights the great potential of this technology. Similar preclinical efforts are currently expanding CRISPR therapies to other hematologic disorders such as primary immunodeficiencies. In the neuromuscular field, the versatility of CRISPR/Cas9 has been instrumental for the generation of new cellular and animal models of Duchenne muscular dystrophy (DMD), offering innovative platforms to speed up preclinical development of therapeutic solutions. Several corrective interventions have been proposed to genetically restore dystrophin production using the CRISPR toolbox and have demonstrated promising results in different DMD animal models. Although these advances represent a significant step forward to the clinical translation of CRISPR/Cas9 therapies to DMD, there are still many hurdles to overcome, such as in vivo delivery methods associated with high viral vector doses, together with safety and immunological concerns. Collectively, the results obtained in the hematological and neuromuscular fields emphasize the transformative impact of CRISPR/Cas9 for patients affected by these debilitating conditions. As each field suffers from different and specific challenges, the clinical translation of CRISPR therapies may progress differentially depending on the genetic disorder. Ongoing investigations and clinical trials will address risks and limitations of these therapies, including long-term efficacy, potential genotoxicity, and adverse immune reactions. This review provides insights into the diverse applications of CRISPR-based technologies in both preclinical and clinical settings for monogenic blood disorders and muscular dystrophy and compare advances in both fields while highlighting current trends, difficulties, and challenges to overcome.

Scalable noninvasive amplicon-based precision sequencing (SNAPseq) for genetic diagnosis and screening of ß-thalassemia and sickle cell disease using a next-generation sequencing platform.

ß-hemoglobinopathies such as ß-thalassemia (BT) and Sickle cell disease (SCD) are inherited monogenic blood disorders with significant global burden. Hence, early and affordable diagnosis can alleviate morbidity and reduce mortality given the lack of effective cure. Currently, Sanger sequencing is considered to be the gold standard genetic test for BT and SCD, but it has a very low throughput requiring multiple amplicons and more sequencing reactions to cover the entire HBB gene. To address this, we have demonstrated an extraction-free single amplicon-based approach for screening the entire ß-globin gene with clinical samples using Scalable noninvasive amplicon-based precision sequencing (SNAPseq) assay catalyzing with next-generation sequencing (NGS). We optimized the assay using noninvasive buccal swab samples and simple finger prick blood for direct amplification with crude lysates. SNAPseq demonstrates high sensitivity and specificity, having a 100% agreement with Sanger sequencing. Furthermore, to facilitate seamless reporting, we have created a much simpler automated pipeline with comprehensive resources for pathogenic mutations in BT and SCD through data integration after systematic classification of variants according to ACMG and AMP guidelines. To the best of our knowledge, this is the first report of the NGS-based high throughput SNAPseq approach for the detection of both BT and SCD in a single assay with high sensitivity in an automated pipeline.

Base editing of the HBG promoter induces potent fetal hemoglobin expression with no detectable off-target mutations in human HSCs.

Reactivating silenced γ-globin expression through the disruption of repressive regulatory domains offers a therapeutic strategy for treating ß-hemoglobinopathies. Here, we used transformer base editor (tBE), a recently developed cytosine base editor with no detectable off-target mutations, to disrupt transcription-factor-binding motifs in hematopoietic stem cells. By performing functional screening of six motifs with tBE, we found that directly disrupting the BCL11A-binding motif in HBG1/2 promoters triggered the highest γ-globin expression. Via a side-by-side comparison with other clinical and preclinical strategies using Cas9 nuclease or conventional BEs (ABE8e and hA3A-BE3), we found that tBE-mediated disruption of the BCL11A-binding motif at the HBG1/2 promoters triggered the highest fetal hemoglobin in healthy and ß-thalassemia patient hematopoietic stem/progenitor cells while exhibiting no detectable DNA or RNA off-target mutations. Durable therapeutic editing by tBE persisted in repopulating hematopoietic stem cells, demonstrating that tBE-mediated editing in HBG1/2 promoters is a safe and effective strategy for treating ß-hemoglobinopathies.

miR-365-3p mediates BCL11A and SOX6 erythroid-specific coregulation: A new player in HbF activation.

Hemoglobin switching is a complex biological process not yet fully elucidated. The mechanism regulating the suppression of fetal hemoglobin (HbF) expression is of particular interest because of the positive impact of HbF on the course of diseases such as ß-thalassemia and sickle cell disease, hereditary hemoglobin disorders that affect the health of countless individuals worldwide. Several transcription factors have been implicated in the control of HbF, of which BCL11A has emerged as a major player in HbF silencing. SOX6 has also been implicated in silencing HbF and is critical to the silencing of the mouse embryonic hemoglobins. BCL11A and SOX6 are co-expressed and physically interact in the erythroid compartment during differentiation. In this study, we observe that BCL11A knockout leads to post-transcriptional downregulation of SOX6 through activation of microRNA (miR)-365-3p. Downregulating SOX6 by transient ectopic expression of miR-365-3p or gene editing activates embryonic and fetal ß-like globin gene expression in erythroid cells. The synchronized expression of BCL11A and SOX6 is crucial for hemoglobin switching. In this study, we identified a BCL11A/miR-365-3p/SOX6 evolutionarily conserved pathway, providing insights into the regulation of the embryonic and fetal globin genes suggesting new targets for treating ß-hemoglobinopathies.

Molecular basis of a high Hb A2/Hb Fß-thalassemia trait: a retrospective analysis, genotype-phenotype interaction, diagnostic implication, and identification of a novel interaction with α-globin gene triplication.

Background: ß 0-thalassemia deletion removing 5´ß-globin promoter usually presents phenotype with high hemoglobin (Hb) A2 and Hb F levels. We report the molecular characteristics and phenotype-genotype correlation in a large cohort of the ß 0-thalassemia with 3.4 kb deletion. Methods: A total of 148 subjects, including 127 heterozygotes, 20 Hb E-ß-thalassemia patients, and a double heterozygote with α-globin gene triplication, were recruited. Hb and DNA analysis were performed to identify thalassemia mutations and four high Hb F single nucleotide polymorphisms (SNPs) including four base pair deletion (-AGCA) at A γ-globin promoter, rs5006884 on OR51B6 gene, -158 G γ-XmnI, BCL11A binding motifs (TGGTCA) between 3´A γ-globin gene and 5´Î´-globin gene. Results: It was found that heterozygous ß 0-thalassemia and Hb E-ß 0-thalassemia with 3.4 kb deletion had significantly higher Hb, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin and Hb F values as compared with those with other mutations. Co-inheritance of heterozygous ß 0-thalassemia with 3.4 kb deletion and α-thalassemia was associated with even higher MCV and MCH values. The Hb E-ß 0-thalassemia patients carried a non-transfusion-dependent thalassemia phenotype with an average Hb of around 10 g/dL without blood transfusion. A hitherto undescribed double heterozygous ß 0-thalassemia with 3.4 kb deletion and α-globin gene triplication presented as a plain ß-thalassemia trait. Most of the subjects had wild-type sequences for the four high Hb F SNPs examined. No significant difference in Hb F was observed between those of subjects with and without these SNPs. Removal of the 5´ß-globin promoter may likely be responsible for this unusual phenotype. Conclusions: The results indicate that ß 0-thalassemia with 3.4 kb deletion is a mild ß-thalassemia allele. This information should be provided at genetic counseling and prenatal thalassemia diagnosis.

Gene Therapy for ß-Hemoglobinopathies: From Discovery to Clinical Trials.

Investigations to understand the function and control of the globin genes have led to some of the most exciting molecular discoveries and biomedical breakthroughs of the 20th and 21st centuries. Extensive characterization of the globin gene locus, accompanied by pioneering work on the utilization of viruses as human gene delivery tools in human hematopoietic stem and progenitor cells (HPSCs), has led to transformative and successful therapies via autologous hematopoietic stem-cell transplant with gene therapy (HSCT-GT). Due to the advanced understanding of the ß-globin gene cluster, the first diseases considered for autologous HSCT-GT were two prevalent ß-hemoglobinopathies: sickle cell disease and ß-thalassemia, both affecting functional ß-globin chains and leading to substantial morbidity. Both conditions are suitable for allogeneic HSCT; however, this therapy comes with serious risks and is most effective using an HLA-matched family donor (which is not available for most patients) to obtain optimal therapeutic and safe benefits. Transplants from unrelated or haplo-identical donors carry higher risks, although they are progressively improving. Conversely, HSCT-GT utilizes the patient's own HSPCs, broadening access to more patients. Several gene therapy clinical trials have been reported to have achieved significant disease improvement, and more are underway. Based on the safety and the therapeutic success of autologous HSCT-GT, the U.S. Food and Drug Administration (FDA) in 2022 approved an HSCT-GT for ß-thalassemia (Zynteglo™). This review illuminates the ß-globin gene research journey, adversities faced, and achievements reached; it highlights important molecular and genetic findings of the ß-globin locus, describes the predominant globin vectors, and concludes by describing promising results from clinical trials for both sickle cell disease and ß-thalassemia.

Epigenetic Regulation of ß-Globin Genes and the Potential to Treat Hemoglobinopathies through Epigenome Editing.

Beta-like globin gene expression is developmentally regulated during life by transcription factors, chromatin looping and epigenome modifications of the ß-globin locus. Epigenome modifications, such as histone methylation/demethylation and acetylation/deacetylation and DNA methylation, are associated with up- or down-regulation of gene expression. The understanding of these mechanisms and their outcome in gene expression has paved the way to the development of new therapeutic strategies for treating various diseases, such as ß-hemoglobinopathies. Histone deacetylase and DNA methyl-transferase inhibitors are currently being tested in clinical trials for hemoglobinopathies patients. However, these approaches are often uncertain, non-specific and their global effect poses serious safety concerns. Epigenome editing is a recently developed and promising tool that consists of a DNA recognition domain (zinc finger, transcription activator-like effector or dead clustered regularly interspaced short palindromic repeats Cas9) fused to the catalytic domain of a chromatin-modifying enzyme. It offers a more specific targeting of disease-related genes (e.g., the ability to reactivate the fetal γ-globin genes and improve the hemoglobinopathy phenotype) and it facilitates the development of scarless gene therapy approaches. Here, we summarize the mechanisms of epigenome regulation of the ß-globin locus, and we discuss the application of epigenome editing for the treatment of hemoglobinopathies.

The heme-regulated inhibitor kinase requires dimerization for heme-sensing activity.

The heme-regulated inhibitor (HRI) is a heme-sensing kinase that regulates mRNA translation in erythroid cells. In heme deficiency, HRI is activated to phosphorylate eukaryotic initiation factor 2α and halt production of globins, thus avoiding accumulation of heme-free globin chains. HRI is inhibited by heme via binding to one or two heme-binding domains within the HRI N-terminal and kinase domains. HRI has recently been found to inhibit fetal hemoglobin (HbF) production in adult erythroid cells. Depletion of HRI increases HbF production, presenting a therapeutically exploitable target for the treatment of patients with sickle cell disease or thalassemia, which benefit from elevated HbF levels. HRI is known to be an oligomeric enzyme that is activated through autophosphorylation, although the exact nature of the HRI oligomer, its relation to autophosphorylation, and its mode of heme regulation remain unclear. Here, we employ biochemical and biophysical studies to demonstrate that HRI forms a dimeric species that is not dependent on autophosphorylation, the C-terminal coiled-coil domain in HRI is essential for dimer formation, and dimer formation facilitates efficient autophosphorylation and activation of HRI. We also employ kinetic studies to demonstrate that the primary avenue by which heme inhibits HRI is through the heme-binding site within the kinase domain, and that this inhibition is relatively independent of binding of ATP and eukaryotic initiation factor 2α substrates. Together, these studies highlight the mode of heme inhibition and the importance of dimerization in human HRI heme-sensing activity.

Effective therapies for sickle cell disease: are we there yet?

Sickle cell disease (SCD) is a common genetic blood disorder associated with acute and chronic pain, progressive multiorgan damage, and early mortality. Recent advances in technologies to manipulate the human genome, a century of research and the development of techniques enabling the isolation, efficient genetic modification, and reimplantation of autologous patient hematopoietic stem cells (HSCs), mean that curing most patients with SCD could soon be a reality in wealthy countries. In parallel, ongoing research is pursuing more facile treatments, such as in-vivo-delivered genetic therapies and new drugs that can eventually be administered in low- and middle-income countries where most SCD patients reside.

Development of a double shmiR lentivirus effectively targeting both BCL11A and ZNF410 for enhanced induction of fetal hemoglobin to treat ß-hemoglobinopathies.

A promising treatment for ß-hemoglobinopathies is the de-repression of γ-globin expression leading to increased fetal hemoglobin (HbF) by targeting BCL11A. Here, we aim to improve a lentivirus vector (LV) containing a single BCL11A shmiR (SS) to further increase γ-globin induction. We engineered a novel LV to express two shmiRs simultaneously targeting BCL11A and the γ-globin repressor ZNF410. Erythroid cells derived from human HSCs transduced with the double shmiR (DS) showed up to a 70% reduction of both BCL11A and ZNF410 proteins. There was a consistent and significant additional 10% increase in HbF compared to targeting BCL11A alone in erythroid cells. Erythrocytes differentiated from SCD HSCs transduced with the DS demonstrated significantly reduced in vitro sickling phenotype compared to the SS. Erythrocytes differentiated from transduced HSCs from ß-thalassemia major patients demonstrated improved globin chain balance by increased γ-globin with reduced microcytosis. Reconstitution of DS-transduced cells from Berkeley SCD mice was associated with a statistically larger reduction in peripheral blood hemolysis markers compared with the SS vector. Overall, these results indicate that the DS LV targeting BCL11A and ZNF410 can enhance HbF induction for treating ß-hemoglobinopathies and could be used as a model to simultaneously and efficiently target multiple gene products.

CRISPR/Cas9-based multiplex genome editing of BCL11A and HBG efficiently induces fetal hemoglobin expression.

Beta-hemoglobinopathies are caused by mutations in the ß-globin gene. One strategy to cure this disease relies on re-activating the γ-globin expression. BCL11A is an important transcription factor that suppresses the γ-globin expression, which makes it one of the most promising therapeutic targets in ß-hemoglobinopathies. Here, we performed single-gene editing and multiplex gene editing via CRISPR/Cas9 technology to edit BCL11A erythroid-specific enhancer and BCL11A binding site on γ-globin gene promoter in HUDEP-2 cells and adult human CD34+ cells. Multiplex gene editing led to higher γ-globin expression than single-gene editing without inhibiting erythroid differentiation. By further optimizing the on-target DNA editing efficiency of multiplex gene editing, the percentage of F-cells exceeded 50% in HUDEP-2 cells. Amplicon deep sequencing and whole genome sequencing were used to detect the editing frequency of on- and potential off-target sites in CD34+ cells. No off-target mutations were detected, suggesting its accuracy in HSPCs. In summary, our study provides a new approach which can be used for the treatment of ß-hemoglobinopathies in the future.

Acyclovir induces fetal hemoglobin via downregulation of γ-globin repressors, BCL11A and SOX6 trans-acting factors.

Pharmacological reactivation of developmentally silenced fetal hemoglobin (HbF) is an attractive approach to ameliorate the clinical manifestations of ß-thalassemia and sickle cell anemia. Hydroxyurea, the only HbF inducer, has obtained regulatory approval. However, hydroxyurea non-responders and associated myelosuppression making its widespread use undesirable. A high level of HbF with safe and effective agents remains an elusive therapeutic goal for this global health burden. This study demonstrated the effect of acyclovir on γ-globin expression and erythropoiesis, associated with increased HbF production. In vitro, human erythroleukemia cells and human CD34+ erythroid progenitors, and in vivo ß-YAC transgenic mice were used as experimental models. We found that acyclovir significantly induces expression of the γ-globin gene and HbF synthesis in CD34+ erythroid progenitors, without affecting terminal erythroid differentiation and erythroid cell proliferation. In contrast to other HbF inducers, no associated cytotoxicity with acyclovir was observed. Further, we reported the effect of acyclovir on γ-globin gene transcriptional regulators including BCL11A, FOP1, KLF1 SOX6, and GATA-1. Significant downregulation of the γ-globin repressors BCL11A and SOX6 was observed at both mRNA and protein levels. Whereas, GATA-1, a master erythroid transcription factor, was upregulated in acyclovir treated human CD34+ erythroid culture. Similarly, the HbF inducing effect of acyclovir in ß-YAC transgenic mice revealed a good in vitro correlation, with a substantial increase in fetal globin mRNA, and F cells population. These findings collectively suggest acyclovir as an effective HbF inducer and pave the way to evaluate its clinical efficacy in treating ß-globin disorders.

Reverse Phase-high-performance Liquid Chromatography (RP-HPLC) Analysis of Globin Chains from Human Erythroid Cells.

ß-hemoglobinopathies are severe genetic disorders characterized either by the abnormal synthesis of the adult ß-globin chains of the hemoglobin (Hb) tetramer (ßS-globin chains) in sickle cell disease (SCD) or by the reduced ß-globin production in ß-thalassemia. The identification and quantification of globin chains are crucial for the diagnosis of these diseases and for testing new therapeutic approaches aimed at correcting the ß-hemoglobinopathy phenotype. Conventional techniques to detect the different Hb molecules include cellulose-acetate electrophoresis (CEA), capillary electrophoresis (CE), isoelectric focusing (IEF), and cation-exchange-HPLC (CE-HPLC). However, these methods cannot distinguish the different globin chains and precisely determine their relative expression. We have set up a high-resolution and reproducible reverse phase-HPLC (RP-HPLC) to detect and identify the globin chains composing the hemoglobin tetramers based on their different hydrophobic properties. RP-HPLC mobile phases are composed of acetonitrile (ACN) that creates a hydrophobic environment and trifluoroacetic acid (TFA), which breaks the heme group within the Hb tetramers releasing individual globin chains. Hb-containing lysates are loaded onto the AerisTM 3.6-µm WIDEPORE C4 200 Å LC Column and a gradient of increasing hydrophobicity of the mobile phase over time allows globin chain separation. The relative amount of globin chains is measured at a wavelength (λ) of 220 nm. This protocol is designed for evaluating globin chains in (i) red blood cells (RBCs) obtained from human peripheral blood, (ii) RBCs in vitro differentiated from hematopoietic stem/progenitor cells (HSPCs), and (iii) burst-forming unit-erythroid (BFU-E), i.e., erythroid progenitors obtained in vitro from human peripheral blood or in vitro cultured HSPCs. This technique allows to precisely identify the different globin chains and obtain a relative quantification. RP-HPLC can be used to confirm the diagnosis of ß-hemoglobinopathies, to evaluate the disease severity and validate novel approaches for the treatment of these diseases.

Genome Editing for ß-Hemoglobinopathies: Advances and Challenges.

ß-hemoglobinopathies are the most common genetic disorders worldwide and are caused by mutations affecting the production or the structure of adult hemoglobin. Patients affected by these diseases suffer from anemia, impaired oxygen delivery to tissues, and multi-organ damage. In the absence of a compatible donor for allogeneic bone marrow transplantation, the lifelong therapeutic options are symptomatic care, red blood cell transfusions and pharmacological treatments. The last decades of research established lentiviral-mediated gene therapy as an efficacious therapeutic strategy. However, this approach is highly expensive and associated with a variable outcome depending on the effectiveness of the viral vector and the quality of the cell product. In the last years, genome editing emerged as a valuable tool for the development of curative strategies for ß-hemoglobinopathies. Moreover, due to the wide range of its applications, genome editing has been extensively used to study regulatory mechanisms underlying globin gene regulation allowing the identification of novel genetic and pharmacological targets. In this work, we review the current advances and challenges of genome editing approaches to ß-hemoglobinopathies. Special focus has been directed towards strategies aimed at correcting the defective ß-globin gene or at inducing fetal hemoglobin (HbF), which are in an advanced state of clinical development.

The methyltransferase PRMT1 regulates γ-globin translation.

Induction of fetal hemoglobin to overcome adult ß-globin gene deficiency is an effective therapeutic strategy to ameliorate human ß-hemoglobinopathies. Previous work has revealed that fetal γ-globin can be translationally induced via integrated stress signaling, but other studies have indicated that activating stress may eventually suppress γ-globin expression transcriptionally. The mechanism by which γ-globin expression is regulated at the translational level remains largely unknown, limiting our ability to determine whether activating stress is a realistic therapeutic option for these disorders. In this study, we performed a functional CRISPR screen targeting protein arginine methyltransferases (PRMTs) to look for changes in γ-globin expression in K562 cells. We not only discovered that several specific PRMTs may block γ-globin transcription, but also revealed PRMT1 as a unique family member that is able to suppress γ-globin synthesis specifically at the translational level. We further identified that a non-AUG uORF within the 5' untranslated region of γ-globin serves as a barrier for translation, which is bypassed upon PRMT1 deficiency. Finally, we found that this novel mechanism of γ-globin suppression could be pharmacologically targeted by the PRMT1 inhibitor, furamidine dihydrochloride. These data raise new questions regarding methyltransferase function and may offer a new therapeutic direction for ß-hemoglobinopathies.

Targeted Protein Degradation as a Promising Tool for Epigenetic Upregulation of Fetal Hemoglobin.

The level of fetal hemoglobin (HbF) is an important disease modifier for ß-thalassemia and sickle cell disease patients. Indeed, genetic tinkering with the HbF repression machinery has demonstrated great potential for disease mitigation. Such genetic treatments are costly and the high incidence of ß-hemoglobinopathies in low-income countries, therefore, calls for the development of affordable, off-the-shelf, oral treatments. The use of PROTAC (PRoteolysis TArgeting Chimeras) technology to influence the epigenetic mechanisms involved in HbF suppression may provide a solution. In this minireview, we briefly explain the HbF repression network highlighting the epigenetic factors that could be targeted for degradation by PROTACs. We hope that this review will inspire clinicians, molecular and chemical biologists to collaborate and contribute to this fascinating field, which should ultimately deliver drugs that reactivate HbF expression with high specificity and low toxicity.

Comparative analysis of lentiviral gene transfer approaches designed to promote fetal hemoglobin production for the treatment of ß-hemoglobinopathies.

ß-Hemoglobinopathies are among the most common single-gene disorders and are caused by different mutations in the ß-globin gene. Recent curative therapeutic approaches for these disorders utilize lentiviral vectors (LVs) to introduce a functional copy of the ß-globin gene into the patient's hematopoietic stem cells. Alternatively, fetal hemoglobin (HbF) can reduce or even prevent the symptoms of disease when expressed in adults. Thus, induction of HbF by means of LVs and other molecular approaches has become an alternative treatment of ß-hemoglobinopathies. Here, we performed a head-to-head comparative analysis of HbF-inducing LVs encoding for: 1) IGF2BP1, 2) miRNA-embedded shRNA (shmiR) sequences specific for the γ-globin repressor protein BCL11A, and 3) γ-globin gene. Furthermore, two novel baboon envelope proteins (BaEV)-LVs were compared to the commonly used vesicular-stomatitis-virus glycoprotein (VSV-G)-LVs. Therapeutic levels of HbF were achieved for all VSV-G-LV approaches, from a therapeutic level of 20% using γ-globin LVs to 50% for both IGF2BP1 and BCL11A-shmiR LVs. Contrarily, BaEV-LVs conferred lower HbF expression with a peak level of 13%, however, this could still ameliorate symptoms of disease. From this thorough comparative analysis of independent HbF-inducing LV strategies, we conclude that HbF-inducing VSV-G-LVs represent a promising alternative to ß-globin gene addition for patients with ß-hemoglobinopathies.

ß-Hemoglobinopathies: The Test Bench for Genome Editing-Based Therapeutic Strategies.

Hemoglobin is a tetrameric protein composed of two α and two ß chains, each containing a heme group that reversibly binds oxygen. The composition of hemoglobin changes during development in order to fulfill the need of the growing organism, stably maintaining a balanced production of α-like and ß-like chains in a 1:1 ratio. Adult hemoglobin (HbA) is composed of two α and two ß subunits (α2ß2 tetramer), whereas fetal hemoglobin (HbF) is composed of two γ and two α subunits (α2γ2 tetramer). Qualitative or quantitative defects in ß-globin production cause two of the most common monogenic-inherited disorders: ß-thalassemia and sickle cell disease. The high frequency of these diseases and the relative accessibility of hematopoietic stem cells make them an ideal candidate for therapeutic interventions based on genome editing. These strategies move in two directions: the correction of the disease-causing mutation and the reactivation of the expression of HbF in adult cells, in the attempt to recreate the effect of hereditary persistence of fetal hemoglobin (HPFH) natural mutations, which mitigate the severity of ß-hemoglobinopathies. Both lines of research rely on the knowledge gained so far on the regulatory mechanisms controlling the differential expression of globin genes during development.